Genome-Wide Screening of Genes Required for Glycosylphosphatidylinositol Biosynthesis
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{"title"=>"Genome-wide screening of genes required for glycosylphosphatidylinositol biosynthesis", "type"=>"journal", "authors"=>[{"first_name"=>"Yao", "last_name"=>"Rong", "scopus_author_id"=>"56949985400"}, {"first_name"=>"Shota", "last_name"=>"Nakamura", "scopus_author_id"=>"35315778200"}, {"first_name"=>"Tetsuya", "last_name"=>"Hirata", "scopus_author_id"=>"55845290900"}, {"first_name"=>"Daisuke", "last_name"=>"Motooka", "scopus_author_id"=>"35315737900"}, {"first_name"=>"Yi Shi", "last_name"=>"Liu", "scopus_author_id"=>"56949377300"}, {"first_name"=>"Zeng An", "last_name"=>"He", "scopus_author_id"=>"56949452800"}, {"first_name"=>"Xiao Dong", "last_name"=>"Gao", "scopus_author_id"=>"55712058400"}, {"first_name"=>"Yusuke", "last_name"=>"Maeda", "scopus_author_id"=>"35376663600"}, {"first_name"=>"Taroh", "last_name"=>"Kinoshita", "scopus_author_id"=>"35399558000"}, {"first_name"=>"Morihisa", "last_name"=>"Fujita", "scopus_author_id"=>"7403990557"}], "year"=>2015, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"84946551855", "pui"=>"606743095", "doi"=>"10.1371/journal.pone.0138553", "issn"=>"19326203", "scopus"=>"2-s2.0-84946551855", "pmid"=>"26383639"}, "id"=>"02ad0812-c5e4-38df-abd1-eb0571bd3bf2", "abstract"=>"Glycosylphosphatidylinositol (GPI) is synthesized and transferred to proteins in the endo-plasmic reticulum (ER). GPI-anchored proteins are then transported from the ER to the plasma membrane through the Golgi apparatus. To date, at least 17 steps have been identi-fied to be required for the GPI biosynthetic pathway. Here, we aimed to establish a compre-hensive screening method to identify genes involved in GPI biosynthesis using mammalian haploid screens. Human haploid cells were mutagenized by the integration of gene trap vectors into the genome. Mutagenized cells were then treated with a bacterial pore-forming toxin, aerolysin, which binds to GPI-anchored proteins for targeting to the cell membrane. Cells that showed low surface expression of CD59, a GPI-anchored protein, were further enriched for. Gene trap insertion sites in the non-selected population and in the enriched population were determined by deep sequencing. This screening enriched 23 gene regions among the 26 known GPI biosynthetic genes, which when mutated are expected to decrease the surface expression of GPI-anchored proteins. Our results indicate that the for-ward genetic approach using haploid cells is a useful and powerful technique to identify fac-tors involved in phenotypes of interest.", "link"=>"http://www.mendeley.com/research/genomewide-screening-genes-required-glycosylphosphatidylinositol-biosynthesis", "reader_count"=>14, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Librarian"=>1, "Student > Doctoral Student"=>3, "Researcher"=>3, "Student > Ph. D. Student"=>1, "Student > Postgraduate"=>1, "Student > Master"=>2, "Professor"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Librarian"=>1, "Student > Doctoral Student"=>3, "Researcher"=>3, "Student > Ph. D. Student"=>1, "Student > Postgraduate"=>1, "Student > Master"=>2, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>5, "Materials Science"=>1, "Agricultural and Biological Sciences"=>3, "Medicine and Dentistry"=>2, "Computer Science"=>1, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Materials Science"=>{"Materials Science"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>3}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>5}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"Netherlands"=>1, "United States"=>1, "Japan"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/2277131", "https://ndownloader.figshare.com/files/2277132", "https://ndownloader.figshare.com/files/2277133", "https://ndownloader.figshare.com/files/2277134", "https://ndownloader.figshare.com/files/2277135", "https://ndownloader.figshare.com/files/2277136"], "description"=>"<div><p>Glycosylphosphatidylinositol (GPI) is synthesized and transferred to proteins in the endoplasmic reticulum (ER). GPI-anchored proteins are then transported from the ER to the plasma membrane through the Golgi apparatus. To date, at least 17 steps have been identified to be required for the GPI biosynthetic pathway. Here, we aimed to establish a comprehensive screening method to identify genes involved in GPI biosynthesis using mammalian haploid screens. Human haploid cells were mutagenized by the integration of gene trap vectors into the genome. Mutagenized cells were then treated with a bacterial pore-forming toxin, aerolysin, which binds to GPI-anchored proteins for targeting to the cell membrane. Cells that showed low surface expression of CD59, a GPI-anchored protein, were further enriched for. Gene trap insertion sites in the non-selected population and in the enriched population were determined by deep sequencing. This screening enriched 23 gene regions among the 26 known GPI biosynthetic genes, which when mutated are expected to decrease the surface expression of GPI-anchored proteins. Our results indicate that the forward genetic approach using haploid cells is a useful and powerful technique to identify factors involved in phenotypes of interest.</p></div>", "links"=>[], "tags"=>["gene trap insertion sites", "surface expression", "Glycosylphosphatidylinositol Biosynthesis Glycosylphosphatidylinositol", "protein", "gene trap vectors", "GPI biosynthetic genes", "screening", "membrane", "er", "GPI biosynthetic pathway", "23 gene regions", "Human haploid cells"], "article_id"=>1548560, "categories"=>["Uncategorised"], "users"=>["Yao Rong", "Shota Nakamura", "Tetsuya Hirata", "Daisuke Motooka", "Yi-Shi Liu", "Zeng-An He", "Xiao-Dong Gao", "Yusuke Maeda", "Taroh Kinoshita", "Morihisa Fujita"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0138553.s001", "https://dx.doi.org/10.1371/journal.pone.0138553.s002", "https://dx.doi.org/10.1371/journal.pone.0138553.s003", "https://dx.doi.org/10.1371/journal.pone.0138553.s004", "https://dx.doi.org/10.1371/journal.pone.0138553.s005", "https://dx.doi.org/10.1371/journal.pone.0138553.s006"], "stats"=>{"downloads"=>18, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Genome_Wide_Screening_of_Genes_Required_for_Glycosylphosphatidylinositol_Biosynthesis_/1548560", "title"=>"Genome-Wide Screening of Genes Required for Glycosylphosphatidylinositol Biosynthesis", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-09-18 02:51:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/2277117"], "description"=>"<p>A. After the enrichment of GPI-negative cells, surface GPI-APs in parental HAP1 cells (WT), bulk population of enriched gene-trapped HAP1 cells (HAP1-GT), and the HAP1-GT transfected with Flp recombinase (FlpE) expression plasmid were stained with fluorescent-aerolysin (FLAER), anti-CD59 antibody, or anti-CD55/DAF antibody. A phycoerythrin (PE)-conjugated secondary antibody was used to stain CD59 and DAF. FLAER was detected by flow cytometry using the FITC channel. Arrows indicate cells recovered GPI-APs after FlpE expression. B. Schematic of the elimination of the gene trap vector from the genome by the action of Flp recombinase. Gene splicing and transcription are impaired following insertion of the gene trap vector into the intronic region of a gene in the forward direction. After flipping out of the gene trap vector by Flp recombinase, gene splicing is normalized.</p>", "links"=>[], "tags"=>["gene trap insertion sites", "surface expression", "Glycosylphosphatidylinositol Biosynthesis Glycosylphosphatidylinositol", "protein", "gene trap vectors", "GPI biosynthetic genes", "screening", "membrane", "er", "GPI biosynthetic pathway", "23 gene regions", "Human haploid cells"], "article_id"=>1548546, "categories"=>["Uncategorised"], "users"=>["Yao Rong", "Shota Nakamura", "Tetsuya Hirata", "Daisuke Motooka", "Yi-Shi Liu", "Zeng-An He", "Xiao-Dong Gao", "Yusuke Maeda", "Taroh Kinoshita", "Morihisa Fujita"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0138553.g002", "stats"=>{"downloads"=>11, "page_views"=>87, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Enrichment_and_characterization_of_GPI_negative_HAP1_cells_/1548546", "title"=>"Enrichment and characterization of GPI-negative HAP1 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-09-18 02:51:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/2277120"], "description"=>"<p>A. Defective CD59 expression in HAP1-GT clone 3 (C3) and restoration by FlpE expression. Parental HAP1 cells (WT), HAP1-GT-C3 cells, and FlpE-transfected HAP1-GT-C3 were stained with an anti-CD59 antibody, followed by PE-conjugated anti-mouse IgG. B. Determination of the gene-trapped insertion site in HAP1-GT-C3. Schematic of an insertion site in <i>PIGP</i>. PIGP protein forms a complex with PIGA, PIGC, PIGH, PIGQ, PIGY, and DPM2 and acts as a GPI-GnT, which is the first reaction for GPI biosynthesis. C. Restoration of CD59 expression in HAP1-GT-C3 by the transfection of <i>PIGP</i>. HAP1-GT-C3 cells were electroporated with a PIGP-Flag-expressing plasmid. The surface expression of CD59 was detected in parental HAP1 cells, HAP1-GT-C3, and PIGP-Flag-transfected C3 cells.</p>", "links"=>[], "tags"=>["gene trap insertion sites", "surface expression", "Glycosylphosphatidylinositol Biosynthesis Glycosylphosphatidylinositol", "protein", "gene trap vectors", "GPI biosynthetic genes", "screening", "membrane", "er", "GPI biosynthetic pathway", "23 gene regions", "Human haploid cells"], "article_id"=>1548549, "categories"=>["Uncategorised"], "users"=>["Yao Rong", "Shota Nakamura", "Tetsuya Hirata", "Daisuke Motooka", "Yi-Shi Liu", "Zeng-An He", "Xiao-Dong Gao", "Yusuke Maeda", "Taroh Kinoshita", "Morihisa Fujita"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0138553.g003", "stats"=>{"downloads"=>7, "page_views"=>28, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Isolation_of_a_mutant_cell_line_defective_in_GPI_biosynthesis_/1548549", "title"=>"Isolation of a mutant cell line defective in GPI biosynthesis.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-09-18 02:51:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/2277122"], "description"=>"<p>The significance of the enrichment of gene trap insertions in enriched GPI-negative cells compared with the non-selected population was plotted as a bubble plot. The horizontal line shows the chromosomal position of the genes, and the vertical line the significance of enrichment of each gene (<i>P</i>-value). The size of the bubble shows the number of inactivating insertion sites in enriched GPI-negative populations. Genes significantly enriched in the GPI-negative population (<i>P</i><0.001) are colored. Yellow bubbles indicate genes encoding the biosynthesis of GPI intermediates in the ER, green bubbles the genes required for GPI-transamidation, orange bubbles the genes involved in Dol-P-Man synthesis and utilization, and pink bubbles the gene involved in GPI-AP remodeling. The bubbles of PIGM and PGAP3 (arrows) were close to the significance limit.</p>", "links"=>[], "tags"=>["gene trap insertion sites", "surface expression", "Glycosylphosphatidylinositol Biosynthesis Glycosylphosphatidylinositol", "protein", "gene trap vectors", "GPI biosynthetic genes", "screening", "membrane", "er", "GPI biosynthetic pathway", "23 gene regions", "Human haploid cells"], "article_id"=>1548551, "categories"=>["Uncategorised"], "users"=>["Yao Rong", "Shota Nakamura", "Tetsuya Hirata", "Daisuke Motooka", "Yi-Shi Liu", "Zeng-An He", "Xiao-Dong Gao", "Yusuke Maeda", "Taroh Kinoshita", "Morihisa Fujita"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0138553.g004", "stats"=>{"downloads"=>1, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Haploid_genetic_screening_for_factors_required_for_GPI_biosynthesis_/1548551", "title"=>"Haploid genetic screening for factors required for GPI biosynthesis.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-09-18 02:51:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/2277124"], "description"=>"<p>Insertion sites of PIGS (A), PIGW (B) and PIGP (C) gene regions in the non-selected and enriched GPI-negative populations. The schematic was constructed using CLC genomics workbench software. Insertions in 5’-region of PIGS gene are shown in enlarged red square. Forward insertions and reverse insertions are shown in green and red, respectively.</p>", "links"=>[], "tags"=>["gene trap insertion sites", "surface expression", "Glycosylphosphatidylinositol Biosynthesis Glycosylphosphatidylinositol", "protein", "gene trap vectors", "GPI biosynthetic genes", "screening", "membrane", "er", "GPI biosynthetic pathway", "23 gene regions", "Human haploid cells"], "article_id"=>1548553, "categories"=>["Uncategorised"], "users"=>["Yao Rong", "Shota Nakamura", "Tetsuya Hirata", "Daisuke Motooka", "Yi-Shi Liu", "Zeng-An He", "Xiao-Dong Gao", "Yusuke Maeda", "Taroh Kinoshita", "Morihisa Fujita"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0138553.g005", "stats"=>{"downloads"=>2, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Insertion_sites_in_GPI_biosynthetic_gene_loci_/1548553", "title"=>"Insertion sites in GPI biosynthetic gene loci.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-09-18 02:51:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/2277128"], "description"=>"<p>GPI is biosynthesized in the ER from PI by the stepwise addition of sugars, an acyl-chain and phospho-ethanolamines. After GPI is transferred to a protein, GPI moieties are remodeled during transport. Genes colored in blue (<i>italic</i>) have been shown to decrease surface expression of GPI-APs in mutant cells. Genes significantly enriched and genes weakly enriched in this study are highlighted in yellow and light yellow, respectively.</p>", "links"=>[], "tags"=>["gene trap insertion sites", "surface expression", "Glycosylphosphatidylinositol Biosynthesis Glycosylphosphatidylinositol", "protein", "gene trap vectors", "GPI biosynthetic genes", "screening", "membrane", "er", "GPI biosynthetic pathway", "23 gene regions", "Human haploid cells"], "article_id"=>1548557, "categories"=>["Uncategorised"], "users"=>["Yao Rong", "Shota Nakamura", "Tetsuya Hirata", "Daisuke Motooka", "Yi-Shi Liu", "Zeng-An He", "Xiao-Dong Gao", "Yusuke Maeda", "Taroh Kinoshita", "Morihisa Fujita"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0138553.g006", "stats"=>{"downloads"=>6, "page_views"=>32, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Biosynthesis_remodeling_and_transport_of_GPI_APs_in_mammalian_cells_/1548557", "title"=>"Biosynthesis, remodeling, and transport of GPI-APs in mammalian cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-09-18 02:51:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/2277114"], "description"=>"<p>A. Structure of the gene trap vector and strategy for enrichment of GPI-negative mutant cells. Retrovirus-based gene trap vectors containing splice acceptor site (SA) and polyadenylation signal (PolyA) were infected into HAP1 cells. The 3′ flippase recognition target (FRT) site of the provirus is copied into the 5′ long terminal repeat (LTR). Mutagenized cells (6 × 10<sup>7</sup> cells) were selected with 6 μg/ml blasticidin for 1 week. To eliminate the possibility that GPI-negative mutant cells were lost during the screening procedure, the 4 times of infected cells (2.4 × 10<sup>8</sup> cells) were used for the treatment with 0.2 nM proaerolysin for 1 day. After the first treatment, the survived cells were harvested and cultured in new plates. Then, 6 × 10<sup>7</sup> cells were treated with 0.2 nM proaerolysin for 1 day again. Resistant cells were proliferated and stained with an anti-CD59 antibody and CD59-negative cells were further enriched by cell sorting. Ψ, packaging signal; PGKpro, PGK promoter; CMV pro, CMV promoter; BSD, blasticidin S-deaminase gene. B. Sensitivity of HAP1 cells to proaerolysin. HAP1 cells were treated with indicated concentrations of proaerolysin (nM) for 3 h. After changing to medium without aerolysin, cell viability was measured by the WST-1 assay and shown in % viability. Viability of cells without proaerolysin treatment was 100%. Data are means ± standard deviation (n = 4).</p>", "links"=>[], "tags"=>["gene trap insertion sites", "surface expression", "Glycosylphosphatidylinositol Biosynthesis Glycosylphosphatidylinositol", "protein", "gene trap vectors", "GPI biosynthetic genes", "screening", "membrane", "er", "GPI biosynthetic pathway", "23 gene regions", "Human haploid cells"], "article_id"=>1548543, "categories"=>["Uncategorised"], "users"=>["Yao Rong", "Shota Nakamura", "Tetsuya Hirata", "Daisuke Motooka", "Yi-Shi Liu", "Zeng-An He", "Xiao-Dong Gao", "Yusuke Maeda", "Taroh Kinoshita", "Morihisa Fujita"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0138553.g001", "stats"=>{"downloads"=>2, "page_views"=>20, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Haploid_genetic_screening_to_reveal_factors_required_for_GPI_biosynthesis_using_aerolysin_sensitivity_/1548543", "title"=>"Haploid genetic screening to reveal factors required for GPI biosynthesis using aerolysin sensitivity.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-09-18 02:51:49"}

PMC Usage Stats | Further Information

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{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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