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Mewis K.,Genome Science and Technology Program | Armstrong Z.,Genome Science and Technology Program | Song Y.C.,UBC Graduate Program in Bioinformatics | Withers S.G.,Genome Science and Technology Program | And 2 more authors.
Journal of Biotechnology | Year: 2013

Functional metagenomics has emerged as a powerful method for gene model validation and enzyme discovery from natural and human engineered ecosystems. Here we report development of a high-throughput functional metagenomic screen incorporating bioinformatic and biochemical analyses features. A fosmid library containing 6144 clones sourced from a mining bioremediation system was screened for cellulase activity using 2,4-dinitrophenyl β-cellobioside, a previously proven cellulose model substrate. Fifteen active clones were recovered and fully sequenced revealing 9 unique clones with the ability to hydrolyse 1,4-β-. d-glucosidic linkages. Transposon mutagenesis identified genes belonging to glycoside hydrolase (GH) 1, 3, or 5 as necessary for mediating this activity. Reference trees for GH 1, 3, and 5 families were generated from sequences in the CAZy database for automated phylogenetic analysis of fosmid end and active clone sequences revealing known and novel cellulase encoding genes. Active cellulase genes recovered in functional screens were subcloned into inducible high copy plasmids, expressed and purified to determine enzymatic properties including thermostability, pH optima, and substrate specificity. The workflow described here provides a general paradigm for recovery and characterization of microbially derived genes and gene products based on genetic logic and contemporary screening technologies developed for model organismal systems. © 2013 The Authors. Source


de Melo J.,Johns | Blackshaw S.,Johns | Blackshaw S.,Center for High throughput Biology | Blackshaw S.,Institute for Cell Engineering
Journal of Visualized Experiments | Year: 2011

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation. We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability. © JoVE 2006-2011 All Rights Reserved. Source


Chen S.,University of Washington | Blackshaw S.,Center for High throughput Biology | Blackshaw S.,Johns Hopkins University
Development | Year: 2011

The mammalian retina is a tractable model system for analyzing transcriptional networks that guide neural development. Spalt family zinc-finger transcription factors play a crucial role in photoreceptor specification in Drosophila, but their role in mammalian retinal development has not been investigated. In this study, we show that that the spalt homolog Sall3 is prominently expressed in developing cone photoreceptors and horizontal interneurons of the mouse retina and in a subset of cone bipolar cells. We find that Sall3 is both necessary and sufficient to activate the expression of multiple cone-specific genes, and that Sall3 protein is selectively bound to the promoter regions of these genes. Notably, Sall3 shows more prominent expression in short wavelength-sensitive cones than in medium wavelength-sensitive cones, and that Sall3 selectively activates expression of the short but not the medium wavelength-sensitive cone opsin gene. We further observe that Sall3 regulates the differentiation of horizontal interneurons, which form direct synaptic contacts with cone photoreceptors. Loss of function of Sall3 eliminates expression of the horizontal cell-specific transcription factor Lhx1, resulting in a radial displacement of horizontal cells that partially phenocopies the loss of function of Lhx1. These findings not only demonstrate that Spalt family transcription factors play a conserved role in regulating photoreceptor development in insects and mammals, but also identify Sall3 as a factor that regulates terminal differentiation of both cone photoreceptors and their postsynaptic partners. © 2011. Published by The Company of Biologists Ltd. Source


Qiao Y.,University of British Columbia | Qiao Y.,Child and Family Research Institute | Harvard C.,University of British Columbia | Tyson C.,Cytogenetics Laboratory | And 7 more authors.
Human Genetics | Year: 2010

Array CGH enables the detection of pathogenic copy number variants (CNVs) in 5-15% of individuals with intellectual disability (ID), making it a promising tool for uncovering ID candidate genes. However, most CNVs encompass multiple genes, making it difficult to identify key disease gene(s) underlying ID etiology. Using array CGH we identified 47 previously unreported unique CNVs in 45/255 probands. We prioritized ID candidate genes using five bioinformatic gene prioritization web tools. Gene priority lists were created by comparing integral genes from each CNV from our ID cohort with sets of training genes specific either to ID or randomly selected. Our findings suggest that different training sets alter gene prioritization only moderately; however, only the ID gene training set resulted in significant enrichment of genes with nervous system function (19%) in prioritized versus non-prioritized genes from the same de novo CNVs (7%, p < 0.05). This enrichment further increased to 31% when the five web tools were used in concert and included genes within mitogen-activated protein kinase (MAPK) and neuroactive ligand-receptor interaction pathways. Gene prioritization web tools enrich for genes with relevant function in ID and more readily facilitate the selection of ID candidate genes for functional studies, particularly for large CNVs. © 2010 Springer-Verlag. Source


Newman R.H.,North Carolina A&T State University | Rho H.-S.,Center for High throughput Biology | Woodard C.,Center for High throughput Biology | Neiswinger J.,Center for High throughput Biology | And 24 more authors.
Molecular Systems Biology | Year: 2013

The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)-based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase-substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high-quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high-resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B-cell receptor signaling. Overall, these studies provide global insights into kinase-mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans. © 2013 EMBO and Macmillan Publishers Limited. Source

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