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1.  Cytochrome b Divergence between Avian Sister Species Is Linked to Generation Length and Body Mass 
PLoS ONE  2014;9(2):e85006.
It is increasingly realised that the molecular clock does not tick at a constant rate. Rather, mitochondrial mutation rates are influenced by factors such as generation length and body mass. This has implications for the use of genetic data in species delimitation. It could be that speciation, as recognised by avian taxonomists, is associated with a certain minimum genetic distance between sister taxa, in which case we would predict no difference in the cytochrome b divergence of sister taxa according to the species' body size or generation time. Alternatively, if what taxonomists recognise as speciation has tended to be associated with the passage of a minimum amount of time since divergence, then there might be less genetic divergence between sister taxa with slower mutation rates, namely those that are heavier and/or with longer generation times. After excluding non-flying species, we analysed a database of over 600 avian sister species pairs, and found that species pairs with longer generation lengths (which tend to be the larger species) showed less cytochrome b divergence. This finding cautions against using any simple unitary criterion of genetic divergence to delimit species.
doi:10.1371/journal.pone.0085006
PMCID: PMC3914784  PMID: 24505250
2.  The Vertebrate Genome Annotation browser 10 years on 
Nucleic Acids Research  2013;42(D1):D771-D779.
The Vertebrate Genome Annotation (VEGA) database (http://vega.sanger.ac.uk), initially designed as a community resource for browsing manual annotation of the human genome project, now contains five reference genomes (human, mouse, zebrafish, pig and rat). Its introduction pages have been redesigned to enable the user to easily navigate between whole genomes and smaller multi-species haplotypic regions of interest such as the major histocompatibility complex. The VEGA browser is unique in that annotation is updated via the Human And Vertebrate Analysis aNd Annotation (HAVANA) update track every 2 weeks, allowing single gene updates to be made publicly available to the research community quickly. The user can now access different haplotypic subregions more easily, such as those from the non-obese diabetic mouse, and display them in a more intuitive way using the comparative tools. We also highlight how the user can browse manually annotated updated patches from the Genome Reference Consortium (GRC).
doi:10.1093/nar/gkt1241
PMCID: PMC3964964  PMID: 24316575
3.  Current status and new features of the Consensus Coding Sequence database  
Nucleic Acids Research  2013;42(D1):D865-D872.
The Consensus Coding Sequence (CCDS) project (http://www.ncbi.nlm.nih.gov/CCDS/) is a collaborative effort to maintain a dataset of protein-coding regions that are identically annotated on the human and mouse reference genome assemblies by the National Center for Biotechnology Information (NCBI) and Ensembl genome annotation pipelines. Identical annotations that pass quality assurance tests are tracked with a stable identifier (CCDS ID). Members of the collaboration, who are from NCBI, the Wellcome Trust Sanger Institute and the University of California Santa Cruz, provide coordinated and continuous review of the dataset to ensure high-quality CCDS representations. We describe here the current status and recent growth in the CCDS dataset, as well as recent changes to the CCDS web and FTP sites. These changes include more explicit reporting about the NCBI and Ensembl annotation releases being compared, new search and display options, the addition of biologically descriptive information and our approach to representing genes for which support evidence is incomplete. We also present a summary of recent and future curation targets.
doi:10.1093/nar/gkt1059
PMCID: PMC3965069  PMID: 24217909
4.  GRASP [Genomic Resource Access for Stoichioproteomics]: comparative explorations of the atomic content of 12 Drosophila proteomes 
BMC Genomics  2013;14:599.
Background
“Stoichioproteomics” relates the elemental composition of proteins and proteomes to variation in the physiological and ecological environment. To help harness and explore the wealth of hypotheses made possible under this framework, we introduce GRASP (http://www.graspdb.net), a public bioinformatic knowledgebase containing information on the frequencies of 20 amino acids and atomic composition of their side chains. GRASP integrates comparative protein composition data with annotation data from multiple public databases. Currently, GRASP includes information on proteins of 12 sequenced Drosophila (fruit fly) proteomes, which will be expanded to include increasingly diverse organisms over time. In this paper we illustrate the potential of GRASP for testing stoichioproteomic hypotheses by conducting an exploratory investigation into the composition of 12 Drosophila proteomes, testing the prediction that protein atomic content is associated with species ecology and with protein expression levels.
Results
Elements varied predictably along multivariate axes. Species were broadly similar, with the D. willistoni proteome a clear outlier. As expected, individual protein atomic content within proteomes was influenced by protein function and amino acid biochemistry. Evolution in elemental composition across the phylogeny followed less predictable patterns, but was associated with broad ecological variation in diet. Using expression data available for D. melanogaster, we found evidence consistent with selection for efficient usage of elements within the proteome: as expected, nitrogen content was reduced in highly expressed proteins in most tissues, most strongly in the gut, where nutrients are assimilated, and least strongly in the germline.
Conclusions
The patterns identified here using GRASP provide a foundation on which to base future research into the evolution of atomic composition in Drosophila and other taxa.
doi:10.1186/1471-2164-14-599
PMCID: PMC3844568  PMID: 24007337
Bioinformatics; Comparative-phylogenetic analysis; Ecological stoichiometry; Material costs; Nutrient limitation; Proteomics
5.  The non-obese diabetic mouse sequence, annotation and variation resource: an aid for investigating type 1 diabetes 
Model organisms are becoming increasingly important for the study of complex diseases such as type 1 diabetes (T1D). The non-obese diabetic (NOD) mouse is an experimental model for T1D having been bred to develop the disease spontaneously in a process that is similar to humans. Genetic analysis of the NOD mouse has identified around 50 disease loci, which have the nomenclature Idd for insulin-dependent diabetes, distributed across at least 11 different chromosomes. In total, 21 Idd regions across 6 chromosomes, that are major contributors to T1D susceptibility or resistance, were selected for finished sequencing and annotation at the Wellcome Trust Sanger Institute. Here we describe the generation of 40.4 mega base-pairs of finished sequence from 289 bacterial artificial chromosomes for the NOD mouse. Manual annotation has identified 738 genes in the diabetes sensitive NOD mouse and 765 genes in homologous regions of the diabetes resistant C57BL/6J reference mouse across 19 candidate Idd regions. This has allowed us to call variation consequences between homologous exonic sequences for all annotated regions in the two mouse strains. We demonstrate the importance of this resource further by illustrating the technical difficulties that regions of inter-strain structural variation between the NOD mouse and the C57BL/6J reference mouse can cause for current next generation sequencing and assembly techniques. Furthermore, we have established that the variation rate in the Idd regions is 2.3 times higher than the mean found for the whole genome assembly for the NOD/ShiLtJ genome, which we suggest reflects the fact that positive selection for functional variation in immune genes is beneficial in regard to host defence. In summary, we provide an important resource, which aids the analysis of potential causative genes involved in T1D susceptibility.
Database URLs: http://www.sanger.ac.uk/resources/mouse/nod/; http://vega-previous.sanger.ac.uk/info/data/mouse_regions.html
doi:10.1093/database/bat032
PMCID: PMC3668384  PMID: 23729657
6.  Structural and functional annotation of the porcine immunome 
BMC Genomics  2013;14:332.
Background
The domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems.
Results
The Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome.
Conclusions
This extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig’s adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response.
doi:10.1186/1471-2164-14-332
PMCID: PMC3658956  PMID: 23676093
Immune response; Porcine; Genome annotation; Co-expression network; Phylogenetic analysis; Accelerated evolution
7.  Sequencing and comparative analysis of the gorilla MHC genomic sequence 
Major histocompatibility complex (MHC) genes play a critical role in vertebrate immune response and because the MHC is linked to a significant number of auto-immune and other diseases it is of great medical interest. Here we describe the clone-based sequencing and subsequent annotation of the MHC region of the gorilla genome. Because the MHC is subject to extensive variation, both structural and sequence-wise, it is not readily amenable to study in whole genome shotgun sequence such as the recently published gorilla genome. The variation of the MHC also makes it of evolutionary interest and therefore we analyse the sequence in the context of human and chimpanzee. In our comparisons with human and re-annotated chimpanzee MHC sequence we find that gorilla has a trimodular RCCX cluster, versus the reference human bimodular cluster, and additional copies of Class I (pseudo)genes between Gogo-K and Gogo-A (the orthologues of HLA-K and -A). We also find that Gogo-H (and Patr-H) is coding versus the HLA-H pseudogene and, conversely, there is a Gogo-DQB2 pseudogene versus the HLA-DQB2 coding gene. Our analysis, which is freely available through the VEGA genome browser, provides the research community with a comprehensive dataset for comparative and evolutionary research of the MHC.
doi:10.1093/database/bat011
PMCID: PMC3626023  PMID: 23589541
8.  Microtubule plus-end tracking protein CLASP2 regulates neuronal polarity and synaptic function 
Microtubule organization and dynamics are essential during axon and dendrite formation and maintenance in neurons. However, little is known about the regulation of microtubule dynamics during synaptic development and function in mammalian neurons. Here, we present evidence that the microtubule plus-end tracking protein CLASP2 (cytoplasmic linker associated protein 2) is a key regulator of axon and dendrite outgrowth that leads to functional alterations in synaptic activity and formation. We found that CLASP2 protein levels steadily increase throughout neuronal development in the mouse brain and are specifically enriched at the growth cones of extending neurites. The shRNA-mediated knockdown of CLASP2 in primary mouse neurons decreased axon and dendritic length whereas overexpression of human CLASP2 caused the formation of multiple axons, enhanced dendritic branching, and Golgi condensation, implicating CLASP2 in neuronal morphogenesis. In addition, the CLASP2-induced morphological changes led to significant functional alterations in synaptic transmission. CLASP2 overexpression produced a large increase in spontaneous miniature event frequency that was specific to excitatory neurotransmitter release. The changes in presynaptic activity produced by CLASP2 overexpression were accompanied by increases in presynaptic terminal circumference, total synapse number and a selective increase in presynaptic proteins that are involved in neurotransmitter release. Also, we found a smaller increase in miniature event amplitude that was accompanied by an increase in postsynaptic surface expression of GluA1 receptor localization. Together, these results provide evidence for involvement of the microtubule plus-end tracking protein CLASP2 in cytoskeleton-related mechanisms underlying neuronal polarity and interplay between the microtubule stabilization and synapse formation and activity.
doi:10.1523/JNEUROSCI.2108-12.2012
PMCID: PMC3489028  PMID: 23035100
CLASP2; Golgi; axons; synapses; dendrites
9.  Myristoylation exerts direct and allosteric effects on Gα conformation and dynamics in solution 
Biochemistry  2012;51(9):1911-1924.
Coupling of heterotrimeric G proteins to activated G protein-coupled receptors results in nucleotide exchange on the Gα subunit, which in turn decreases its affinity for both Gβγ and activated receptors. N-terminal myristoylation of Gα subunits aids in membrane localization of inactive G proteins. Despite the presence of the covalently attached myristoyl group, Gα proteins are highly soluble after GTP binding. This study investigated factors facilitating the solubility of the activated, myristoylated protein. In doing so, we also identified myristoylation-dependent differences in regions of Gα known to play important roles in interactions with receptors, effectors, and nucleotide binding. Amide-hydrogen deuterium exchange and site-directed fluorescence of activated proteins revealed a solvent-protected amino terminus which was enhanced by myristoylation. Furthermore, fluorescence quenching confirmed that the myristoylated amino terminus lies in close proximity to the Switch II region in the activated protein. Myristoylation also stabilized the interaction between the guanine ring and the base of the α5 helix which contacts bound nucleotide. The allosteric effects of myristoylation on protein structure, function, and localization indicate that the myristoylated amino terminus of Gαi functions as a myristoyl switch, with implications for myristoylation in the stabilization of nucleotide binding and in the spatial regulation of G protein signaling.
doi:10.1021/bi201472c
PMCID: PMC3312377  PMID: 22329346
10.  Homeostatic regulation of AMPA receptor trafficking and degradation by light-controlled single synaptic activation 
Neuron  2011;72(5):806-818.
During homeostatic adjustment in response to alterations in neuronal activity, synaptic expression of AMPA receptors (AMPARs) is globally tuned up- or down so that the neuronal activity is restored to a physiological range. Given that a central neuron receives multiple presynaptic inputs, whether and how AMPAR synaptic expression is homeostatically regulated at individual synapses remains unclear. In cultured hippocampal neurons, we report that when activity of an individual presynaptic terminal is selectively elevated by light-controlled excitation, AMPAR abundance at the excited synapses is selectively down-regulated in an NMDAR-dependent manner. The reduction in surface AMPARs is accompanied by enhanced receptor endocytosis and dependent on proteasomal activity. Synaptic activation also leads to a site-specific increase in the ubiquitin ligase Nedd4 and polyubiquitination levels, consistent with AMPAR ubiquitination and degradation in the spine. These results indicate that AMPAR accumulation at individual synapses is subject to autonomous homeostatic regulation in response to synaptic activity.
doi:10.1016/j.neuron.2011.10.011
PMCID: PMC3240854  PMID: 22153376
11.  Accelerated Calvarial Healing in Mice Lacking Toll-Like Receptor 4 
PLoS ONE  2012;7(10):e46945.
The bone and immune systems are closely interconnected. The immediate inflammatory response after fracture is known to trigger a healing cascade which plays an important role in bone repair. Toll-like receptor 4 (TLR4) is a member of a highly conserved receptor family and is a critical activator of the innate immune response after tissue injury. TLR4 signaling has been shown to regulate the systemic inflammatory response induced by exposed bone components during long-bone fracture. Here we tested the hypothesis that TLR4 activation affects the healing of calvarial defects. A 1.8 mm diameter calvarial defect was created in wild-type (WT) and TLR4 knockout (TLR4−/−) mice. Bone healing was tested using radiographic, histologic and gene expression analyses. Radiographic and histomorphometric analyses revealed that calvarial healing was accelerated in TLR4−/− mice. More bone was observed in TLR4−/− mice compared to WT mice at postoperative days 7 and 14, although comparable healing was achieved in both groups by day 21. Bone remodeling was detected in both groups on postoperative day 28. In TLR4−/− mice compared to WT mice, gene expression analysis revealed that higher expression levels of IL-1β, IL-6, TNF-α,TGF-β1, TGF-β3, PDGF and RANKL and lower expression level of RANK were detected at earlier time points (≤ postoperative 4 days); while higher expression levels of IL-1β and lower expression levels of VEGF, RANK, RANKL and OPG were detected at late time points (> postoperative 4 days). This study provides evidence of accelerated bone healing in TLR4−/− mice with earlier and higher expression of inflammatory cytokines and with increased osteoclastic activity. Further work is required to determine if this is due to inflammation driven by TLR4 activation.
doi:10.1371/journal.pone.0046945
PMCID: PMC3468586  PMID: 23071670
12.  Nedd4-mediated AMPA receptor ubiquitination regulates receptor turnover and trafficking 
Journal of neurochemistry  2011;119(1):27-39.
AMPA receptors (AMPARs) are the primary mediators of excitatory synaptic transmission in the brain. Alterations in AMPAR localization and turnover have been considered critical mechanisms underpinning synaptic plasticity and higher brain functions, but the molecular processes that control AMPAR trafficking and stability are still not fully understood. Here, we report that mammalian AMPARs are subject to ubiquitination in neurons and in transfected heterologous cells. Ubiquitination facilitates AMPAR endocytosis, leading to a reduction in AMPAR cell-surface localization and total receptor abundance. Mutation of lysine residues to arginine residues at the GluA1 C-terminus dramatically reduces GluA1 ubiquitination and abolishes ubiquitin-dependent GluA1 internalization and degradation, indicating that the lysine residues, particularly K868, are sites of ubiquitination. We also find that the E3 ligase Nedd4 is enriched in synaptosomes and co-localizes and associates with AMPARs in neurons. Nedd4 expression leads to AMPAR ubiquitination, leading to reduced AMPAR surface expression and suppressed excitatory synaptic transmission. Conversely, knockdown of Nedd4 by specific siRNAs abolishes AMPAR ubiquitination. These data indicate that Nedd4 is the E3 ubiquitin ligase responsible for AMPAR ubiquitination, a modification that regulates multiple aspects of AMPAR molecular biology including trafficking, localization and stability.
doi:10.1111/j.1471-4159.2011.07221.x
PMCID: PMC3110981  PMID: 21338354
glutamate receptors; AMPA receptors; Nedd4; E3 ligase; ubiquitination; trafficking
13.  AMPA Receptor Trafficking in Homeostatic Synaptic Plasticity: Functional Molecules and Signaling Cascades 
Neural Plasticity  2012;2012:825364.
Homeostatic synaptic plasticity is a negative-feedback response employed to compensate for functional disturbances in the nervous system. Typically, synaptic activity is strengthened when neuronal firing is chronically suppressed or weakened when neuronal activity is chronically elevated. At both the whole cell and entire network levels, activity manipulation leads to a global up- or downscaling of the transmission efficacy of all synapses. However, the homeostatic response can also be induced locally at subcellular regions or individual synapses. Homeostatic synaptic scaling is expressed mainly via the regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking and synaptic expression. Here we review the recently identified functional molecules and signaling pathways that are involved in homeostatic plasticity, especially the homeostatic regulation of AMPAR localization at excitatory synapses.
doi:10.1155/2012/825364
PMCID: PMC3359728  PMID: 22655210
14.  Larger testes are associated with a higher level of polyandry, but a smaller ejaculate volume, across bushcricket species (Tettigoniidae) 
Biology Letters  2010;7(2):261-264.
While early models of ejaculate allocation predicted that both relative testes and ejaculate size should increase with sperm competition intensity across species, recent models predict that ejaculate size may actually decrease as testes size and sperm competition intensity increase, owing to the confounding effect of potential male mating rate. A recent study demonstrated that ejaculate volume decreased in relation to increased polyandry across bushcricket species, but testes mass was not measured. Here, we recorded testis mass for 21 bushcricket species, while ejaculate (ampulla) mass, nuptial gift mass, sperm number and polyandry data were largely obtained from the literature. Using phylogenetic-comparative analyses, we found that testis mass increased with the degree of polyandry, but decreased with increasing ejaculate mass. We found no significant relationship between testis mass and either sperm number or nuptial gift mass. While these results are consistent with recent models of ejaculate allocation, they could alternatively be driven by substances in the ejaculate that affect the degree of polyandry and/or by a trade-off between resources spent on testes mass versus non-sperm components of the ejaculate.
doi:10.1098/rsbl.2010.0840
PMCID: PMC3061181  PMID: 21068028
sperm competition; testes size; ejaculate size; polyandry; sexual selection
15.  Community gene annotation in practice 
Manual annotation of genomic data is extremely valuable to produce an accurate reference gene set but is expensive compared with automatic methods and so has been limited to model organisms. Annotation tools that have been developed at the Wellcome Trust Sanger Institute (WTSI, http://www.sanger.ac.uk/.) are being used to fill that gap, as they can be used remotely and so open up viable community annotation collaborations. We introduce the ‘Blessed’ annotator and ‘Gatekeeper’ approach to Community Annotation using the Otterlace/ZMap genome annotation tool. We also describe the strategies adopted for annotation consistency, quality control and viewing of the annotation.
Database URL: http://vega.sanger.ac.uk/index.html
doi:10.1093/database/bas009
PMCID: PMC3308165  PMID: 22434843
16.  Distribution of HSV-1 and VZV in Ganglia of the Human Head and Neck 
The Journal of infectious diseases  2009;200(12):1901-1906.
The distribution of the neurotropic alphaherpesviruses (HSV-1, HSV-2, and VZV) was determined in autonomic and sensory ganglia of the head and neck from formalin-fixed human cadavers. HSV-1 and VZV DNA were found in 18/58 and 16/58 trigeminal, 23/58 and 11/58 pterygopalatine, 25/60 and 14/60 ciliary, 25/48 and 11/48 geniculate, 15/50 and 8/50 otic, 14/47 and 4/47 submandibular, 18/58 and 10/58 superior cervical, and 12/36 and 1/36 nodose ganglia, respectively. HSV-2 was not detected in any site. Viral DNA positivity and location were independently distributed among autonomic and sensory ganglia of the human head and neck.
doi:10.1086/648474
PMCID: PMC2782560  PMID: 19919304
Herpes simplex virus (HSV); varicella-zoster virus (VZV); human ganglia; formalin-fixed tissue, DNA
19.  A NOVEL SHORT HAIRPIN RNA (SHRNA) EXPRESSION SYSTEM PROMOTES SOX9-DEPENDENT GENE SILENCING 
Plasmid  2009;62(1):50-55.
Cartilage development and function are dependent on a temporally integrated program of gene expression. With the advent of RNA interference (RNAi), artificial control of these complex programs becomes a possibility, limited only by the ability to regulate and express the RNAi. Using existing methods for production of RNAi’s, we have constructed a plasmid-based short hairpin RNA (shRNA) expression system under control of the human pol III H1 promoter and supplemented this promoter with DNA binding sites for the cartilage-specific transcription factor Sox9. The resulting shRNA expression system displays robust, Sox9-dependent gene silencing. Dependence on Sox9 expression was confirmed by electrophoretic mobility shift assays. The ability of the system to regulate heterologously expressed Sox9 was demonstrated by Western blot, as a function of both Sox9 to shRNA ratio, as well as time from transfection. This novel expression system supports auto-regulatory gene silencing, providing a tissue-specific feedback mechanism for temporal control of gene expression. Its applications for both basic mechanistic studies and therapeutic purposes should facilitate the design and implementation of innovative tissue engineering strategies.
doi:10.1016/j.plasmid.2009.04.001
PMCID: PMC2760318  PMID: 19389425
Sox9; shRNA; tissue-specific; regulated expression; inducible gene silencing
20.  Manual annotation and analysis of the defensin gene cluster in the C57BL/6J mouse reference genome 
BMC Genomics  2009;10:606.
Background
Host defense peptides are a critical component of the innate immune system. Human alpha- and beta-defensin genes are subject to copy number variation (CNV) and historically the organization of mouse alpha-defensin genes has been poorly defined. Here we present the first full manual genomic annotation of the mouse defensin region on Chromosome 8 of the reference strain C57BL/6J, and the analysis of the orthologous regions of the human and rat genomes. Problems were identified with the reference assemblies of all three genomes. Defensins have been studied for over two decades and their naming has become a critical issue due to incorrect identification of defensin genes derived from different mouse strains and the duplicated nature of this region.
Results
The defensin gene cluster region on mouse Chromosome 8 A2 contains 98 gene loci: 53 are likely active defensin genes and 22 defensin pseudogenes. Several TATA box motifs were found for human and mouse defensin genes that likely impact gene expression. Three novel defensin genes belonging to the Cryptdin Related Sequences (CRS) family were identified. All additional mouse defensin loci on Chromosomes 1, 2 and 14 were annotated and unusual splice variants identified. Comparison of the mouse alpha-defensins in the three main mouse reference gene sets Ensembl, Mouse Genome Informatics (MGI), and NCBI RefSeq reveals significant inconsistencies in annotation and nomenclature. We are collaborating with the Mouse Genome Nomenclature Committee (MGNC) to establish a standardized naming scheme for alpha-defensins.
Conclusions
Prior to this analysis, there was no reliable reference gene set available for the mouse strain C57BL/6J defensin genes, demonstrating that manual intervention is still critical for the annotation of complex gene families and heavily duplicated regions. Accurate gene annotation is facilitated by the annotation of pseudogenes and regulatory elements. Manually curated gene models will be incorporated into the Ensembl and Consensus Coding Sequence (CCDS) reference sets. Elucidation of the genomic structure of this complex gene cluster on the mouse reference sequence, and adoption of a clear and unambiguous naming scheme, will provide a valuable tool to support studies on the evolution, regulatory mechanisms and biological functions of defensins in vivo.
doi:10.1186/1471-2164-10-606
PMCID: PMC2807441  PMID: 20003482
21.  Accessing out-of-hours care following implementation of the GMS contract: an observational study 
Background
There is widespread concern that the quality of out-of-hours primary care for patients with complex needs may be at risk now that the new general medical services contract (GMS) has been implemented.
Aim
To explore changes in the use of out-of-hours services around the time of implementation of the new contract for patients with complex needs, using patients with cancer as an example.
Design of study
Longitudinal observational study.
Setting
Out-of-hours primary care provider covering Devon (adult population 900 000), UK.
Method
Two, 1-year periods corresponding to pre- (April 2003 to March 2004) and post-contract implementation (October 2004 to September 2005) were sampled. Call rates per 1000 of the adult population (age ≥16 years) were calculated for all calls (any cause) and cancer-related calls. Anonymised outcome and process measures data were extracted.
Results
Although overall call rates per 1000 population had increased by 26% (185 pre-contract to 233 post-contract), the proportion of cancer-related calls remained relatively constant (2.08% versus 1.96%). Around half (56%) of these callers had advanced cancer needs (including palliative care). By post-contract, the time taken to triage had significantly increased (P<0.001). Although the proportions admitted to hospital or receiving a home visit remained constant, calls where a special message was sent by the out-of-hours clinician to the in-hours team had decreased (P<0.001).
Conclusion
The demand for out-of-hours care for patients with cancer did not alter disproportionately after implementation of the contract. While potential quality indicators (for example, hospital admissions, home visiting rates) remained constant, potentially adverse changes to triage time and communication between out-of-hours and in-hours clinicians were observed. Quality standards and provider databases require further refinement to capture elements of care relevant to patients with complex needs.
doi:10.3399/bjgp08X280191
PMCID: PMC2435660  PMID: 18482487
cancer care; cohort study; healthcare delivery; out-of-hours medical care; primary health care
22.  DNA sequence of human chromosome 17 and analysis of rearrangement in the human lineage 
Zody, Michael C. | Garber, Manuel | Adams, David J. | Sharpe, Ted | Harrow, Jennifer | Lupski, James R. | Nicholson, Christine | Searle, Steven M. | Wilming, Laurens | Young, Sarah K. | Abouelleil, Amr | Allen, Nicole R. | Bi, Weimin | Bloom, Toby | Borowsky, Mark L. | Bugalter, Boris E. | Butler, Jonathan | Chang, Jean L. | Chen, Chao-Kung | Cook, April | Corum, Benjamin | Cuomo, Christina A. | de Jong, Pieter J. | DeCaprio, David | Dewar, Ken | FitzGerald, Michael | Gilbert, James | Gibson, Richard | Gnerre, Sante | Goldstein, Steven | Grafham, Darren V. | Grocock, Russell | Hafez, Nabil | Hagopian, Daniel S. | Hart, Elizabeth | Norman, Catherine Hosage | Humphray, Sean | Jaffe, David B. | Jones, Matt | Kamal, Michael | Khodiyar, Varsha K. | LaButti, Kurt | Laird, Gavin | Lehoczky, Jessica | Liu, Xiaohong | Lokyitsang, Tashi | Loveland, Jane | Lui, Annie | Macdonald, Pendexter | Major, John E. | Matthews, Lucy | Mauceli, Evan | McCarroll, Steven A. | Mihalev, Atanas H. | Mudge, Jonathan | Nguyen, Cindy | Nicol, Robert | O'Leary, Sinéad B. | Osoegawa, Kazutoyo | Schwartz, David C. | Shaw-Smith, Charles | Stankiewicz, Pawel | Steward, Charles | Swarbreck, David | Venkataraman, Vijay | Whittaker, Charles A. | Yang, Xiaoping | Zimmer, Andrew R. | Bradley, Allan | Hubbard, Tim | Birren, Bruce W. | Rogers, Jane | Lander, Eric S. | Nusbaum, Chad
Nature  2006;440(7087):1045-1049.
Chromosome 17 is unusual among the human chromosomes in many respects. It is the largest human autosome with orthology to only a single mouse chromosome1, mapping entirely to the distal half of mouse chromosome 11. Chromosome 17 is rich in protein-coding genes, having the second highest gene density in the genome2,3. It is also enriched in segmental duplications, ranking third in density among the autosomes4. Here we report a finished sequence for human chromosome 17, as well as a structural comparison with the finished sequence for mouse chromosome 11, the first finished mouse chromosome. Comparison of the orthologous regions reveals striking differences. In contrast to the typical pattern seen in mammalian evolution5,6, the human sequence has undergone extensive intrachromosomal rearrangement, whereas the mouse sequence has been remarkably stable. Moreover, although the human sequence has a high density of segmental duplication, the mouse sequence has a very low density. Notably, these segmental duplications correspond closely to the sites of structural rearrangement, demonstrating a link between duplication and rearrangement. Examination of the main classes of duplicated segments provides insight into the dynamics underlying expansion of chromosome-specific, low-copy repeats in the human genome.
doi:10.1038/nature04689
PMCID: PMC2610434  PMID: 16625196
23.  Transforming Growth Factor β1 regulates fibronectin isoform expression and splicing factor SRp40 expression during ATDC5 chondrogenic maturation 
Experimental cell research  2007;313(8):1518-1532.
Fibronectin (FN) isoform expression is altered during chondrocyte commitment and maturation, with cartilage favoring expression of FN isoforms that include the type II repeat extra domain B (EDB) but exclude extra domain A (EDA). We and others have hypothesized that the regulated splicing of FN mRNAs is necessary for progression of chondrogenesis. To test this, we treated the pre-chondrogenic cell line ATDC5 with transforming growth factor -β1, which has been shown to modulate expression of the EDA and EDB exons, as well as the late markers of chondrocyte maturation; it also slightly accelerates the early acquisition of a sulfated proteoglycan matrix without affecting cell proliferation. When chondrocytes are treated with TGF-β1, the EDA exon is preferentially excluded at all times whereas the EDB exon is relatively depleted at early times. This regulated alternative splicing of FN correlates with regulation of alternative splicing of SRp40, a splicing factor facilitating inclusion of the EDA exon. To determine if overexpression of the SRp40 isoforms altered FN and FN EDA organization, cDNAs encoding these isoforms were overexpressed in ATDC5 cells. Overexpression of the long-form of SRp40 yielded a FN organization similar to TGF-β1 treatment; whereas overexpression of the short form of SRp40 (which facilitates EDA inclusion) increased formation of long-thick FN fibrils. Therefore, we conclude that the effects of TGF-β1 on FN splicing during chondrogenesis may be largely dependent on its effect on SRp40 isoform expression.
doi:10.1016/j.yexcr.2007.01.028
PMCID: PMC1920702  PMID: 17391668
Fibronectin; TGF-β1; SRp40; alternative splicing; collagen type II; collagen type I; Alcian blue
24.  Variation analysis and gene annotation of eight MHC haplotypes: The MHC Haplotype Project 
Immunogenetics  2008;60(1):1-18.
The human major histocompatibility complex (MHC) is contained within about 4 Mb on the short arm of chromosome 6 and is recognised as the most variable region in the human genome. The primary aim of the MHC Haplotype Project was to provide a comprehensively annotated reference sequence of a single, human leukocyte antigen-homozygous MHC haplotype and to use it as a basis against which variations could be assessed from seven other similarly homozygous cell lines, representative of the most common MHC haplotypes in the European population. Comparison of the haplotype sequences, including four haplotypes not previously analysed, resulted in the identification of >44,000 variations, both substitutions and indels (insertions and deletions), which have been submitted to the dbSNP database. The gene annotation uncovered haplotype-specific differences and confirmed the presence of more than 300 loci, including over 160 protein-coding genes. Combined analysis of the variation and annotation datasets revealed 122 gene loci with coding substitutions of which 97 were non-synonymous. The haplotype (A3-B7-DR15; PGF cell line) designated as the new MHC reference sequence, has been incorporated into the human genome assembly (NCBI35 and subsequent builds), and constitutes the largest single-haplotype sequence of the human genome to date. The extensive variation and annotation data derived from the analysis of seven further haplotypes have been made publicly available and provide a framework and resource for future association studies of all MHC-associated diseases and transplant medicine.
doi:10.1007/s00251-007-0262-2
PMCID: PMC2206249  PMID: 18193213
Major histocompatibility complex; Haplotype; Polymorphism; Retroelement; Genetic predisposition to disease; Population genetics
25.  Lessons learned from the initial sequencing of the pig genome: comparative analysis of an 8 Mb region of pig chromosome 17 
Genome Biology  2007;8(8):R168.
The sequencing, annotation and comparative analysis of an 8Mb region of pig chromosome 17 allows the coverage and quality of the pig genome sequencing project to be assessed
Background
We describe here the sequencing, annotation and comparative analysis of an 8 Mb region of pig chromosome 17, which provides a useful test region to assess coverage and quality for the pig genome sequencing project. We report our findings comparing the annotation of draft sequence assembled at different depths of coverage.
Results
Within this region we annotated 71 loci, of which 53 are orthologous to human known coding genes. When compared to the syntenic regions in human (20q13.13-q13.33) and mouse (chromosome 2, 167.5 Mb-178.3 Mb), this region was found to be highly conserved with respect to gene order. The most notable difference between the three species is the presence of a large expansion of zinc finger coding genes and pseudogenes on mouse chromosome 2 between Edn3 and Phactr3 that is absent from pig and human. All of our annotation has been made publicly available in the Vertebrate Genome Annotation browser, VEGA. We assessed the impact of coverage on sequence assembly across this region and found, as expected, that increased sequence depth resulted in fewer, longer contigs. One-third of our annotated loci could not be fully re-aligned back to the low coverage version of the sequence, principally because the transcripts are fragmented over several contigs.
Conclusion
We have demonstrated the considerable advantages of sequencing at increased read depths and discuss the implications that lower coverage sequence may have on subsequent comparative and functional studies, particularly those involving complex loci such as GNAS.
doi:10.1186/gb-2007-8-8-r168
PMCID: PMC2374978  PMID: 17705864

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