pgbkt7 brct Search Results


pgbkt7 brct  (TaKaRa)
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TaKaRa pgbkt7 brct
BRCA1 variant mapping. ( a ) Overview of BRCA1 variants from amino acid residues 1396–1863. ( b ) Depiction of BRCA1 protein domains and motifs in the aa 1,315–1,863 region and the variants that have been tested by transcriptional assays in previous (blue) or the current (red) studies ( c ) Depiction of BRCA1 constructs used in this study and the locations of the variants tested. ( d ) VarCall analysis of missense variants in the carboxy-terminal region (aa 1,315–1,863 of the BRCA1 protein). Transcriptional assays were performed for 98 missense variants in the aa 1,395–1,864 context (9 of these represent retests), 10 missense variants were tested by transcriptional assays in the aa 1,315–1,863 context (3 variants found in the population, 7 variants not currently known to occur in the population). Transcriptional assays were performed using a luciferase reporter system where 293T cells were co-transfected with a reporter plasmid, pG5Luc, which contains a firefly luciferase gene under the control of five GAL4 binding sites, a pCDNA3 plasmid coding for either wild-type or variant BRCA1 constructs fused to the GAL4 DNA-binding domain, and an internal control containing a Renilla luciferase gene constitutively expressed (described in Carvalho et al. (2009)) . Results of these transcriptional assays were analysed using VarCall to predict the likelihood of pathogenicity. In addition, the coiled-coil domain and secondary structures of the <t>BRCT</t> domain were overlaid on the results. See for additional details.
Pgbkt7 Brct, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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BRCA1 variant mapping. ( a ) Overview of BRCA1 variants from amino acid residues 1396–1863. ( b ) Depiction of BRCA1 protein domains and motifs in the aa 1,315–1,863 region and the variants that have been tested by transcriptional assays in previous (blue) or the current (red) studies ( c ) Depiction of BRCA1 constructs used in this study and the locations of the variants tested. ( d ) VarCall analysis of missense variants in the carboxy-terminal region (aa 1,315–1,863 of the BRCA1 protein). Transcriptional assays were performed for 98 missense variants in the aa 1,395–1,864 context (9 of these represent retests), 10 missense variants were tested by transcriptional assays in the aa 1,315–1,863 context (3 variants found in the population, 7 variants not currently known to occur in the population). Transcriptional assays were performed using a luciferase reporter system where 293T cells were co-transfected with a reporter plasmid, pG5Luc, which contains a firefly luciferase gene under the control of five GAL4 binding sites, a pCDNA3 plasmid coding for either wild-type or variant BRCA1 constructs fused to the GAL4 DNA-binding domain, and an internal control containing a Renilla luciferase gene constitutively expressed (described in Carvalho et al. (2009)) . Results of these transcriptional assays were analysed using VarCall to predict the likelihood of pathogenicity. In addition, the coiled-coil domain and secondary structures of the BRCT domain were overlaid on the results. See for additional details.

Journal: NPJ Genomic Medicine

Article Title: Functional assays provide a robust tool for the clinical annotation of genetic variants of uncertain significance

doi: 10.1038/npjgenmed.2016.1

Figure Lengend Snippet: BRCA1 variant mapping. ( a ) Overview of BRCA1 variants from amino acid residues 1396–1863. ( b ) Depiction of BRCA1 protein domains and motifs in the aa 1,315–1,863 region and the variants that have been tested by transcriptional assays in previous (blue) or the current (red) studies ( c ) Depiction of BRCA1 constructs used in this study and the locations of the variants tested. ( d ) VarCall analysis of missense variants in the carboxy-terminal region (aa 1,315–1,863 of the BRCA1 protein). Transcriptional assays were performed for 98 missense variants in the aa 1,395–1,864 context (9 of these represent retests), 10 missense variants were tested by transcriptional assays in the aa 1,315–1,863 context (3 variants found in the population, 7 variants not currently known to occur in the population). Transcriptional assays were performed using a luciferase reporter system where 293T cells were co-transfected with a reporter plasmid, pG5Luc, which contains a firefly luciferase gene under the control of five GAL4 binding sites, a pCDNA3 plasmid coding for either wild-type or variant BRCA1 constructs fused to the GAL4 DNA-binding domain, and an internal control containing a Renilla luciferase gene constitutively expressed (described in Carvalho et al. (2009)) . Results of these transcriptional assays were analysed using VarCall to predict the likelihood of pathogenicity. In addition, the coiled-coil domain and secondary structures of the BRCT domain were overlaid on the results. See for additional details.

Article Snippet: Fragments coding for the tandem BRCT domains of MCPH1 (aa 649–832) and MDC1 (aa 1,894–2,079) were obtained by PCR amplification ( ) and cloned into the pGBKT7 or pGADT7 vectors (Clontech) as fusions to the GAL4 DBD or AD, respectively. pGBKT7 BRCT, pGADT7 BRCT or empty pGBKT7 were transformed in the Y2HGold Saccharomyces cerevisiae strain and plated on dropout medium lacking Tryptophan (SD-Trp) or Leucine (-Leu) and number of colonies was scored.

Techniques: Variant Assay, Construct, Luciferase, Transfection, Plasmid Preparation, Binding Assay

Mutagenesis screen to identify loss of function missense variants in MCPH1 and MDC1 tandem BRCT domains. ( a ) Expression in yeast of proteins containing the tandem BRCT of MCPH1 or MDC1 fused to the GAL4 DNA-binding domain (DBD), but not to the activation domain (AD) of GAL4, lead to growth inhibition and a small colony phenotype. Libraries of mutagenised constructs coding for the tandem BRCTs of MCPH1 or MDC1 were transformed into yeast and transformants with regular size colonies (carrying loss of function mutants) were isolated and sequenced to identify residues that disrupt the BRCT function. ( b ) Loss of function variants (red) cluster around the BRCT phosphopeptide-binding pocket in MCPH1 (PDB ID: 3U3Z) and MDC1 (PDB ID: 2AZM). ( c ) MCPH1 BRCT variants found in cancer samples and predicted to impair function, as inferred by BRCA1 data sets, (R693H and W815R) abrogate the small-colony phenotype, whereas a predicted neutral mutation (N661S) does not.

Journal: NPJ Genomic Medicine

Article Title: Functional assays provide a robust tool for the clinical annotation of genetic variants of uncertain significance

doi: 10.1038/npjgenmed.2016.1

Figure Lengend Snippet: Mutagenesis screen to identify loss of function missense variants in MCPH1 and MDC1 tandem BRCT domains. ( a ) Expression in yeast of proteins containing the tandem BRCT of MCPH1 or MDC1 fused to the GAL4 DNA-binding domain (DBD), but not to the activation domain (AD) of GAL4, lead to growth inhibition and a small colony phenotype. Libraries of mutagenised constructs coding for the tandem BRCTs of MCPH1 or MDC1 were transformed into yeast and transformants with regular size colonies (carrying loss of function mutants) were isolated and sequenced to identify residues that disrupt the BRCT function. ( b ) Loss of function variants (red) cluster around the BRCT phosphopeptide-binding pocket in MCPH1 (PDB ID: 3U3Z) and MDC1 (PDB ID: 2AZM). ( c ) MCPH1 BRCT variants found in cancer samples and predicted to impair function, as inferred by BRCA1 data sets, (R693H and W815R) abrogate the small-colony phenotype, whereas a predicted neutral mutation (N661S) does not.

Article Snippet: Fragments coding for the tandem BRCT domains of MCPH1 (aa 649–832) and MDC1 (aa 1,894–2,079) were obtained by PCR amplification ( ) and cloned into the pGBKT7 or pGADT7 vectors (Clontech) as fusions to the GAL4 DBD or AD, respectively. pGBKT7 BRCT, pGADT7 BRCT or empty pGBKT7 were transformed in the Y2HGold Saccharomyces cerevisiae strain and plated on dropout medium lacking Tryptophan (SD-Trp) or Leucine (-Leu) and number of colonies was scored.

Techniques: Mutagenesis, Expressing, Binding Assay, Activation Assay, Inhibition, Construct, Transformation Assay, Isolation