Molecular Biology and Biochemistry, Department of

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The Homeobox Genes of Caenorhabditis elegans and Insights into Their Spatio-Temporal Expression Dynamics during Embryogenesis

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2015
Abstract: 

Homeobox genes play crucial roles for the development of multicellular eukaryotes. We have generated a revised list of all homeobox genes for Caenorhabditis elegans and provide a nomenclature for the previously unnamed ones. We show that, out of 103 homeobox genes, 70 are co-orthologous to human homeobox genes. 14 are highly divergent, lacking an obvious ortholog even in other Caenorhabditis species. One of these homeobox genes encodes 12 homeodomains, while three other highly divergent homeobox genes encode a novel type of double homeodomain, termed HOCHOB. To understand how transcription factors regulate cell fate during development, precise spatio-temporal expression data need to be obtained. Using a new imaging framework that we developed, Endrov, we have generated spatio-temporal expression profiles during embryogenesis of over 60 homeobox genes, as well as a number of other developmental control genes using GFP reporters. We used dynamic feedback during recording to automatically adjust the camera exposure time in order to increase the dynamic range beyond the limitations of the camera. We have applied the new framework to examine homeobox gene expression patterns and provide an analysis of these patterns. The methods we developed to analyze and quantify expression data are not only suitable for C. elegans, but can be applied to other model systems or even to tissue culture systems.

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Article
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Genome-Wide Association Study (GWAS) for Growth Rate and Age at Sexual Maturation in Atlantic Salmon (Salmo salar)

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2015
Abstract: 

Early sexual maturation is considered a serious drawback for Atlantic salmon aquaculture as it retards growth, increases production times and affects flesh quality. Although both growth and sexual maturation are thought to be complex processes controlled by several genetic and environmental factors, selection for these traits has been continuously accomplished since the beginning of Atlantic salmon selective breeding programs. In this genome-wide association study (GWAS) we used a 6.5K single-nucleotide polymorphism (SNP) array to genotype ∼480 individuals from the Cermaq Canada broodstock program and search for SNPs associated with growth and age at sexual maturation. Using a mixed model approach we identified markers showing a significant association with growth, grilsing (early sexual maturation) and late sexual maturation. The most significant associations were found for grilsing, with markers located in Ssa10, Ssa02, Ssa13, Ssa25 and Ssa12, and for late maturation with markers located in Ssa28, Ssa01 and Ssa21. A lower level of association was detected with growth on Ssa13. Candidate genes, which were linked to these genetic markers, were identified and some of them show a direct relationship with developmental processes, especially for those in association with sexual maturation. However, the relatively low power to detect genetic markers associated with growth (days to 5 kg) in this GWAS indicates the need to use a higher density SNP array in order to overcome the low levels of linkage disequilibrium observed in Atlantic salmon before the information can be incorporated into a selective breeding program.

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Article
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Do Housekeeping Genes Exist?

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2015
Abstract: 

The searching of human housekeeping (HK) genes has been a long quest since the emergence of transcriptomics, and is instrumental for us to understand the structure of genome and the fundamentals of biological processes. The resolved genes are frequently used in evolution studies and as normalization standards in quantitative gene-expression analysis. Within the past 20 years, more than a dozen HK-gene studies have been conducted, yet none of them sampled human tissues completely. We believe an integration of these results will help remove false positive genes owing to the inadequate sampling. Surprisingly, we only find one common gene across 15 examined HK-gene datasets comprising 187 different tissue and cell types. Our subsequent analyses suggest that it might not be appropriate to rigidly define HK genes as expressed in all tissue types that have diverse developmental, physiological, and pathological states. It might be beneficial to use more robustly identified HK functions for filtering criteria, in which the representing genes can be a subset of genome. These genes are not necessarily the same, and perhaps need not to be the same, everywhere in our body.

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Article
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G-Quadruplex Structures Formed by Expanded Hexanucleotide Repeat RNA and DNA from the Neurodegenerative Disease-Linked C9orf72 Gene Efficiently Sequester and Activate Heme

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2014-09-10
Abstract: 

The expansion of a (G4C2)n repeat within the human C9orf72 gene has been causally linked to a number of neurodegenerative diseases, most notably familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies have shown that the repeat expansion alters gene function in four ways, disrupting the gene's normal cellular roles and introducing toxic gain of function at the level of both DNA and RNA. (G4C2)n DNA, as well as the RNA transcribed from it, are found to fold into four-stranded G-quadruplex structures. It has been shown that the toxicity of the RNA G-quadruplexes, often localized in intracellular RNA foci, lies in their ability to sequester many important RNA binding proteins. Herein we propose that a distinct toxic property of such RNA and DNA G-quadruplexes from the C9orf72 gene may arise from their ability to bind and oxidatively activate cellular heme. We show that G-quadruplexes formed by both (G4C2)4 RNA and DNA not only complex tightly with heme but also enhance its intrinsic peroxidase and oxidase propensities. By contrast, the antisense (C4G2)4 RNA and DNA neither bind heme nor influence its oxidative activity. Curiously, the ability of C9orf72 DNA and transcripts to bind and activate heme mirror similar properties that have been reported for the Aβ peptide and its oligomers in Alzheimer's disease neurons. It is therefore conceivable that C9orf72 RNA G-quadruplex tangles play roles in sequestering intracellular heme and promoting oxidative damage in ALS and FTD analogous to those proposed for Aβ peptide and its tangles in Alzheimer's Disease. Given that neurodegenerative diseases in general are characterized by mitochondrial and respiratory malfunctions, the role of C9orf72 DNA and RNA in heme sequestration as well as its inappropriate activation in ALS and FTD neurons may warrant examination.

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Article
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Genetic Spectrum of Autosomal Recessive Non-Syndromic Hearing Loss in Pakistani Families

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2014-06-20
Abstract: 

The frequency of inherited bilateral autosomal recessive non-syndromic hearing loss (ARNSHL) in Pakistan is 1.6/1000 individuals. More than 50% of the families carry mutations in GJB2 while mutations in MYO15A account for about 5% of recessive deafness. In the present study a cohort of 30 ARNSHL families was initially screened for mutations in GJB2 and MYO15A. Homozygosity mapping was performed by employing whole genome single nucleotide polymorphism (SNP) genotyping in the families that did not carry mutations in GJB2 or MYO15A. Mutation analysis was performed for the known ARNSHL genes present in the homozygous regions to determine the causative mutations. This allowed the identification of a causative mutation in all the 30 families including 9 novel mutations, which were identified in 9 different families (GJB2 (c.598G>A, p.Gly200Arg); MYO15A (c.9948G>A, p.Gln3316Gln; c.3866+1G>A; c.8767C>T, p.Arg2923* and c.8222T>C, p.Phe2741Ser), TMC1 (c.362+18A>G), BSND (c.97G>C, p.Val33Leu), TMPRSS3 (c.726C>G, p.Cys242Trp) and MSRB3 (c.20T>G, p.Leu7Arg)). Furthermore, 12 recurrent mutations were detected in 21 other families. The 21 identified mutations included 10 (48%) missense changes, 4 (19%) nonsense mutations, 3 (14%) intronic mutations, 2 (9%) splice site mutations and 2 (9%) frameshift mutations. GJB2 accounted for 53% of the families, while mutations in MYO15A were the second most frequent (13%) cause of ARNSHL in these 30 families. The identification of novel as well as recurrent mutations in the present study increases the spectrum of mutations in known deafness genes which could lead to the identification of novel founder mutations and population specific mutated deafness genes causative of ARNSHL. These results provide detailed genetic information that has potential diagnostic implication in the establishment of cost-efficient allele-specific analysis of frequently occurring variants in combination with other reported mutations in Pakistani populations.

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Article
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Regulation of Mitotic Cytoskeleton Dynamics and Cytokinesis by Integrin-Linked Kinase in Retinoblastoma Cells

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2014-06-09
Abstract: 

During cell division integrin-linked kinase (ILK) has been shown to regulate microtubule dynamics and centrosome clustering, processes involved in cell cycle progression, and malignant transformation. In this study, we examine the effects of downregulating ILK on mitotic function in human retinoblastoma cell lines. These retinal cancer cells, caused by the loss of function of two gene alleles (Rb1) that encode the retinoblastoma tumour suppressor, have elevated expression of ILK. Here we show that inhibition of ILK activity results in a concentration-dependent increase in nuclear area and multinucleated cells. Moreover, inhibition of ILK activity and expression increased the accumulation of multinucleated cells over time. In these cells, aberrant cytokinesis and karyokinesis correlate with altered mitotic spindle organization, decreased levels of cortical F-actin and centrosome de-clustering. Centrosome de-clustering, induced by ILK siRNA, was rescued in FLAG-ILK expressing Y79 cells as compared to those expressing FLAG-tag alone. Inhibition of ILK increased the proportion of cells exhibiting mitotic spindles and caused a significant G2/M arrest as early as 24 hours after exposure to QLT-0267. Live cell analysis indicate ILK downregulation causes an increase in multipolar anaphases and failed cytokinesis (bipolar and multipolar) of viable cells. These studies extend those indicating a critical function for ILK in mitotic cytoskeletal organization and describe a novel role for ILK in cytokinesis of Rb deficient cells.

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Article
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Pharmacological Inhibition of O-GlcNAcase (OGA) Prevents Cognitive Decline and Amyloid Plaque Formation in Bigenic Tau/APP Mutant Mice

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2014
Abstract: 

Background

Amyloid plaques and neurofibrillary tangles (NFTs) are the defining pathological hallmarks of Alzheimer’s disease (AD). Increasing the quantity of the O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification of nuclear and cytoplasmic proteins slows neurodegeneration and blocks the formation of NFTs in a tauopathy mouse model. It remains unknown, however, if O-GlcNAc can influence the formation of amyloid plaques in the presence of tau pathology.

Results

We treated double transgenic TAPP mice, which express both mutant human tau and amyloid precursor protein (APP), with a highly selective orally bioavailable inhibitor of the enzyme responsible for removing O-GlcNAc (OGA) to increase O-GlcNAc in the brain. We find that increased O-GlcNAc levels block cognitive decline in the TAPP mice and this effect parallels decreased β-amyloid peptide levels and decreased levels of amyloid plaques.

Conclusions

This study indicates that increased O-GlcNAc can influence β-amyloid pathology in the presence of tau pathology. The findings provide good support for OGA as a promising therapeutic target to alter disease progression in Alzheimer disease.

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Article
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Whole-Genome Sequencing Of Mesorhizobium huakuii 7653R Provides Molecular Insights into Host Specificity and Symbiosis Island Dynamics

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2014
Abstract: 

Background

Evidence based on genomic sequences is urgently needed to confirm the phylogenetic relationship between Mesorhizobium strain MAFF303099 and M. huakuii. To define underlying causes for the rather striking difference in host specificity between M. huakuii strain 7653R and MAFF303099, several probable determinants also require comparison at the genomic level. An improved understanding of mobile genetic elements that can be integrated into the main chromosomes of Mesorhizobium to form genomic islands would enrich our knowledge of how genome dynamics may contribute to Mesorhizobium evolution in general.

Results

In this study, we sequenced the complete genome of 7653R and compared it with five other Mesorhizobium genomes. Genomes of 7653R and MAFF303099 were found to share a large set of orthologs and, most importantly, a conserved chromosomal backbone and even larger perfectly conserved synteny blocks. We also identified candidate molecular differences responsible for the different host specificities of these two strains. Finally, we reconstructed an ancestral Mesorhizobium genomic island that has evolved into diverse forms in different Mesorhizobium species.

Conclusions

Our ortholog and synteny analyses firmly establish MAFF303099 as a strain of M. huakuii. Differences in nodulation factors and secretion systems T3SS, T4SS, and T6SS may be responsible for the unique host specificities of 7653R and MAFF303099 strains. The plasmids of 7653R may have arisen by excision of the original genomic island from the 7653R chromosome.

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Article
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Genome-wide variations in a natural isolate of the nematode Caenorhabditis elegans

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2014
Abstract: 

Background

Increasing genetic and phenotypic differences found among natural isolates of C. elegans have encouraged researchers to explore the natural variation of this nematode species.

Results

Here we report on the identification of genomic differences between the reference strain N2 and the Hawaiian strain CB4856, one of the most genetically distant strains from N2. To identify both small- and large-scale genomic variations (GVs), we have sequenced the CB4856 genome using both Roche 454 (~400 bps single reads) and Illumina GA DNA sequencing methods (101 bps paired-end reads). Compared to previously described variants (available in WormBase), our effort uncovered twice as many single nucleotide variants (SNVs) and increased the number of small InDels almost 20-fold. Moreover, we identified and validated large insertions, most of which range from 150 bps to 1.2 kb in length in the CB4856 strain. Identified GVs had a widespread impact on protein-coding sequences, including 585 single-copy genes that have associated severe phenotypes of reduced viability in RNAi and genetics studies. Sixty of these genes are homologs of human genes associated with diseases. Furthermore, our work confirms previously identified GVs associated with differences in behavioural and biological traits between the N2 and CB4856 strains.

Conclusions

The identified GVs provide a rich resource for future studies that aim to explain the genetic basis for other trait differences between the N2 and CB4856 strains.

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Article
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Bridging the Gap: A Canadian Perspective on Translational Kidney Research

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2014
Abstract: 

Purpose of review

Chronic kidney disease affects approximately 3 million Canadians. Ongoing investment in high quality kidney research is needed to improve the care of patients with kidney disease. The barriers to translating such research are discussed in this review.

Sources of information

Personal knowledge, research funding body websites, and published reports.

Findings

In this review, we discuss the meaning of the term translational research and present some of the programs aimed at ensuring efficient translation of scientific discoveries with a discussion of the barriers to translation. We highlight some successes and barriers to kidney research translation using recent examples of research in Canadian nephrology. We present the following examples of kidney research: (1) research aimed at identifying the causative genes for inherited kidney diseases; (2) recent discoveries in cell-based therapies for kidney disease; (3) an examination of the impact of acute kidney injury in renal transplant patients; and (4) the development of a kidney failure risk equation to improve prognosis accuracy.

Limitations

This review focuses on research conducted by the authors.

Implications

The process of research translation is prolonged and challenging and therefore requires resources, patience, and careful planning. With increased awareness and understanding of the barriers to research translation, researchers and funding bodies can work together to increase the rate at which important research findings reach clinical practice and improve the care of patients with kidney disease.

Document type: 
Article
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