Structured Review

Stratagene polymerase chain reaction pcr fragment
Polymerase Chain Reaction Pcr Fragment, supplied by Stratagene, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 88 stars, based on 1 article reviews
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Polymerase Chain Reaction:

Article Title: Functional Significance of the Alternative Transcript Processing of the Arabidopsis Floral Promoter FCA
Article Snippet: .. A polymerase chain reaction (PCR) fragment containing the 3′ region of FCA was amplified from a pBluescript IISK− plasmid containing the 9763-bp FCA gene using the primer 5′-AAGAATAAATCTAGAGGTACATGAGACGAG-3′ (which contains an XbaI site after the stop codon of FCA) and the T7 primer (Stratagene). .. This was cloned into the pUC18-GUS vector as an XbaI-KpnI fragment to produce the construct pUC18-GUS-FCA-3′.

Amplification:

Article Title: Functional Significance of the Alternative Transcript Processing of the Arabidopsis Floral Promoter FCA
Article Snippet: .. A polymerase chain reaction (PCR) fragment containing the 3′ region of FCA was amplified from a pBluescript IISK− plasmid containing the 9763-bp FCA gene using the primer 5′-AAGAATAAATCTAGAGGTACATGAGACGAG-3′ (which contains an XbaI site after the stop codon of FCA) and the T7 primer (Stratagene). .. This was cloned into the pUC18-GUS vector as an XbaI-KpnI fragment to produce the construct pUC18-GUS-FCA-3′.

Plasmid Preparation:

Article Title: Functional Significance of the Alternative Transcript Processing of the Arabidopsis Floral Promoter FCA
Article Snippet: .. A polymerase chain reaction (PCR) fragment containing the 3′ region of FCA was amplified from a pBluescript IISK− plasmid containing the 9763-bp FCA gene using the primer 5′-AAGAATAAATCTAGAGGTACATGAGACGAG-3′ (which contains an XbaI site after the stop codon of FCA) and the T7 primer (Stratagene). .. This was cloned into the pUC18-GUS vector as an XbaI-KpnI fragment to produce the construct pUC18-GUS-FCA-3′.

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  • 85
    Stratagene mouse dmrt7 cdna fragment
    Expression of <t>Dmrt7</t> mRNA and Protein (A) RT-PCR analysis of Dmrt7 mRNA from ten organs of adult mouse. <t>cDNA</t> from each organ was amplified with primers specific for Dmrt7 (top row) and β- actin (bottom row). (B) Dmrt7 mRNA expression during the first round of spermatogenesis. cDNAs obtained from testis at the indicated days after birth were amplified as in (A). (C) DMRT7 protein expression. Immunofluorescence of testis sections from 6-wk-old male stained with antibody to DMRT7 (green) and DAPI (blue). (D) DMRT7 subcellular localization to XY body. Testis sections from 6-wk-old male stained with antibodies to DMRT7 (red) and SUMO-1 (green). SUMO-1 is localized to the XY body. Right-most panel shows merge of other two panels. Inserts show higher magnification of pachytene spermatocytes with XY bodies.
    Mouse Dmrt7 Cdna Fragment, supplied by Stratagene, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Stratagene rt pcr fragment sizes
    <t>RT-PCR</t> comparison of E. coli MG1655 LSMMG (LS) and control (C) samples. Template RNA was isolated from mid-logarithmic cells grown in MOPS (left panel) or LB (right panel) medium. LSMMG up- or down-regulation of each putative operon, determined by functional genomic analysis, is indicated below the gel image. Lanes: M, Hi-Lo <t>DNA</t> markers (Minnesota Molecular, Inc); hdeAB ; flgBCDE ; cusCF ; tdcDEFG ; rpsF-priB-rpsR-rplI . See materials and methods for details.
    Rt Pcr Fragment Sizes, supplied by Stratagene, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene vinculin
    A <t>vinculin</t> mutant unable to associate with the Arp2/3 complex is impaired in lamellipodial extension and spreading on fibronectin. Vinculin null cells reexpressing vector alone (Vin−/−), WT or mutant vinculin (P878A) were allowed to spread onto 50 μg/ml of FN for 120 min (A) and then examined by immunofluorescence with antibodies against vinculin or using Alexa-conjugated phalloidin. The white arrows indicate the transfected cells. Bars, 20 μm. (B) the extent of spreading was quantified by expressing the ratio ± SD of the length/width of 50 cells at the 120-min time point. The WT reexpressing cells are significantly more spread than either the null or mutant reexpressers (P
    Vinculin, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Stratagene native β globin orf
    The sequence of the <t>ORF</t> determines the extension of the AUG-proximity effect. ( A ) Physical maps of the α-globin and the hybrid β/α- and α/β/α-globin mRNAs studied. Dark-gray rectangles represent α-globin exons and light-gray rectangles represent <t>β-globin</t> sequences. Translation initiation codons (AUG) and native termination codons (UAA) or premature translation termination codons (PTCs) are depicted by heavy black vertical lines. The name of each mRNA is indicated to its right. ( B ) Representative RPA using RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The identification of α-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the left of the autoradiograph. Levels of α-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations from three independent experiments). mRNA levels are normalized to the expression level of the wild-type mRNA (αWT). A 2-fold RNA sample (2x αWT) from HeLa cells transfected with a αWT gene was also analyzed to demonstrate that the experimental RPA was carried out in probe excess. P -values were estimated using a student's t -test. Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) Schematic representation of the studied β-globin and hybrid α/β- and β/α/β-globin mRNAs, using the same depiction code as for (A) (see above). ( D ) Illustrative RPA of RNA from HeLa cells transiently transfected with the constructs identified beneath each lane. Identification of the β-globin and puro R protected fragments is indicated to the left of the autoradiograph. Average levels (and standard deviations from three independent experiments) of β-globin mRNA (plotted above each respective lane) were measured relatively to the puro R mRNA as in (B) (see above). Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control.
    Native β Globin Orf, supplied by Stratagene, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Expression of Dmrt7 mRNA and Protein (A) RT-PCR analysis of Dmrt7 mRNA from ten organs of adult mouse. cDNA from each organ was amplified with primers specific for Dmrt7 (top row) and β- actin (bottom row). (B) Dmrt7 mRNA expression during the first round of spermatogenesis. cDNAs obtained from testis at the indicated days after birth were amplified as in (A). (C) DMRT7 protein expression. Immunofluorescence of testis sections from 6-wk-old male stained with antibody to DMRT7 (green) and DAPI (blue). (D) DMRT7 subcellular localization to XY body. Testis sections from 6-wk-old male stained with antibodies to DMRT7 (red) and SUMO-1 (green). SUMO-1 is localized to the XY body. Right-most panel shows merge of other two panels. Inserts show higher magnification of pachytene spermatocytes with XY bodies.

    Journal: PLoS Genetics

    Article Title: A Mammal-Specific Doublesex Homolog Associates with Male Sex Chromatin and Is Required for Male Meiosis

    doi: 10.1371/journal.pgen.0030062

    Figure Lengend Snippet: Expression of Dmrt7 mRNA and Protein (A) RT-PCR analysis of Dmrt7 mRNA from ten organs of adult mouse. cDNA from each organ was amplified with primers specific for Dmrt7 (top row) and β- actin (bottom row). (B) Dmrt7 mRNA expression during the first round of spermatogenesis. cDNAs obtained from testis at the indicated days after birth were amplified as in (A). (C) DMRT7 protein expression. Immunofluorescence of testis sections from 6-wk-old male stained with antibody to DMRT7 (green) and DAPI (blue). (D) DMRT7 subcellular localization to XY body. Testis sections from 6-wk-old male stained with antibodies to DMRT7 (red) and SUMO-1 (green). SUMO-1 is localized to the XY body. Right-most panel shows merge of other two panels. Inserts show higher magnification of pachytene spermatocytes with XY bodies.

    Article Snippet: A mouse Dmrt7 cDNA fragment containing sequences from exon 8 was used to screen a mouse BAC library from the 129/SvJ strain (Stratagene, http://www.stratagene.com ), and clones containing promoter sequences were isolated and sequenced to obtain Dmrt7 genomic sequence.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Immunofluorescence, Staining

    RT-PCR comparison of E. coli MG1655 LSMMG (LS) and control (C) samples. Template RNA was isolated from mid-logarithmic cells grown in MOPS (left panel) or LB (right panel) medium. LSMMG up- or down-regulation of each putative operon, determined by functional genomic analysis, is indicated below the gel image. Lanes: M, Hi-Lo DNA markers (Minnesota Molecular, Inc); hdeAB ; flgBCDE ; cusCF ; tdcDEFG ; rpsF-priB-rpsR-rplI . See materials and methods for details.

    Journal: BMC Microbiology

    Article Title: Characterization of Escherichia coli MG1655 grown in a low-shear modeled microgravity environment

    doi: 10.1186/1471-2180-7-15

    Figure Lengend Snippet: RT-PCR comparison of E. coli MG1655 LSMMG (LS) and control (C) samples. Template RNA was isolated from mid-logarithmic cells grown in MOPS (left panel) or LB (right panel) medium. LSMMG up- or down-regulation of each putative operon, determined by functional genomic analysis, is indicated below the gel image. Lanes: M, Hi-Lo DNA markers (Minnesota Molecular, Inc); hdeAB ; flgBCDE ; cusCF ; tdcDEFG ; rpsF-priB-rpsR-rplI . See materials and methods for details.

    Article Snippet: RT-PCR fragment sizes were determined by comparison to Kb DNA ladder (Stratagene, Cedar Creek, TX) for subsequent comparison to predicted fragment sizes.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Functional Assay

    A vinculin mutant unable to associate with the Arp2/3 complex is impaired in lamellipodial extension and spreading on fibronectin. Vinculin null cells reexpressing vector alone (Vin−/−), WT or mutant vinculin (P878A) were allowed to spread onto 50 μg/ml of FN for 120 min (A) and then examined by immunofluorescence with antibodies against vinculin or using Alexa-conjugated phalloidin. The white arrows indicate the transfected cells. Bars, 20 μm. (B) the extent of spreading was quantified by expressing the ratio ± SD of the length/width of 50 cells at the 120-min time point. The WT reexpressing cells are significantly more spread than either the null or mutant reexpressers (P

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: A vinculin mutant unable to associate with the Arp2/3 complex is impaired in lamellipodial extension and spreading on fibronectin. Vinculin null cells reexpressing vector alone (Vin−/−), WT or mutant vinculin (P878A) were allowed to spread onto 50 μg/ml of FN for 120 min (A) and then examined by immunofluorescence with antibodies against vinculin or using Alexa-conjugated phalloidin. The white arrows indicate the transfected cells. Bars, 20 μm. (B) the extent of spreading was quantified by expressing the ratio ± SD of the length/width of 50 cells at the 120-min time point. The WT reexpressing cells are significantly more spread than either the null or mutant reexpressers (P

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Mutagenesis, Plasmid Preparation, Immunofluorescence, Transfection, Expressing

    Recruitment of the Arp2/3 complex to vinculin is not required for cell adhesion or cell migration. Vinculin-null cells reexpressing vector alone (Vin−/−), WT vinculin, or mutant vinculin (P878A) were examined for the ability to adhere to 50 μg/ml FN (A) or migrate through Transwell filters (B). The mean number of cells expressing vector alone that migrated through the filter was set to 100%. The relative number of cells/field ± SEM was calculated from the means of three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: Recruitment of the Arp2/3 complex to vinculin is not required for cell adhesion or cell migration. Vinculin-null cells reexpressing vector alone (Vin−/−), WT vinculin, or mutant vinculin (P878A) were examined for the ability to adhere to 50 μg/ml FN (A) or migrate through Transwell filters (B). The mean number of cells expressing vector alone that migrated through the filter was set to 100%. The relative number of cells/field ± SEM was calculated from the means of three independent experiments.

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Migration, Plasmid Preparation, Mutagenesis, Expressing

    PI3K is required for recruitment of the Arp2/3 complex to vinculin. Effect of inhibition of PI3K by wortmannin (A) on recruitment of the Arp2/3 complex to vinculin. Serum-starved A431 cells were pretreated with DMSO or 100 nM wortmannin (WM) for 30 min and then left resting (0) or stimulated with EGF for the times indicated (min). Vinculin was immunoprecipitated and blotted for vinculin or p34-Arc as described in Fig. 1 .

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: PI3K is required for recruitment of the Arp2/3 complex to vinculin. Effect of inhibition of PI3K by wortmannin (A) on recruitment of the Arp2/3 complex to vinculin. Serum-starved A431 cells were pretreated with DMSO or 100 nM wortmannin (WM) for 30 min and then left resting (0) or stimulated with EGF for the times indicated (min). Vinculin was immunoprecipitated and blotted for vinculin or p34-Arc as described in Fig. 1 .

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Inhibition, Immunoprecipitation

    Binding of the Arp2/3 complex to vinculin is direct . Vinculin fusion proteins attached to beads were incubated with 2 μg of purified, human platelet Arp2/3 complex for 1.5 h at 4°C. The beads were washed and the pellets were examined for the ability of the fusion proteins to bind the Arp2/3 complex. Purified Arp2/3 denotes a sample of Arp2/3 complex purified from platelets.

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: Binding of the Arp2/3 complex to vinculin is direct . Vinculin fusion proteins attached to beads were incubated with 2 μg of purified, human platelet Arp2/3 complex for 1.5 h at 4°C. The beads were washed and the pellets were examined for the ability of the fusion proteins to bind the Arp2/3 complex. Purified Arp2/3 denotes a sample of Arp2/3 complex purified from platelets.

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Binding Assay, Incubation, Purification

    Recruitment of the Arp2/3 complex to sites of integrin aggregation. (A) REF52 cells expressing GFP or p34GFP were plated on FN-coated coverslips for 1.5 h at 37°C. (B) Serum-starved Swiss3T3 cells were plated on FN-coated coverslips, and microinjected with 61LRac and C3. The cells were examined by immunofluorescence using antibodies against vinculin and the Arp3 subunit of the Arp2/3 complex. Sites of colocalization of the Arp2/3 complex with vinculin (focal complexes) are indicated with arrows in A. Bar, 20 μm. (C and D) Hela cells expressing GFP or p34GFP were plated on collagen and presented with beads coated with fibronectin (FN) or poly-lysine (PL beads) for 20 min (C and D marked early) or 35 min (D, late). Cells were stained for β1 integrins or HLA. The percentage of beads able to recruit the β1 integrins (gray bars), the GFP fusion proteins (black bars), and/or HLA (white bars) was scored using immunofluorescence. In some experiments, cells were preincubated with 100 nM wortmannin (D, wortmannin) for 30 min before incubation with the beads.

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: Recruitment of the Arp2/3 complex to sites of integrin aggregation. (A) REF52 cells expressing GFP or p34GFP were plated on FN-coated coverslips for 1.5 h at 37°C. (B) Serum-starved Swiss3T3 cells were plated on FN-coated coverslips, and microinjected with 61LRac and C3. The cells were examined by immunofluorescence using antibodies against vinculin and the Arp3 subunit of the Arp2/3 complex. Sites of colocalization of the Arp2/3 complex with vinculin (focal complexes) are indicated with arrows in A. Bar, 20 μm. (C and D) Hela cells expressing GFP or p34GFP were plated on collagen and presented with beads coated with fibronectin (FN) or poly-lysine (PL beads) for 20 min (C and D marked early) or 35 min (D, late). Cells were stained for β1 integrins or HLA. The percentage of beads able to recruit the β1 integrins (gray bars), the GFP fusion proteins (black bars), and/or HLA (white bars) was scored using immunofluorescence. In some experiments, cells were preincubated with 100 nM wortmannin (D, wortmannin) for 30 min before incubation with the beads.

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Expressing, Immunofluorescence, Staining, Incubation

    Rac1 is necessary and sufficient for recruitment of the Arp2/3 complex to vinculin. (A) Effect of dominant negative Rac1 on the recruitment of proteins to vinculin. A431 cells were transiently transfected with vector alone (MOCK) or myc-tagged N17Rac1 (N17Rac1), allowed to recover for 12 h, and then serum starved for an additional 18–24 h in DME. Cells were stimulated with 100 ng/ml of EGF as in Fig. 1 for the times given (min), and vinculin immunoprecipitated. (B) Effect of constitutively active Rac1 on the recruitment of proteins to vinculin. A431 cells were transiently transfected with AU-tagged L61Rac1 (L61), allowed to recover for 12h, and then serum starved for an additional 18–24 h in DME. Vinculin immunoprecipitates were blotted for p34. The graphs beneath each panel indicate the levels of GTP-bound Rac1. Each number represents the mean fold activation ± SEM in Rac1 activity over unstimulated cells from three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: Rac1 is necessary and sufficient for recruitment of the Arp2/3 complex to vinculin. (A) Effect of dominant negative Rac1 on the recruitment of proteins to vinculin. A431 cells were transiently transfected with vector alone (MOCK) or myc-tagged N17Rac1 (N17Rac1), allowed to recover for 12 h, and then serum starved for an additional 18–24 h in DME. Cells were stimulated with 100 ng/ml of EGF as in Fig. 1 for the times given (min), and vinculin immunoprecipitated. (B) Effect of constitutively active Rac1 on the recruitment of proteins to vinculin. A431 cells were transiently transfected with AU-tagged L61Rac1 (L61), allowed to recover for 12h, and then serum starved for an additional 18–24 h in DME. Vinculin immunoprecipitates were blotted for p34. The graphs beneath each panel indicate the levels of GTP-bound Rac1. Each number represents the mean fold activation ± SEM in Rac1 activity over unstimulated cells from three independent experiments.

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Dominant Negative Mutation, Transfection, Plasmid Preparation, Immunoprecipitation, Activation Assay, Activity Assay

    PIP 2 and GST-VCA induce binding of the Arp2/3 complex to vinculin. Serum-starved A431 cells were stimulated with buffer alone or with 100 ng/ml EGF for 5 min. The cells were lysed in 500 μl vol as described in the text, and the lysates were incubated in the presence of no exogenous proteins or 30 μg of GST, 10 mM PIP 2 , 10 mM PIP 2 +30 μg GST–VCA, or 30 μg of GST–VCA for 20 min at 4°C. Vinculin immunoprecipitates were prepared and analyzed as described above. In three independent experiments, compared with the level of Arp2/3 binding to vinculin in response to 5 min EGF stimulation (100%), the level of Arp2/3 complex recruitment in response to the various stimuli was 13% ± 7% (PIP 2 ), 27% ± 15% (GST–VCA), and 94% ± 11% (PIP 2 + GST–VCA).

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: PIP 2 and GST-VCA induce binding of the Arp2/3 complex to vinculin. Serum-starved A431 cells were stimulated with buffer alone or with 100 ng/ml EGF for 5 min. The cells were lysed in 500 μl vol as described in the text, and the lysates were incubated in the presence of no exogenous proteins or 30 μg of GST, 10 mM PIP 2 , 10 mM PIP 2 +30 μg GST–VCA, or 30 μg of GST–VCA for 20 min at 4°C. Vinculin immunoprecipitates were prepared and analyzed as described above. In three independent experiments, compared with the level of Arp2/3 binding to vinculin in response to 5 min EGF stimulation (100%), the level of Arp2/3 complex recruitment in response to the various stimuli was 13% ± 7% (PIP 2 ), 27% ± 15% (GST–VCA), and 94% ± 11% (PIP 2 + GST–VCA).

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Binding Assay, Incubation

    Factors that trigger membrane protrusion induce the association of the Arp2/3 complex with vinculin. (A and B) Growth factor treatment of cells. Serum-starved A431 cells were stimulated with buffer (0) or human EGF (100 ng/ml) for the times indicated (min). The cells were lysed and vinculin or talin was immunoprecipitated. 20% of resulting immunoprecipitates were subjected to Western blot analysis with antibodies against vinculin (A) or talin (B), 10% was used for the actin blots (A and B, bottom), and 70% was used for the blots of p34 subunit of the Arp2/3 complex (p34, bottom). LYS denotes a sample of whole cell lysate. (C) Cells spreading on FN. Hs68, human foreskin fibroblasts, were plated into each well of a FN-coated 6-well plate in serum-free DME. The cells were centrifuged onto the dish and allowed to recover at 37°C for the times indicated. The cells were lysed, vinculin was immunoprecipitated, and cells were blotted as described in A and B. ST, stationary cells that had been plated for 4 h on a similar concentration of FN.

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: Factors that trigger membrane protrusion induce the association of the Arp2/3 complex with vinculin. (A and B) Growth factor treatment of cells. Serum-starved A431 cells were stimulated with buffer (0) or human EGF (100 ng/ml) for the times indicated (min). The cells were lysed and vinculin or talin was immunoprecipitated. 20% of resulting immunoprecipitates were subjected to Western blot analysis with antibodies against vinculin (A) or talin (B), 10% was used for the actin blots (A and B, bottom), and 70% was used for the blots of p34 subunit of the Arp2/3 complex (p34, bottom). LYS denotes a sample of whole cell lysate. (C) Cells spreading on FN. Hs68, human foreskin fibroblasts, were plated into each well of a FN-coated 6-well plate in serum-free DME. The cells were centrifuged onto the dish and allowed to recover at 37°C for the times indicated. The cells were lysed, vinculin was immunoprecipitated, and cells were blotted as described in A and B. ST, stationary cells that had been plated for 4 h on a similar concentration of FN.

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Immunoprecipitation, Western Blot, Concentration Assay

    Mapping the Arp2/3 complex binding site of vinculin. (A) Linear schematic is shown of vinculin and the fragments of vinculin as fusion proteins of GST (GST–VIN). (B and C) Association of the Arp2/3 complex with GST–VIN proteins. Thirty micrograms of fusion protein attached to beads were incubated for 1.5 h in the absence (−) or presence (+) of 10 μl of a crude platelet fraction. The beads were sedimented, washed, and subjected to Western blot analysis as described. Note that in C, the binding of the Arp2/3 complex to vinculin fragments does not involve binding to actin. (D) Substitution of proline 878 or 876 with alanine prevents binding of the Arp2/3 complex to vinculin in vitro. 30 μg of GST or GST fusions of VIN 811–881, VIN 811–881 with a proline to alanine mutation at 876 (P876A) or at 878 (P878A) were examined for their ability to retrieve the Arp2/3 complex, ponsin, vinexin, or VASP from cell lysates obtained from platelets, MDCK, C2C12, or A431 cells, respectively. Different cell types were used for preparing the lysates because of failure to detect the antigens in some cell types. (E) Full-length vinculin harboring a proline to alanine mutation at 878 prevents recruitment of the Arp2/3 complex, but has no effect on actin binding. Serum-starved, Vin−/− MEFs expressing an empty vector (−/−), full-length vinculin (WT), or full-length vinculin with a P878A substitution (P878A) were left resting (−) or stimulated (+) with 10% FBS for 5 min, vinculin immunoprecipitates were obtained and analyzed.

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: Mapping the Arp2/3 complex binding site of vinculin. (A) Linear schematic is shown of vinculin and the fragments of vinculin as fusion proteins of GST (GST–VIN). (B and C) Association of the Arp2/3 complex with GST–VIN proteins. Thirty micrograms of fusion protein attached to beads were incubated for 1.5 h in the absence (−) or presence (+) of 10 μl of a crude platelet fraction. The beads were sedimented, washed, and subjected to Western blot analysis as described. Note that in C, the binding of the Arp2/3 complex to vinculin fragments does not involve binding to actin. (D) Substitution of proline 878 or 876 with alanine prevents binding of the Arp2/3 complex to vinculin in vitro. 30 μg of GST or GST fusions of VIN 811–881, VIN 811–881 with a proline to alanine mutation at 876 (P876A) or at 878 (P878A) were examined for their ability to retrieve the Arp2/3 complex, ponsin, vinexin, or VASP from cell lysates obtained from platelets, MDCK, C2C12, or A431 cells, respectively. Different cell types were used for preparing the lysates because of failure to detect the antigens in some cell types. (E) Full-length vinculin harboring a proline to alanine mutation at 878 prevents recruitment of the Arp2/3 complex, but has no effect on actin binding. Serum-starved, Vin−/− MEFs expressing an empty vector (−/−), full-length vinculin (WT), or full-length vinculin with a P878A substitution (P878A) were left resting (−) or stimulated (+) with 10% FBS for 5 min, vinculin immunoprecipitates were obtained and analyzed.

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Binding Assay, Incubation, Western Blot, In Vitro, Mutagenesis, Expressing, Plasmid Preparation

    Mutation of the proline 878 ablates recruitment of the Arp2/3 complex to FN-coated beads. Vinculin-null cells reexpressing WT vinculin or vinculin with a mutated Arp2/3 complex binding site (P878A) were presented with beads coated with FN (50 μg/ml) for 15 min. The percentage of beads able to recruit vinculin (light gray) and p34GFP (dark gray) was scored by immunofluorescence microscopy.

    Journal: The Journal of Cell Biology

    Article Title: Recruitment of the Arp2/3 complex to vinculin

    doi: 10.1083/jcb.200206043

    Figure Lengend Snippet: Mutation of the proline 878 ablates recruitment of the Arp2/3 complex to FN-coated beads. Vinculin-null cells reexpressing WT vinculin or vinculin with a mutated Arp2/3 complex binding site (P878A) were presented with beads coated with FN (50 μg/ml) for 15 min. The percentage of beads able to recruit vinculin (light gray) and p34GFP (dark gray) was scored by immunofluorescence microscopy.

    Article Snippet: All other fusion proteins containing fragments of vinculin were constructed by PCR amplification of the DNA of interest and subcloning the resulting DNA into pGEX-T. To obtain GST–P876A and GST–P878A, site-specific mutagenesis was carried out using the GST–VIN 811–881 template according to the Quickchange Manual (Stratagene).

    Techniques: Mutagenesis, Binding Assay, Immunofluorescence, Microscopy

    The sequence of the ORF determines the extension of the AUG-proximity effect. ( A ) Physical maps of the α-globin and the hybrid β/α- and α/β/α-globin mRNAs studied. Dark-gray rectangles represent α-globin exons and light-gray rectangles represent β-globin sequences. Translation initiation codons (AUG) and native termination codons (UAA) or premature translation termination codons (PTCs) are depicted by heavy black vertical lines. The name of each mRNA is indicated to its right. ( B ) Representative RPA using RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The identification of α-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the left of the autoradiograph. Levels of α-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations from three independent experiments). mRNA levels are normalized to the expression level of the wild-type mRNA (αWT). A 2-fold RNA sample (2x αWT) from HeLa cells transfected with a αWT gene was also analyzed to demonstrate that the experimental RPA was carried out in probe excess. P -values were estimated using a student's t -test. Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) Schematic representation of the studied β-globin and hybrid α/β- and β/α/β-globin mRNAs, using the same depiction code as for (A) (see above). ( D ) Illustrative RPA of RNA from HeLa cells transiently transfected with the constructs identified beneath each lane. Identification of the β-globin and puro R protected fragments is indicated to the left of the autoradiograph. Average levels (and standard deviations from three independent experiments) of β-globin mRNA (plotted above each respective lane) were measured relatively to the puro R mRNA as in (B) (see above). Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control.

    Journal: Nucleic Acids Research

    Article Title: Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity

    doi: 10.1093/nar/gkv588

    Figure Lengend Snippet: The sequence of the ORF determines the extension of the AUG-proximity effect. ( A ) Physical maps of the α-globin and the hybrid β/α- and α/β/α-globin mRNAs studied. Dark-gray rectangles represent α-globin exons and light-gray rectangles represent β-globin sequences. Translation initiation codons (AUG) and native termination codons (UAA) or premature translation termination codons (PTCs) are depicted by heavy black vertical lines. The name of each mRNA is indicated to its right. ( B ) Representative RPA using RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The identification of α-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the left of the autoradiograph. Levels of α-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations from three independent experiments). mRNA levels are normalized to the expression level of the wild-type mRNA (αWT). A 2-fold RNA sample (2x αWT) from HeLa cells transfected with a αWT gene was also analyzed to demonstrate that the experimental RPA was carried out in probe excess. P -values were estimated using a student's t -test. Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) Schematic representation of the studied β-globin and hybrid α/β- and β/α/β-globin mRNAs, using the same depiction code as for (A) (see above). ( D ) Illustrative RPA of RNA from HeLa cells transiently transfected with the constructs identified beneath each lane. Identification of the β-globin and puro R protected fragments is indicated to the left of the autoradiograph. Average levels (and standard deviations from three independent experiments) of β-globin mRNA (plotted above each respective lane) were measured relatively to the puro R mRNA as in (B) (see above). Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control.

    Article Snippet: The βWT-pk, β23-pk and β39-pk gene variants were constructed by replacing the first 19 codons of a native β-globin ORF by a 19-codon sequence resulting in a pseudoknot structure in the mRNA , using the ExSite PCR-Based Mutagenesis Kit (Stratagene) as indicated by the manufacturer, with mutagenic primers #83 and #84, and the βWT and β39 genes as DNA templates, or primers #83 and #85 using the β23 gene as DNA template.

    Techniques: Sequencing, Recombinase Polymerase Amplification, Isolation, Transfection, Construct, Autoradiography, Expressing

    The boundary of the AUG-proximity effect for NMD resistance is determined by the mRNA secondary structure. ( A ) Schematic representation of the studied human β-globin mRNAs. Heavy black vertical lines indicate the positions of translation initiation codons (AUG) and natural (UAA) or premature termination codons (PTCs). Diagonally-striped rectangles represent 25 consecutive ‘CAA’ triplets [(CAA) 25 ]. The identification of each transcript is indicated to the right. ( B ) Representative RPA of RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The identification of β-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the left of the autoradiograph. Levels of β-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations from three independent experiments) normalized to the expression level of βWT mRNA. A 2-fold RNA sample (2x βWT) from HeLa cells transfected with the βWT gene was also analyzed to ascertain that the RPA was carried out in probe excess. P -values were determined using a student's t -test. Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) Schematic representation of the studied human α-globin mRNAs, using the same depiction code as for (A) (see above). Loop shapes in dark rectangles represent pseudoknot (pk) structures. The alteration of the two potential re-initiating AUGs, at codons 32 and 76, to ACG triplets is indicated by short and thick vertical lines (32–76Met→Thr). The identification of each mRNA is indicated to the right of the diagram. ( D ) Representative RPA using RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The positions of α-globin and the puromycin-resistance (puro R ) protected fragments are indicated to the right of the autoradiograph. Resulting levels of α-globin mRNA were quantified as in (B). Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( E ) Luminometry assay to test translation re-initiation after translation of a highly structured (pseudoknot; pk) or unstructured [(CAA) 26 ] ORF. The 5′-part of α-globin gene upstream of codon 32 (AUG), was fused with the firefly luciferase ORF at its translation initiation codon. Volume-shaped rectangles represent luciferase exons, dark-gray rectangles represent α-globin sequences. The initiation or potential re-initiation, as well as termination [native (UAA) or premature (UAG)] codons, are represented as black vertical lines. A control construct was created, in which the α-globin translation initiation codon was mutated to AUA allowing to control for the maximum of luciferase activity. HeLa cells were transiently cotransfected with firefly and Renilla luciferase-containing plasmids and protein levels were analyzed as above for Figure 2C .

    Journal: Nucleic Acids Research

    Article Title: Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity

    doi: 10.1093/nar/gkv588

    Figure Lengend Snippet: The boundary of the AUG-proximity effect for NMD resistance is determined by the mRNA secondary structure. ( A ) Schematic representation of the studied human β-globin mRNAs. Heavy black vertical lines indicate the positions of translation initiation codons (AUG) and natural (UAA) or premature termination codons (PTCs). Diagonally-striped rectangles represent 25 consecutive ‘CAA’ triplets [(CAA) 25 ]. The identification of each transcript is indicated to the right. ( B ) Representative RPA of RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The identification of β-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the left of the autoradiograph. Levels of β-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations from three independent experiments) normalized to the expression level of βWT mRNA. A 2-fold RNA sample (2x βWT) from HeLa cells transfected with the βWT gene was also analyzed to ascertain that the RPA was carried out in probe excess. P -values were determined using a student's t -test. Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) Schematic representation of the studied human α-globin mRNAs, using the same depiction code as for (A) (see above). Loop shapes in dark rectangles represent pseudoknot (pk) structures. The alteration of the two potential re-initiating AUGs, at codons 32 and 76, to ACG triplets is indicated by short and thick vertical lines (32–76Met→Thr). The identification of each mRNA is indicated to the right of the diagram. ( D ) Representative RPA using RNA isolated from HeLa cells transiently transfected with the constructs specified beneath each lane. The positions of α-globin and the puromycin-resistance (puro R ) protected fragments are indicated to the right of the autoradiograph. Resulting levels of α-globin mRNA were quantified as in (B). Except otherwise indicated, P -values refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( E ) Luminometry assay to test translation re-initiation after translation of a highly structured (pseudoknot; pk) or unstructured [(CAA) 26 ] ORF. The 5′-part of α-globin gene upstream of codon 32 (AUG), was fused with the firefly luciferase ORF at its translation initiation codon. Volume-shaped rectangles represent luciferase exons, dark-gray rectangles represent α-globin sequences. The initiation or potential re-initiation, as well as termination [native (UAA) or premature (UAG)] codons, are represented as black vertical lines. A control construct was created, in which the α-globin translation initiation codon was mutated to AUA allowing to control for the maximum of luciferase activity. HeLa cells were transiently cotransfected with firefly and Renilla luciferase-containing plasmids and protein levels were analyzed as above for Figure 2C .

    Article Snippet: The βWT-pk, β23-pk and β39-pk gene variants were constructed by replacing the first 19 codons of a native β-globin ORF by a 19-codon sequence resulting in a pseudoknot structure in the mRNA , using the ExSite PCR-Based Mutagenesis Kit (Stratagene) as indicated by the manufacturer, with mutagenic primers #83 and #84, and the βWT and β39 genes as DNA templates, or primers #83 and #85 using the β23 gene as DNA template.

    Techniques: Cellular Antioxidant Activity Assay, Recombinase Polymerase Amplification, Isolation, Transfection, Construct, Autoradiography, Expressing, Luciferase, Activity Assay

    Contrary to what occurs in β-globin mRNA, α-globin mRNAs allow for efficient translation re-initiation, but only partially contributes to NMD inhibition. ( A ) Schematic representation of the studied human α-globin mRNAs. Vertical lines represent translation initiation (AUG) or termination [native (UAA) or premature (PTC)] codons. The conversion of the two potential re-initiation AUGs, at codons 32 and 76, to ACG codons is indicated by short and thick vertical lines. The name of each transcript is specified on the right. ( B ) A representative RPA of RNA isolated from HeLa cells transiently transfected with each gene construct is shown. Identification of the α-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the right of the autoradiograph. Levels of α-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations) normalized to the expression level of the αWT gene. A 2-fold RNA sample (2x αWT) from HeLa cells transfected with a αWT gene was also analyzed to demonstrate that the experimental RPA was carried out in probe excess. For each case, three independent experiments were performed. P -values were determined using a student's t -test and refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) The 5′-part of α-globin gene upstream of codon 32 (AUG), or upstream of codon 55 (AUG) of β-globin gene, was fused with the firefly luciferase open reading frame (ORF). Volume-shaped rectangles represent luciferase ORFs, dark-gray rectangles represent α-globin exons and light-gray rectangles represent β-globin exons. The initiation or potential re-initiation, as well as termination [native (UAA) or premature (PTC)] codons, are represented as black vertical lines. A positive control construct was created for each hybrid gene, in which the translation initiation codon for α- or β-globin gene was mutated to AUA. HeLa cells were transiently cotransfected with firefly and Renilla luciferase-containing plasmids. Luciferase activity was analyzed 16 h after transfection. In parallel, both firefly and Renilla luciferase mRNA levels were determined by semi-quantitative RT-PCR. The activities of firefly and Renilla luciferase were calculated relative to their respective mRNA levels (Relative mRNA Levels, left bars), and then the resulting values for firefly luciferase activity/mRNA were quantified relative to those of Renilla luciferase activity/mRNA. The obtained luciferase relative activities from hybrid mRNAs were normalized to the relative activity of the uncoupled firefly luciferase (Luc). The average values and standard deviations from three independent experiments corresponding to three independent sets of transfections are shown.

    Journal: Nucleic Acids Research

    Article Title: Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity

    doi: 10.1093/nar/gkv588

    Figure Lengend Snippet: Contrary to what occurs in β-globin mRNA, α-globin mRNAs allow for efficient translation re-initiation, but only partially contributes to NMD inhibition. ( A ) Schematic representation of the studied human α-globin mRNAs. Vertical lines represent translation initiation (AUG) or termination [native (UAA) or premature (PTC)] codons. The conversion of the two potential re-initiation AUGs, at codons 32 and 76, to ACG codons is indicated by short and thick vertical lines. The name of each transcript is specified on the right. ( B ) A representative RPA of RNA isolated from HeLa cells transiently transfected with each gene construct is shown. Identification of the α-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the right of the autoradiograph. Levels of α-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations) normalized to the expression level of the αWT gene. A 2-fold RNA sample (2x αWT) from HeLa cells transfected with a αWT gene was also analyzed to demonstrate that the experimental RPA was carried out in probe excess. For each case, three independent experiments were performed. P -values were determined using a student's t -test and refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) The 5′-part of α-globin gene upstream of codon 32 (AUG), or upstream of codon 55 (AUG) of β-globin gene, was fused with the firefly luciferase open reading frame (ORF). Volume-shaped rectangles represent luciferase ORFs, dark-gray rectangles represent α-globin exons and light-gray rectangles represent β-globin exons. The initiation or potential re-initiation, as well as termination [native (UAA) or premature (PTC)] codons, are represented as black vertical lines. A positive control construct was created for each hybrid gene, in which the translation initiation codon for α- or β-globin gene was mutated to AUA. HeLa cells were transiently cotransfected with firefly and Renilla luciferase-containing plasmids. Luciferase activity was analyzed 16 h after transfection. In parallel, both firefly and Renilla luciferase mRNA levels were determined by semi-quantitative RT-PCR. The activities of firefly and Renilla luciferase were calculated relative to their respective mRNA levels (Relative mRNA Levels, left bars), and then the resulting values for firefly luciferase activity/mRNA were quantified relative to those of Renilla luciferase activity/mRNA. The obtained luciferase relative activities from hybrid mRNAs were normalized to the relative activity of the uncoupled firefly luciferase (Luc). The average values and standard deviations from three independent experiments corresponding to three independent sets of transfections are shown.

    Article Snippet: The βWT-pk, β23-pk and β39-pk gene variants were constructed by replacing the first 19 codons of a native β-globin ORF by a 19-codon sequence resulting in a pseudoknot structure in the mRNA , using the ExSite PCR-Based Mutagenesis Kit (Stratagene) as indicated by the manufacturer, with mutagenic primers #83 and #84, and the βWT and β39 genes as DNA templates, or primers #83 and #85 using the β23 gene as DNA template.

    Techniques: Inhibition, Recombinase Polymerase Amplification, Isolation, Transfection, Construct, Autoradiography, Expressing, Luciferase, Positive Control, Activity Assay, Quantitative RT-PCR

    A model for the effect of an AUG-proximal premature termination codon (PTC or stop codon). ( A ) The effect in the human β-globin transcript. During cap-mediated translation initiation, cytoplasmic poly(A) binding protein 1 (PABPC1) interacts with the eukaryotic initiation factor 4G (eIF4G). This interaction indirectly tethers PABPC1 to the 40S ribosomal subunit via the interaction of eIF4G with eIF3, which interacts with the 40S. The resulting configuration brings PABPC1 into the vicinity of the AUG initiation codon as a consequence of 43S scanning. The maintenance of the PABPC1-eIF4G-eIF3 interactions with the 40S during the first steps of translation elongation brings PABPC1 into close proximity with the termination complex at an AUG-proximal PTC. This proximity allows PABPC1 to interact with the release factor eRF3, thus impairing the UPF1-eRF3 interaction, resulting in efficient translation termination and inhibition of NMD—this was called the ‘AUG-proximity effect’. ( B ) In the human α-globin transcript, the AUG-proximity effect is observed for PTCs located further downstream because the open reading frame (ORF) is less structured. The more relaxed structure allows a faster elongation rate resulting in a longer window of the AUG proximity effect. In addition, the brief ORF translation also allows efficient translation re-initiation to occur at codon 32. This re-initiation diminishes the levels of residual exon junction complexes on the mRNA, thus contributing to the overall repression of NMD.

    Journal: Nucleic Acids Research

    Article Title: Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity

    doi: 10.1093/nar/gkv588

    Figure Lengend Snippet: A model for the effect of an AUG-proximal premature termination codon (PTC or stop codon). ( A ) The effect in the human β-globin transcript. During cap-mediated translation initiation, cytoplasmic poly(A) binding protein 1 (PABPC1) interacts with the eukaryotic initiation factor 4G (eIF4G). This interaction indirectly tethers PABPC1 to the 40S ribosomal subunit via the interaction of eIF4G with eIF3, which interacts with the 40S. The resulting configuration brings PABPC1 into the vicinity of the AUG initiation codon as a consequence of 43S scanning. The maintenance of the PABPC1-eIF4G-eIF3 interactions with the 40S during the first steps of translation elongation brings PABPC1 into close proximity with the termination complex at an AUG-proximal PTC. This proximity allows PABPC1 to interact with the release factor eRF3, thus impairing the UPF1-eRF3 interaction, resulting in efficient translation termination and inhibition of NMD—this was called the ‘AUG-proximity effect’. ( B ) In the human α-globin transcript, the AUG-proximity effect is observed for PTCs located further downstream because the open reading frame (ORF) is less structured. The more relaxed structure allows a faster elongation rate resulting in a longer window of the AUG proximity effect. In addition, the brief ORF translation also allows efficient translation re-initiation to occur at codon 32. This re-initiation diminishes the levels of residual exon junction complexes on the mRNA, thus contributing to the overall repression of NMD.

    Article Snippet: The βWT-pk, β23-pk and β39-pk gene variants were constructed by replacing the first 19 codons of a native β-globin ORF by a 19-codon sequence resulting in a pseudoknot structure in the mRNA , using the ExSite PCR-Based Mutagenesis Kit (Stratagene) as indicated by the manufacturer, with mutagenic primers #83 and #84, and the βWT and β39 genes as DNA templates, or primers #83 and #85 using the β23 gene as DNA template.

    Techniques: Binding Assay, Inhibition