Molecular Biology and Biochemistry, Department of

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Derivation of HLA Types from Shotgun Sequence Datasets

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2012
Abstract: 

The human leukocyte antigen (HLA) is key to many aspects of human physiology and medicine. All current sequence-based HLA typing methodologies are targeted approaches requiring the amplification of specific HLA gene segments. Whole genome, exome and transcriptome shotgun sequencing can generate prodigious data but due to the complexity of HLA loci these data have not been immediately informative regarding HLA genotype. We describe HLAminer, a computational method for identifying HLA alleles directly from shotgun sequence datasets (http://www.bcgsc.ca/platform/bioinfo/software/hlaminer webcite). This approach circumvents the additional time and cost of generating HLA-specific data and capitalizes on the increasing accessibility and affordability of massively parallel sequencing.

Document type: 
Article

Coovar: Co-Occurring Variant Analyzer

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2012
Abstract: 

Background

Evaluating the impact of genomic variations (GV) on protein-coding transcripts is an important step in identifying variants of functional significance. Currently available programs for variant annotation depend on external databases or annotate multiple variants affecting the same transcript independently, which limits program use to organisms available in these databases or results in potentially incorrect or incomplete annotations.

Findings

We have developed CooVar (Co-occurring Variant Analyzer), a database-independent program for assessing the impact of GVs on protein-coding transcripts. CooVar takes GVs, reference genome sequence, and protein-coding exons as input and provides annotated GVs and transcripts as output. Other than similar programs, CooVar considers the combined impact of all GVs affecting the same transcript, generating biologically more accurate annotations. CooVar is operated from the command-line and supports standard file formats VCF, GFF/GTF, and GVF, which makes it easy to integrate into existing computational pipelines. We have extensively tested CooVar on worm and human data sets and demonstrate that it generates correct annotations in only a short amount of time.

Conclusions

CooVar is an easy-to-use and lightweight variant annotation tool that considers the combined impact of GVs on protein-coding transcripts. CooVar is freely available at http://genome.sfu.ca/projects/coovar/ webcite.

Document type: 
Article

An Integrated Strategy to Study Muscle Development and Myofilament Structure in Caenorhabditis elegans

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2009-06-26
Abstract: 

A crucial step in the development of muscle cells in all metazoan animals is the assembly and anchorage of the sarcomere, the essential repeat unit responsible for muscle contraction. In Caenorhabditis elegans, many of the critical proteins involved in this process have been uncovered through mutational screens focusing on uncoordinated movement and embryonic arrest phenotypes. We propose that additional sarcomeric proteins exist for which there is a less severe, or entirely different, mutant phenotype produced in their absence. We have used Serial Analysis of Gene Expression (SAGE) to generate a comprehensive profile of late embryonic muscle gene expression. We generated two replicate long SAGE libraries for sorted embryonic muscle cells, identifying 7,974 protein-coding genes. A refined list of 3,577 genes expressed in muscle cells was compiled from the overlap between our SAGE data and available microarray data. Using the genes in our refined list, we have performed two separate RNA interference (RNAi) screens to identify novel genes that play a role in sarcomere assembly and/or maintenance in either embryonic or adult muscle. To identify muscle defects in embryos, we screened specifically for the Pat embryonic arrest phenotype. To visualize muscle defects in adult animals, we fed dsRNA to worms producing a GFP-tagged myosin protein, thus allowing us to analyze their myofilament organization under gene knockdown conditions using fluorescence microscopy. By eliminating or severely reducing the expression of 3,300 genes using RNAi, we identified 122 genes necessary for proper myofilament organization, 108 of which are genes without a previously characterized role in muscle. Many of the genes affecting sarcomere integrity have human homologs for which little or nothing is known.

Document type: 
Article
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Smelt Was the Likely Beneficiary of an Antifreeze Gene Laterally Transferred Between Fishes

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2012
Abstract: 

Background

Type II antifreeze protein (AFP) from the rainbow smelt, Osmerus mordax, is a calcium-dependent C-type lectin homolog, similar to the AFPs from herring and sea raven. While C-type lectins are ubiquitous, type II AFPs are only found in a few species in three widely separated branches of teleost fishes. Furthermore, several other non-homologous AFPs are found in intervening species. We have previously postulated that this sporadic distribution has resulted from lateral gene transfer. The alternative hypothesis, that the AFP evolved from a lectin present in a shared ancestor and that this gene was lost in most species, is not favored because both the exon and intron sequences are highly conserved.

Results

Here we have sequenced and annotated a 160 kb smelt BAC clone containing a centrally-located AFP gene along with 14 other genes. Quantitative PCR indicates that there is but a single copy of this gene within the smelt genome, which is atypical for fish AFP genes. The corresponding syntenic region has been identified and searched in a number of other species and found to be devoid of lectin or AFP sequences. Unlike the introns of the AFP gene, the intronic sequences of the flanking genes are not conserved between species. As well, the rate and pattern of mutation in the AFP gene are radically different from those seen in other smelt and herring genes.

Conclusions

These results provide stand-alone support for an example of lateral gene transfer between vertebrate species. They should further inform the debate about genetically modified organisms by showing that gene transfer between ‘higher’ eukaryotes can occur naturally. Analysis of the syntenic regions from several fishes strongly suggests that the smelt acquired the AFP gene from the herring.

Document type: 
Article

Steroid Hormone Control of Cell Death and Cell Survival: Molecular Insights Using RNAi

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2009-02-13
Abstract: 

The insect steroid hormone ecdysone triggers programmed cell death of obsolete larval tissues during metamorphosis and provides a model system for understanding steroid hormone control of cell death and cell survival. Previous genome-wide expression studies of Drosophila larval salivary glands resulted in the identification of many genes associated with ecdysone-induced cell death and cell survival, but functional verification was lacking. In this study, we test functionally 460 of these genes using RNA interference in ecdysone-treated Drosophila l(2)mbn cells. Cell viability, cell morphology, cell proliferation, and apoptosis assays confirmed the effects of known genes and additionally resulted in the identification of six new pro-death related genes, including sorting nexin-like gene SH3PX1 and Sox box protein Sox14, and 18 new pro-survival genes. Identified genes were further characterized to determine their ecdysone dependency and potential function in cell death regulation. We found that the pro-survival function of five genes (Ras85D, Cp1, CG13784, CG32016, and CG33087), was dependent on ecdysone signaling. The TUNEL assay revealed an additional two genes (Kap-α3 and Smr) with an ecdysone-dependent cell survival function that was associated with reduced cell death. In vitro, Sox14 RNAi reduced the percentage of TUNEL-positive l(2)mbn cells (p<0.05) following ecdysone treatment, and Sox14 overexpression was sufficient to induce apoptosis. In vivo analyses of Sox14-RNAi animals revealed multiple phenotypes characteristic of aberrant or reduced ecdysone signaling, including defects in larval midgut and salivary gland destruction. These studies identify Sox14 as a positive regulator of ecdysone-mediated cell death and provide new insights into the molecular mechanisms underlying the ecdysone signaling network governing cell death and cell survival.

Document type: 
Article
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Chromosomal Differences between European and North American Atlantic salmon Discovered by Linkage Mapping and Supported by Fluorescence in situ Hybridization Analysis

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2012
Abstract: 

BACKGROUND:Geographical isolation has generated a distinct difference between Atlantic salmon of European and North American Atlantic origin. The European Atlantic salmon generally has 29 pairs of chromosomes and 74 chromosome arms whereas it has been reported that the North American Atlantic salmon has 27 chromosome pairs and an NF of 72. In order to predict the major chromosomal rearrangements causing these differences, we constructed a dense linkage map for Atlantic salmon of North American origin and compared it with the well-developed map for European Atlantic salmon.RESULTS:The presented male and female genetic maps for the North American subspecies of Atlantic salmon, contains 3,662 SNPs located on 27 linkage groups. The total lengths of the female and male linkage maps were 2,153cM and 968cM respectively, with males characteristically showing recombination only at the telomeres. We compared these maps with recently published SNP maps from European Atlantic salmon, and predicted three chromosomal reorganization events that we then tested using fluorescence in situ hybridization (FISH) analysis. The proposed rearrangements, which define the differences in the karyotypes of the North American Atlantic salmon relative to the European Atlantic salmon, include the translocation of the p arm of ssa01 to ssa23 and polymorphic fusions: ssa26 with ssa28, and ssa08 with ssa29.CONCLUSIONS:This study identified major chromosomal differences between European and North American Atlantic salmon. However, while gross structural differences were significant, the order of genetic markers at the fine-resolution scale was remarkably conserved. This is a good indication that information from the International Cooperation to Sequence the Atlantic salmon Genome, which is sequencing a European Atlantic salmon, can be transferred to Atlantic salmon from North America.

Document type: 
Article
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Effect of Stress on Viral–Bacterial Synergy in Bovine Respiratory Disease: Novel Mechanisms to Regulate Inflammation

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2006
Abstract: 

The severity of bovine respiratory infections has been linked to a variety offactors, including environmental and nutritional changes, transportation, and socialreorganization of weaned calves. Fatal respiratory infections, however, usually occurwhen a primary viral infection compromises host defences and enhances the severityof a secondary bacterial infection. This viral–bacterial synergy can occur by a numberof different mechanisms and disease challenge models have been developed to analysehost responses during these respiratory infections. A primary bovine herpesvirus-1(BHV-1) respiratory infection followed by a secondary challenge with Mannheimia haemolyticaresults in fatal bovine respiratory disease (BRD) and host responses to these two pathogens have been studied extensively. We used this disease model todemonstrate that stress significantly altered the viral–bacterial synergy resulting infatal BRD. Functional genomic analysis revealed that BHV-1 infection enhanced toll-likereceptors (TLR) expression and increased pro-inflammatory responses whichcontribute to the severity of a Mannheimia haemolytica infection. TLRs play a criticalrole in detecting bacterial infections and inducing pro-inflammatory responses. It isdifficult to understand, however, how stress-induced corticosteroids could enhancethis form of viral–bacterial synergy. Nuclear translocation of the glucocorticoidreceptor activates cell signalling pathways which inhibit both TLR signallingand pro-inflammatory responses. The apparent conundrum between stress-inducedcorticosteroids and enhanced BRD susceptibility is discussed in terms of present data and previous investigations of stress and respiratory disease.

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Article
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Inhibition of Drosophila Wg Signaling Involves Competition between Mad and Armadillo/β-Catenin for dTcf Binding

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2008-12
Abstract: 

Precisely regulated signal transduction pathways are crucial for the regulation of developmental events and prevention of tumorigenesis. Both the Transforming Growth Factor β (TGFβ)/Bone morphogenetic protein (BMP) and Wnt/Wingless (Wg) pathways play essential roles in organismal patterning and growth, and their deregulation can lead to cancers. We describe a mechanism of interaction between Drosophila Wg and BMP signaling in which Wg target gene expression is antagonized by BMP signaling. In vivo, high levels of both an activated BMP receptor and the BMP effector Mad can inhibit the expression of Wg target genes. Conversely, loss of mad can induce Wg target gene expression. In addition, we find that ectopic expression in vivo of the Wg transcription factor dTcf is able to suppress the inhibitory effect caused by ectopic Mad. In vitro binding studies revealed competition for dTcf binding between Mad and the Wnt effector β-catenin/Armadillo (Arm). Our in vivo genetic analyses and target gene studies support a mechanism consistent with the in vitro binding and competition studies, namely that BMP pathway components can repress Wg target gene expression by influencing the binding of Arm and dTcf.

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Article
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An Essential Role for DYF-11/MIP-T3 in Assembling Functional Intraflagellar Transport Complexes

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2008-03
Abstract: 

MIP-T3 is a human protein found previously to associate with microtubules and the kinesin-interacting neuronal protein DISC1 (Disrupted-in-Schizophrenia 1), but whose cellular function(s) remains unknown. Here we demonstrate that the C. elegans MIP-T3 ortholog DYF-11 is an intraflagellar transport (IFT) protein that plays a critical role in assembling functional kinesin motor-IFT particle complexes. We have cloned a loss of function dyf-11 mutant in which several key components of the IFT machinery, including Kinesin-II, as well as IFT subcomplex A and B proteins, fail to enter ciliary axonemes and/or mislocalize, resulting in compromised ciliary structures and sensory functions, and abnormal lipid accumulation. Analyses in different mutant backgrounds further suggest that DYF-11 functions as a novel component of IFT subcomplex B. Consistent with an evolutionarily conserved cilia-associated role, mammalian MIP-T3 localizes to basal bodies and cilia, and zebrafish mipt3 functions synergistically with the Bardet-Biedl syndrome protein Bbs4 to ensure proper gastrulation, a key cilium- and basal body-dependent developmental process. Our findings therefore implicate MIP-T3 in a previously unknown but critical role in cilium biogenesis and further highlight the emerging role of this organelle in vertebrate development.

Document type: 
Article
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Phylogenetic Analysis of the Neks Reveals Early Diversification of Ciliary-Cell Cycle Kinases

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2007-10
Abstract: 

NIMA-related kinases (Neks) have been studied in diverse eukaryotes, including the fungus Aspergillus and the ciliate Tetrahymena. In the former, a single Nek plays an essential role in cell cycle regulation; in the latter, which has more than 30 Neks in its genome, multiple Neks regulate ciliary length. Mammalian genomes encode an intermediate number of Neks, several of which are reported to play roles in cell cycle regulation and/or localize to centrosomes. Previously, we reported that organisms with cilia typically have more Neks than organisms without cilia, but were unable to establish the evolutionary history of the gene family.

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