pbs  (New England Biolabs)


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  • 92

    Structured Review

    New England Biolabs pbs
    Pbs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbs/product/New England Biolabs
    Average 92 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    pbs - by Bioz Stars, 2020-05
    92/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Enhancement of Sleeping Beauty Transposition by CpG Methylation: Possible Role of Heterochromatin Formation
    Article Snippet: .. For the DNA probe of IR/DR-L for the electrophoretic mobility shift assay (EMSA), the HindIII-KpnI fragment of pTransCX- EGFP : Neo was cloned into the HindIII-KpnI site of pBS, resulting in pBS-IR/DR-L. Methylation of plasmid DNAs was performed with SssI CpG methylase (NEB) according to the manufacturer's protocol, followed by purification with a PCR purification kit (Qiagen). .. Complete methylation was confirmed by resistance to digestion with methylation-sensitive enzymes.

    Methylation:

    Article Title: Enhancement of Sleeping Beauty Transposition by CpG Methylation: Possible Role of Heterochromatin Formation
    Article Snippet: .. For the DNA probe of IR/DR-L for the electrophoretic mobility shift assay (EMSA), the HindIII-KpnI fragment of pTransCX- EGFP : Neo was cloned into the HindIII-KpnI site of pBS, resulting in pBS-IR/DR-L. Methylation of plasmid DNAs was performed with SssI CpG methylase (NEB) according to the manufacturer's protocol, followed by purification with a PCR purification kit (Qiagen). .. Complete methylation was confirmed by resistance to digestion with methylation-sensitive enzymes.

    Protein Binding:

    Article Title: Functionalized Tobacco Mosaic Virus Coat Protein Monomers and Oligomers as Nanocarriers for Anti-Cancer Peptides
    Article Snippet: .. The samples were treated overnight at 4 °C in PBS with appropriate combinations of primary antibodies (mouse anti-NRP1 (Evitria) and rabbit anti-MBP (New England Biolabs, E8031S) for detection of CPL fusion protein binding to NRP1; mouse anti-NRP1 (Evitria) and rabbit anti-PlexA1 (Abcam, ab23391) for detection of receptor protein dimer disruption). .. Subsequent steps of the assay were performed according to the manufacturer’s recommendations described in the Duolink In Situ Fluorescence Protocol with components of the Duolink PLA and Duolink In Situ Detection Orange kits (Sigma-Aldrich).

    Electrophoretic Mobility Shift Assay:

    Article Title: Enhancement of Sleeping Beauty Transposition by CpG Methylation: Possible Role of Heterochromatin Formation
    Article Snippet: .. For the DNA probe of IR/DR-L for the electrophoretic mobility shift assay (EMSA), the HindIII-KpnI fragment of pTransCX- EGFP : Neo was cloned into the HindIII-KpnI site of pBS, resulting in pBS-IR/DR-L. Methylation of plasmid DNAs was performed with SssI CpG methylase (NEB) according to the manufacturer's protocol, followed by purification with a PCR purification kit (Qiagen). .. Complete methylation was confirmed by resistance to digestion with methylation-sensitive enzymes.

    Purification:

    Article Title: Enhancement of Sleeping Beauty Transposition by CpG Methylation: Possible Role of Heterochromatin Formation
    Article Snippet: .. For the DNA probe of IR/DR-L for the electrophoretic mobility shift assay (EMSA), the HindIII-KpnI fragment of pTransCX- EGFP : Neo was cloned into the HindIII-KpnI site of pBS, resulting in pBS-IR/DR-L. Methylation of plasmid DNAs was performed with SssI CpG methylase (NEB) according to the manufacturer's protocol, followed by purification with a PCR purification kit (Qiagen). .. Complete methylation was confirmed by resistance to digestion with methylation-sensitive enzymes.

    Article Title: The occurrence of chitin in the hemocytes of invertebrates
    Article Snippet: .. Fixed hemocytes were exposed to three different conditions: (i) phosphate-buffered saline (PBS; 50 mM sodium phosphate, 100 mM sodium chloride, pH 7.4) containing 1 mM sodium ethylenediaminetetraacetic acid, pH 6.0, and 40 units of purified Brugia malayi chitinase (New England BioLabs, Ipswich, MA, USA); (ii) buffer without chitinase; or, (iii) as a control for the effects of the treatment conditions, 40 units of chitinase in the same buffer heated to 75 °C for 20 min (heat-inactivated chitinase). ..

    Incubation:

    Article Title: Temperature-dependent release of ATP from human erythrocytes: mechanism for the control of local tissue perfusion
    Article Snippet: .. After washing with PBS, wells were incubated with rabbit polyclonal antibodies raised against the amino terminus of human CFTR diluted 1:2000 in PBS (New England Biolabs, Hitchin, UK) for 1 h at 37°C and 2 h at 4°C. .. After washing with 0.02% Tween 20 in PBS, wells were treated with human anti-protein A conjugated to horseradish peroxidase (Sigma) diluted to 1:3000 in PBS and incubated at 37°C for 1 h. The reaction was developed using o -phenylenediamine dihydrochloride (SigmaFAST™).

    Article Title: Caspase-9 Activation Results in Downstream Caspase-8 Activation and Bid Cleavage in 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Parkinson's Disease
    Article Snippet: .. After being washed three times in PBS, the cells were incubated with rabbit polyclonal antibodies that recognize the active forms of caspase-3 (1:50; New England Biolabs, Beverly, MA), caspase-9 (1:100; New England Biolabs), and caspase-8 (1:500; SK440, gift from SmithKline Beecham, Philadelphia, PA) at 4°C overnight. .. After being washed with PBS, the cells were incubated with BODIPY FL goat anti-rabbit IgG conjugates (1:200; Molecular Probes, Eugene, OR) at RT for 1 hr.

    Article Title: miR-124-3p acts as a potential marker and suppresses tumor growth in gastric cancer
    Article Snippet: .. To block nonspecific binding, the membranes were incubated with 5% skimmed milk powder in phosphate-buffered saline at room temperature for 1 h. The membranes were then incubated with primary antibodies overnight at 4°C, followed by horseradish peroxidase-labeled secondary antibodies at room temperature for 1 h, and protein bands were detected by electrochemiluminescence (ECL) with ECL western blotting detection reagents (New England BioLabs, Inc., Ipswich, MA, USA). .. The antibodies and their dilutions were the same as those used for the IHC analysis. β-actin (4967S; 1:500; Cell Signaling Technology, Inc., Danvers, MA, USA) was used as a protein-loading control.

    Article Title: Ankyrin facilitates intracellular trafficking of ?1-Na+-K+-ATPase in polarized cells
    Article Snippet: .. Precipitates were washed three times with PBS, resuspended in 40 μl of 0.5% SDS and 1% 2-mercaptoethanol, and incubated at 100°C for 10 min. Insoluble matter was precipitated, and 5 μl of 500 mM sodium citrate (pH 5.5) containing 5 μl endoglycosidase H (New England Biolabs) were added. .. Digestion was carried out for 6 h at 37°C, after which samples were analyzed by SDS-PAGE and Western blotted using anti-GFP antibody.

    other:

    Article Title: Single-cell transcriptomics of the human retinal pigment epithelium and choroid in health and macular degeneration
    Article Snippet: Cryopreserved cells were rapidly thawed in a 37 °C water bath and resuspended to concentrations of 1,000 cells/μL in PBS with 0.04% nonacetylated BSA (New England Biolabs).

    Blocking Assay:

    Article Title: miR-124-3p acts as a potential marker and suppresses tumor growth in gastric cancer
    Article Snippet: .. To block nonspecific binding, the membranes were incubated with 5% skimmed milk powder in phosphate-buffered saline at room temperature for 1 h. The membranes were then incubated with primary antibodies overnight at 4°C, followed by horseradish peroxidase-labeled secondary antibodies at room temperature for 1 h, and protein bands were detected by electrochemiluminescence (ECL) with ECL western blotting detection reagents (New England BioLabs, Inc., Ipswich, MA, USA). .. The antibodies and their dilutions were the same as those used for the IHC analysis. β-actin (4967S; 1:500; Cell Signaling Technology, Inc., Danvers, MA, USA) was used as a protein-loading control.

    Polymerase Chain Reaction:

    Article Title: Enhancement of Sleeping Beauty Transposition by CpG Methylation: Possible Role of Heterochromatin Formation
    Article Snippet: .. For the DNA probe of IR/DR-L for the electrophoretic mobility shift assay (EMSA), the HindIII-KpnI fragment of pTransCX- EGFP : Neo was cloned into the HindIII-KpnI site of pBS, resulting in pBS-IR/DR-L. Methylation of plasmid DNAs was performed with SssI CpG methylase (NEB) according to the manufacturer's protocol, followed by purification with a PCR purification kit (Qiagen). .. Complete methylation was confirmed by resistance to digestion with methylation-sensitive enzymes.

    Western Blot:

    Article Title: miR-124-3p acts as a potential marker and suppresses tumor growth in gastric cancer
    Article Snippet: .. To block nonspecific binding, the membranes were incubated with 5% skimmed milk powder in phosphate-buffered saline at room temperature for 1 h. The membranes were then incubated with primary antibodies overnight at 4°C, followed by horseradish peroxidase-labeled secondary antibodies at room temperature for 1 h, and protein bands were detected by electrochemiluminescence (ECL) with ECL western blotting detection reagents (New England BioLabs, Inc., Ipswich, MA, USA). .. The antibodies and their dilutions were the same as those used for the IHC analysis. β-actin (4967S; 1:500; Cell Signaling Technology, Inc., Danvers, MA, USA) was used as a protein-loading control.

    Binding Assay:

    Article Title: miR-124-3p acts as a potential marker and suppresses tumor growth in gastric cancer
    Article Snippet: .. To block nonspecific binding, the membranes were incubated with 5% skimmed milk powder in phosphate-buffered saline at room temperature for 1 h. The membranes were then incubated with primary antibodies overnight at 4°C, followed by horseradish peroxidase-labeled secondary antibodies at room temperature for 1 h, and protein bands were detected by electrochemiluminescence (ECL) with ECL western blotting detection reagents (New England BioLabs, Inc., Ipswich, MA, USA). .. The antibodies and their dilutions were the same as those used for the IHC analysis. β-actin (4967S; 1:500; Cell Signaling Technology, Inc., Danvers, MA, USA) was used as a protein-loading control.

    Plasmid Preparation:

    Article Title: Enhancement of Sleeping Beauty Transposition by CpG Methylation: Possible Role of Heterochromatin Formation
    Article Snippet: .. For the DNA probe of IR/DR-L for the electrophoretic mobility shift assay (EMSA), the HindIII-KpnI fragment of pTransCX- EGFP : Neo was cloned into the HindIII-KpnI site of pBS, resulting in pBS-IR/DR-L. Methylation of plasmid DNAs was performed with SssI CpG methylase (NEB) according to the manufacturer's protocol, followed by purification with a PCR purification kit (Qiagen). .. Complete methylation was confirmed by resistance to digestion with methylation-sensitive enzymes.

    Electrochemiluminescence:

    Article Title: miR-124-3p acts as a potential marker and suppresses tumor growth in gastric cancer
    Article Snippet: .. To block nonspecific binding, the membranes were incubated with 5% skimmed milk powder in phosphate-buffered saline at room temperature for 1 h. The membranes were then incubated with primary antibodies overnight at 4°C, followed by horseradish peroxidase-labeled secondary antibodies at room temperature for 1 h, and protein bands were detected by electrochemiluminescence (ECL) with ECL western blotting detection reagents (New England BioLabs, Inc., Ipswich, MA, USA). .. The antibodies and their dilutions were the same as those used for the IHC analysis. β-actin (4967S; 1:500; Cell Signaling Technology, Inc., Danvers, MA, USA) was used as a protein-loading control.

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    New England Biolabs pbs sk csx nkx2 5 ref
    Effect of ten mutations on transcriptional activation. 10T1/2 cells were transfected with pcDNA3 expression vectors encoding wild-type or each of ten mutations with the reporter gene ANF-luciferase. When the wild-type <t>CSX/NKX2.5</t> was transfected with the ANF reporter gene, luciferase activity was increased 23-fold compared with cells transfected with the empty expression vector pcDNA3. M112, group 1, and group 2 expression vectors did not activate the ANF promoter. M25 and M198 transactivated the reporter gene similarly to the wild-type CSX/NKX2.5; however, M259 transactivated only 5.2-fold. Another COOH-terminus deletion mutant, CSX/NKX2.5(1–200), transactivated the reporter construct approximately 136-fold. Bars represent means ± SEM of at least three separate transfection assays done in duplicate.
    Pbs Sk Csx Nkx2 5 Ref, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbs sk csx nkx2 5 ref/product/New England Biolabs
    Average 85 stars, based on 1 article reviews
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    New England Biolabs pbs ii k plasmid dna
    Production of joint molecules used to test the branch migration activity of proteins. (A) Joint molecules with a 3′ displaced ssDNA tail are produced by RAD51 using gapped <t>DNA</t> and <t>pBS</t> <t>II</t> K (+) dsDNA linearized with XhoI. (B) Joint molecules with
    Pbs Ii K Plasmid Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs pbs gfp
    The RpoS Ec recognition motif of P ospC lies within a 65-bp core region. The activity levels of P ospC TA (TA), P ospC CG (CG), and P ospC trunc (trunc), and the full-length P ospC , all cloned into <t>pBS(</t> <t>gfp</t> ), were compared after 8 h of growth in either LM5005 (speckled
    Pbs Gfp, supplied by New England Biolabs, 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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    88
    New England Biolabs primer binding site pbs
    (A) The 3′ end of the primer was blocked by the addition of an AZT residue. The ability of wild-type <t>HIV-1</t> RT and the RT variants to remove the blocking AZT residue (deblocking) and extend the freed end of the primer was tested in the presence of 100.0 μM concentrations of each dNTP, 10.0 μM AZTTP, and varying concentrations of ATP (1.0, 2.0, 5.0, and 10.0 mM) as the pyrophosphate donor. The ratio of dTTP:AZTTP remained at 10:1. The locations of the starting <t>PBS</t> primer and the fully extended primer are marked. (B) The gel in panel A was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of ATP present in the reaction mixture. (C) The 3′ end of the primer was blocked by the addition of a ddT residue. The ability of wild-type HIV-1 RT and the RT variants to remove the blocking ddT residue (deblocking) and extend the freed end of the primer was tested in the presence of 100.0 μM concentrations of each dNTP, 10.0 μM ddTTP, and varying concentrations of ATP (1.0, 2.0, 5.0, and 10.0 mM) as the pyrophosphate donor. The ratio of dTTP:ddTTP remained at 10:1. The locations of the starting PBS primer and the fully extended primer are marked. (D) The gel in panel C was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of ATP present in the reaction mixture. Experiments using D4TTP as the blocking group gave similar results.
    Primer Binding Site Pbs, supplied by New England Biolabs, 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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    Average 88 stars, based on 2 article reviews
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    Effect of ten mutations on transcriptional activation. 10T1/2 cells were transfected with pcDNA3 expression vectors encoding wild-type or each of ten mutations with the reporter gene ANF-luciferase. When the wild-type CSX/NKX2.5 was transfected with the ANF reporter gene, luciferase activity was increased 23-fold compared with cells transfected with the empty expression vector pcDNA3. M112, group 1, and group 2 expression vectors did not activate the ANF promoter. M25 and M198 transactivated the reporter gene similarly to the wild-type CSX/NKX2.5; however, M259 transactivated only 5.2-fold. Another COOH-terminus deletion mutant, CSX/NKX2.5(1–200), transactivated the reporter construct approximately 136-fold. Bars represent means ± SEM of at least three separate transfection assays done in duplicate.

    Journal: Journal of Clinical Investigation

    Article Title: Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease

    doi:

    Figure Lengend Snippet: Effect of ten mutations on transcriptional activation. 10T1/2 cells were transfected with pcDNA3 expression vectors encoding wild-type or each of ten mutations with the reporter gene ANF-luciferase. When the wild-type CSX/NKX2.5 was transfected with the ANF reporter gene, luciferase activity was increased 23-fold compared with cells transfected with the empty expression vector pcDNA3. M112, group 1, and group 2 expression vectors did not activate the ANF promoter. M25 and M198 transactivated the reporter gene similarly to the wild-type CSX/NKX2.5; however, M259 transactivated only 5.2-fold. Another COOH-terminus deletion mutant, CSX/NKX2.5(1–200), transactivated the reporter construct approximately 136-fold. Bars represent means ± SEM of at least three separate transfection assays done in duplicate.

    Article Snippet: pBS SK(-)- CSX/NKX2.5 (ref. ) digested with BglI -blunt–ended and EcoRI was ligated into SacI -blunt–ended and EcoRI -digested pMALC2 (New England Biolabs Inc., Beverly, Massachusetts, USA) to construct maltose binding protein–CSX/NKX2.5 (MBP-CSX/NKX2.5).

    Techniques: Activation Assay, Transfection, Expressing, Luciferase, Activity Assay, Plasmid Preparation, Mutagenesis, Construct

    Interaction of group 2 mutants with GATA4 protein. [ 35 S]-labeled wild-type CSX/NKX2.5 and four group 2 mutant proteins were mixed with GST-GATA4 protein (lanes 1–5) or GST alone (lanes 6–10). Bound labeled proteins were resolved on SDS-PAGE and autoradiographed (top panel). Fifty percent input of [ 35 S]-labeled proteins is also shown. Coomassie blue–stained GST-GATA4 (lanes 1–5) or GST (lanes 6–10) fusion proteins are shown (bottom panel).

    Journal: Journal of Clinical Investigation

    Article Title: Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease

    doi:

    Figure Lengend Snippet: Interaction of group 2 mutants with GATA4 protein. [ 35 S]-labeled wild-type CSX/NKX2.5 and four group 2 mutant proteins were mixed with GST-GATA4 protein (lanes 1–5) or GST alone (lanes 6–10). Bound labeled proteins were resolved on SDS-PAGE and autoradiographed (top panel). Fifty percent input of [ 35 S]-labeled proteins is also shown. Coomassie blue–stained GST-GATA4 (lanes 1–5) or GST (lanes 6–10) fusion proteins are shown (bottom panel).

    Article Snippet: pBS SK(-)- CSX/NKX2.5 (ref. ) digested with BglI -blunt–ended and EcoRI was ligated into SacI -blunt–ended and EcoRI -digested pMALC2 (New England Biolabs Inc., Beverly, Massachusetts, USA) to construct maltose binding protein–CSX/NKX2.5 (MBP-CSX/NKX2.5).

    Techniques: Labeling, Mutagenesis, SDS Page, Staining

    Expression of translated products in cells: intron-splicing defect in M112 mutant resulted in poor protein accumulation. ( a ) Wild-type and mutant CSX/NKX2.5 expression vectors were transfected into COS 7 cells, and the protein expression was examined by Western blotting using anti-FLAG Ab approximately 24 hours after transfection (FLAG, top). All mutant proteins except M112 (lane 11, asterisk indicates the expected molecular weight of M112 protein) were detected at the expected molecular weight. GAPDH expression in each lane was also shown (GAPDH, bottom). ( b ) Wild-type and mutant CSX/NKX2.5 proteins accumulated in the nucleus colocalizing with Hoechst nuclear staining (NUC, lower panels). The results presented are wild-type, group 1 (M170), group 2 (M191), group 3 (M198), and group 4 (M25). ( c ) G→T substitution (large arrow) identified in M112 mutant on the CSX/NKX2.5 splicing donor site was examined by RT-PCR. RNA-purified form COS 7 cells transfected with the wild-type and M112 mutant of CSX/NKX2.5 were amplified with two primers spanning the intron. In the wild-type transfectant, the intron was spliced out, resulting in the generation of 240-bp product, whereas in the mutant transfectant, G→T substitution of the first codon of the intron splicing site abolished the normal splicing, resulting in generation of 1,779-bp product. CSX/NKX2.5 protein was encoded by the two exons represented. ( d ) Translated products were examined approximately 24 hours after transfection by Western blotting using anti-FLAG mAb (top) and anti-Csx/Nkx2.5 mAb (bottom). Wild-type CSX/NKX2.5 gene was translated into approximately 42-kDa protein and was detected with anti-FLAG and anti-Csx/Nkx2.5 mAb (lane 1). In contrast, M112 mutant protein that is expected to migrate approximately 29 kDa was not detected in the cell lysate (lane 2). In in vitro transcription and translation system, cDNA produced approximately 42-kDa protein (lane 1), and M112 genomic construct produced about 29-kDa protein (lane 2).

    Journal: Journal of Clinical Investigation

    Article Title: Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease

    doi:

    Figure Lengend Snippet: Expression of translated products in cells: intron-splicing defect in M112 mutant resulted in poor protein accumulation. ( a ) Wild-type and mutant CSX/NKX2.5 expression vectors were transfected into COS 7 cells, and the protein expression was examined by Western blotting using anti-FLAG Ab approximately 24 hours after transfection (FLAG, top). All mutant proteins except M112 (lane 11, asterisk indicates the expected molecular weight of M112 protein) were detected at the expected molecular weight. GAPDH expression in each lane was also shown (GAPDH, bottom). ( b ) Wild-type and mutant CSX/NKX2.5 proteins accumulated in the nucleus colocalizing with Hoechst nuclear staining (NUC, lower panels). The results presented are wild-type, group 1 (M170), group 2 (M191), group 3 (M198), and group 4 (M25). ( c ) G→T substitution (large arrow) identified in M112 mutant on the CSX/NKX2.5 splicing donor site was examined by RT-PCR. RNA-purified form COS 7 cells transfected with the wild-type and M112 mutant of CSX/NKX2.5 were amplified with two primers spanning the intron. In the wild-type transfectant, the intron was spliced out, resulting in the generation of 240-bp product, whereas in the mutant transfectant, G→T substitution of the first codon of the intron splicing site abolished the normal splicing, resulting in generation of 1,779-bp product. CSX/NKX2.5 protein was encoded by the two exons represented. ( d ) Translated products were examined approximately 24 hours after transfection by Western blotting using anti-FLAG mAb (top) and anti-Csx/Nkx2.5 mAb (bottom). Wild-type CSX/NKX2.5 gene was translated into approximately 42-kDa protein and was detected with anti-FLAG and anti-Csx/Nkx2.5 mAb (lane 1). In contrast, M112 mutant protein that is expected to migrate approximately 29 kDa was not detected in the cell lysate (lane 2). In in vitro transcription and translation system, cDNA produced approximately 42-kDa protein (lane 1), and M112 genomic construct produced about 29-kDa protein (lane 2).

    Article Snippet: pBS SK(-)- CSX/NKX2.5 (ref. ) digested with BglI -blunt–ended and EcoRI was ligated into SacI -blunt–ended and EcoRI -digested pMALC2 (New England Biolabs Inc., Beverly, Massachusetts, USA) to construct maltose binding protein–CSX/NKX2.5 (MBP-CSX/NKX2.5).

    Techniques: Expressing, Mutagenesis, Transfection, Western Blot, Molecular Weight, Staining, Reverse Transcription Polymerase Chain Reaction, Purification, Amplification, In Vitro, Produced, Construct

    DNA binding affinity of group 1, 2, and 5 mutant proteins versus wild-type CSX/NKX2.5. ( a ) Sequence of two consensus CSX/NKX2.5 binding sites (–242 bp and –87 bp sites) in rat ANF promoter; the paired binding sites in –242 bp site, and a single binding site in –87 bp site. ( b ) –242 bp site was used for the DNA binding assay (lane 1) mixed with threefold serially increased CSX/NKX2.5 fusion proteins (0.018–4.4 μg/mL of MBP-CSX/NKX2.5 fusion protein) (lanes 2–7). Wild-type CSX/NKX2.5 bound as a monomer (M) as well as a dimer (D) depending on the protein concentration. No shifted bands were observed in groups 1 and 5 (M149, M170, and M112). ( c ) The EMSA of the group 2 mutant proteins that have a single missense mutation in the HD. 178 Thr-Met (M178) mutation was located just before the third helix, and 188 Asn-Lys (M188), 189 Arg-Gly (M189), and 191 Tyr-Cys (M191) were located in the third helix. Two conserved amino acid mutations were identified: 188 Asn ( 51 Asn in HD) is conserved in all the HD protein that is directly bound to the major groove of DNA, and 191 Tyr ( 54 Tyr in HD) is conserved in all NK2 class homeoprotein. All four mutant proteins show dramatically reduced DNA binding affinity compared with the wild-type CSX/NKX2.5. D, dimer; M, monomer; F, free probe. Mutation sites of group 2 are indicated with white asterisks.

    Journal: Journal of Clinical Investigation

    Article Title: Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease

    doi:

    Figure Lengend Snippet: DNA binding affinity of group 1, 2, and 5 mutant proteins versus wild-type CSX/NKX2.5. ( a ) Sequence of two consensus CSX/NKX2.5 binding sites (–242 bp and –87 bp sites) in rat ANF promoter; the paired binding sites in –242 bp site, and a single binding site in –87 bp site. ( b ) –242 bp site was used for the DNA binding assay (lane 1) mixed with threefold serially increased CSX/NKX2.5 fusion proteins (0.018–4.4 μg/mL of MBP-CSX/NKX2.5 fusion protein) (lanes 2–7). Wild-type CSX/NKX2.5 bound as a monomer (M) as well as a dimer (D) depending on the protein concentration. No shifted bands were observed in groups 1 and 5 (M149, M170, and M112). ( c ) The EMSA of the group 2 mutant proteins that have a single missense mutation in the HD. 178 Thr-Met (M178) mutation was located just before the third helix, and 188 Asn-Lys (M188), 189 Arg-Gly (M189), and 191 Tyr-Cys (M191) were located in the third helix. Two conserved amino acid mutations were identified: 188 Asn ( 51 Asn in HD) is conserved in all the HD protein that is directly bound to the major groove of DNA, and 191 Tyr ( 54 Tyr in HD) is conserved in all NK2 class homeoprotein. All four mutant proteins show dramatically reduced DNA binding affinity compared with the wild-type CSX/NKX2.5. D, dimer; M, monomer; F, free probe. Mutation sites of group 2 are indicated with white asterisks.

    Article Snippet: pBS SK(-)- CSX/NKX2.5 (ref. ) digested with BglI -blunt–ended and EcoRI was ligated into SacI -blunt–ended and EcoRI -digested pMALC2 (New England Biolabs Inc., Beverly, Massachusetts, USA) to construct maltose binding protein–CSX/NKX2.5 (MBP-CSX/NKX2.5).

    Techniques: Binding Assay, Mutagenesis, Sequencing, DNA Binding Assay, Protein Concentration

    DNA binding affinity of the group 3 and 4 mutant proteins versus wild-type CSX/NKX2.5. Sequence of the native –242 bp site (top) and a mutated –242 bp binding site (bottom) in the ANF promoter. EMSA of group 3 (M198 and M259) and group 4 (M25, the mutation site is marked with an asterisk) protein compared with that of the wild-type CSX/NKX2.5. Proteins were mixed with probes containing either tandemly repeated binding sites ( a – d ) or single binding site ( e – h ). Lanes showing similar monomer/dimer ratios are indicated with asterisks in the top panels ( a – d ). In all three mutant proteins, binding affinity as a dimer is reduced approximately 3- to 3 2 fold ( b – d versus a ), whereas they show similar DNA binding affinity as wild-type to the single binding site ( f – h versus e ). D, dimer; M, monomer; F, free probe.

    Journal: Journal of Clinical Investigation

    Article Title: Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease

    doi:

    Figure Lengend Snippet: DNA binding affinity of the group 3 and 4 mutant proteins versus wild-type CSX/NKX2.5. Sequence of the native –242 bp site (top) and a mutated –242 bp binding site (bottom) in the ANF promoter. EMSA of group 3 (M198 and M259) and group 4 (M25, the mutation site is marked with an asterisk) protein compared with that of the wild-type CSX/NKX2.5. Proteins were mixed with probes containing either tandemly repeated binding sites ( a – d ) or single binding site ( e – h ). Lanes showing similar monomer/dimer ratios are indicated with asterisks in the top panels ( a – d ). In all three mutant proteins, binding affinity as a dimer is reduced approximately 3- to 3 2 fold ( b – d versus a ), whereas they show similar DNA binding affinity as wild-type to the single binding site ( f – h versus e ). D, dimer; M, monomer; F, free probe.

    Article Snippet: pBS SK(-)- CSX/NKX2.5 (ref. ) digested with BglI -blunt–ended and EcoRI was ligated into SacI -blunt–ended and EcoRI -digested pMALC2 (New England Biolabs Inc., Beverly, Massachusetts, USA) to construct maltose binding protein–CSX/NKX2.5 (MBP-CSX/NKX2.5).

    Techniques: Binding Assay, Mutagenesis, Sequencing

    Inhibition of ANF-luciferase transcriptional activity of wild-type CSX/NKX2.5 by group 1, 2, and 3 mutants but not by group 4 mutant. ( a ) Inhibition of transactivation of the ANF-luciferase reporter gene in the presence of both 1:1 and 2:1 ratio of M170, M189, M191, and M259 expression plasmid to wild-type expression plasmid. 10T1/2 cells were transiently transacted with 1.2 μg of ANF-luciferase(-638), 0.3 μg of RSV–β-galactosidase, 0.7 μg of wild-type CSX/NKX2.5 expression plasmid, and 0.7 μg (hatched bars) or 1.4 μg (filled bars) of mutant expression plasmid or empty pcDNA3 plasmid to adjust the total amount of plasmid. A moderate reduction of luciferase activity was observed in M170 mutants, and a further decrease of luciferase activity was detected in M189, M191, and M250 mutants. In contrast, M25 mutant increased luciferase activity. Results are presented as a percent of the ANF reporter activity when cotransfected with wild-type and mutant plasmid compared with that of the wild type alone (open bar). Bars represent means ± SEM of the means of at least three separate transfection assays done in duplicate. ( b ) Protein-protein interaction of wild-type CSX/NKX2.5 with mutant proteins. MBP-fused wild-type CSX/NKX2.5 protein was mixed with [ 35 S]-labeled wild-type (lane 1) or ten mutants (lanes 2–11). After washing five times with binding buffer, the protein complexes were resolved on SDS-PAGE and autoradiographed (top panel). Group 1 (lanes 2 and 3) and group 5 (lane 11) mutants did not associate with MBP-CSX/NKX2.5, whereas group 2, 3, and 4 mutants associated with MBP-CSX/NKX2.5 (lanes 4–10). Fifty percent input of [ 35 S]-labeled proteins are shown in the middle panel, and Coomassie blue–stained MBP-fused wild-type CSX/NKX2.5 proteins are shown in the bottom panel.

    Journal: Journal of Clinical Investigation

    Article Title: Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease

    doi:

    Figure Lengend Snippet: Inhibition of ANF-luciferase transcriptional activity of wild-type CSX/NKX2.5 by group 1, 2, and 3 mutants but not by group 4 mutant. ( a ) Inhibition of transactivation of the ANF-luciferase reporter gene in the presence of both 1:1 and 2:1 ratio of M170, M189, M191, and M259 expression plasmid to wild-type expression plasmid. 10T1/2 cells were transiently transacted with 1.2 μg of ANF-luciferase(-638), 0.3 μg of RSV–β-galactosidase, 0.7 μg of wild-type CSX/NKX2.5 expression plasmid, and 0.7 μg (hatched bars) or 1.4 μg (filled bars) of mutant expression plasmid or empty pcDNA3 plasmid to adjust the total amount of plasmid. A moderate reduction of luciferase activity was observed in M170 mutants, and a further decrease of luciferase activity was detected in M189, M191, and M250 mutants. In contrast, M25 mutant increased luciferase activity. Results are presented as a percent of the ANF reporter activity when cotransfected with wild-type and mutant plasmid compared with that of the wild type alone (open bar). Bars represent means ± SEM of the means of at least three separate transfection assays done in duplicate. ( b ) Protein-protein interaction of wild-type CSX/NKX2.5 with mutant proteins. MBP-fused wild-type CSX/NKX2.5 protein was mixed with [ 35 S]-labeled wild-type (lane 1) or ten mutants (lanes 2–11). After washing five times with binding buffer, the protein complexes were resolved on SDS-PAGE and autoradiographed (top panel). Group 1 (lanes 2 and 3) and group 5 (lane 11) mutants did not associate with MBP-CSX/NKX2.5, whereas group 2, 3, and 4 mutants associated with MBP-CSX/NKX2.5 (lanes 4–10). Fifty percent input of [ 35 S]-labeled proteins are shown in the middle panel, and Coomassie blue–stained MBP-fused wild-type CSX/NKX2.5 proteins are shown in the bottom panel.

    Article Snippet: pBS SK(-)- CSX/NKX2.5 (ref. ) digested with BglI -blunt–ended and EcoRI was ligated into SacI -blunt–ended and EcoRI -digested pMALC2 (New England Biolabs Inc., Beverly, Massachusetts, USA) to construct maltose binding protein–CSX/NKX2.5 (MBP-CSX/NKX2.5).

    Techniques: Inhibition, Luciferase, Activity Assay, Mutagenesis, Expressing, Plasmid Preparation, Transfection, Labeling, Binding Assay, SDS Page, Staining

    Diagram of CSX/NKX2.5 cDNA with the location of ten mutation sites identified in congenital heart disease. Ten mutation sites (asterisks in Wild) were divided into five groups based on the predicted protein structure: nonsense mutation in the HD (group 1: M149 and M170); missense mutation in the HD (group 2: M178, M188, M189, and M191); premature termination after HD (group 3: M198 and M259); 25 Arg-Cys missense mutation (group 4: M25); and mutation at the intron-splicing donor site (group 5: M112). Phenotypes observed in patients are listed on the left. For example, “11/12” indicates that 11 patients show the phenotype among 12 patients examined. These mutation sites were mapped on CSX/NKX2.5 cDNA, which encodes 324 amino acids including 60 amino acids of HD (shaded box). Nuclear localization signal at the NH 2 -terminus of the HD is indicated (black box). Predicted translated product of M112 mutation in splicing donor site is indicated with a light gray box. AV block, atrioventricular conduction block; ASD, atrial septal defect; VSD, ventricular septal defect; TOF, tetralogy of Fallot; TV, tricuspid valve abnormality; DORV, double outlet right ventricle; NLS, nuclear localization signal; HD, homeodomain.

    Journal: Journal of Clinical Investigation

    Article Title: Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease

    doi:

    Figure Lengend Snippet: Diagram of CSX/NKX2.5 cDNA with the location of ten mutation sites identified in congenital heart disease. Ten mutation sites (asterisks in Wild) were divided into five groups based on the predicted protein structure: nonsense mutation in the HD (group 1: M149 and M170); missense mutation in the HD (group 2: M178, M188, M189, and M191); premature termination after HD (group 3: M198 and M259); 25 Arg-Cys missense mutation (group 4: M25); and mutation at the intron-splicing donor site (group 5: M112). Phenotypes observed in patients are listed on the left. For example, “11/12” indicates that 11 patients show the phenotype among 12 patients examined. These mutation sites were mapped on CSX/NKX2.5 cDNA, which encodes 324 amino acids including 60 amino acids of HD (shaded box). Nuclear localization signal at the NH 2 -terminus of the HD is indicated (black box). Predicted translated product of M112 mutation in splicing donor site is indicated with a light gray box. AV block, atrioventricular conduction block; ASD, atrial septal defect; VSD, ventricular septal defect; TOF, tetralogy of Fallot; TV, tricuspid valve abnormality; DORV, double outlet right ventricle; NLS, nuclear localization signal; HD, homeodomain.

    Article Snippet: pBS SK(-)- CSX/NKX2.5 (ref. ) digested with BglI -blunt–ended and EcoRI was ligated into SacI -blunt–ended and EcoRI -digested pMALC2 (New England Biolabs Inc., Beverly, Massachusetts, USA) to construct maltose binding protein–CSX/NKX2.5 (MBP-CSX/NKX2.5).

    Techniques: Mutagenesis, Blocking Assay

    Production of joint molecules used to test the branch migration activity of proteins. (A) Joint molecules with a 3′ displaced ssDNA tail are produced by RAD51 using gapped DNA and pBS II K (+) dsDNA linearized with XhoI. (B) Joint molecules with

    Journal: Methods (San Diego, Calif.)

    Article Title: Analyzing the Branch Migration Activities of Eukaryotic Proteins

    doi: 10.1016/j.ymeth.2010.02.010

    Figure Lengend Snippet: Production of joint molecules used to test the branch migration activity of proteins. (A) Joint molecules with a 3′ displaced ssDNA tail are produced by RAD51 using gapped DNA and pBS II K (+) dsDNA linearized with XhoI. (B) Joint molecules with

    Article Snippet: Digest 10 μg of pBS II K (+) plasmid DNA with 40 units of AlwNI (NEB) in a 100 μl volume.

    Techniques: Migration, Activity Assay, Produced

    The scheme used to produce gapped DNA. i) pBS II K (+) plasmid DNA is digested with XhoI and AlwNI. ii) The large DNA fragment from this digest is purified by gel electrophoresis in agarose gels, and then iii) annealed to circular ssDNA to generate gapped

    Journal: Methods (San Diego, Calif.)

    Article Title: Analyzing the Branch Migration Activities of Eukaryotic Proteins

    doi: 10.1016/j.ymeth.2010.02.010

    Figure Lengend Snippet: The scheme used to produce gapped DNA. i) pBS II K (+) plasmid DNA is digested with XhoI and AlwNI. ii) The large DNA fragment from this digest is purified by gel electrophoresis in agarose gels, and then iii) annealed to circular ssDNA to generate gapped

    Article Snippet: Digest 10 μg of pBS II K (+) plasmid DNA with 40 units of AlwNI (NEB) in a 100 μl volume.

    Techniques: Plasmid Preparation, Purification, Nucleic Acid Electrophoresis

    Illustration of the 0.8% agarose gel used to purify the large (2065 bp) dsDNA fragment of pBS II K (+) following digestion with XhoI and AlwNI. After electrophoresis, lanes A, C, and E are excised from the gel and stained with ethidium bromide (dashed

    Journal: Methods (San Diego, Calif.)

    Article Title: Analyzing the Branch Migration Activities of Eukaryotic Proteins

    doi: 10.1016/j.ymeth.2010.02.010

    Figure Lengend Snippet: Illustration of the 0.8% agarose gel used to purify the large (2065 bp) dsDNA fragment of pBS II K (+) following digestion with XhoI and AlwNI. After electrophoresis, lanes A, C, and E are excised from the gel and stained with ethidium bromide (dashed

    Article Snippet: Digest 10 μg of pBS II K (+) plasmid DNA with 40 units of AlwNI (NEB) in a 100 μl volume.

    Techniques: Agarose Gel Electrophoresis, Electrophoresis, Staining

    The RpoS Ec recognition motif of P ospC lies within a 65-bp core region. The activity levels of P ospC TA (TA), P ospC CG (CG), and P ospC trunc (trunc), and the full-length P ospC , all cloned into pBS( gfp ), were compared after 8 h of growth in either LM5005 (speckled

    Journal: Journal of Bacteriology

    Article Title: Analysis of Promoter Elements Involved in the Transcriptional Initiation of RpoS-Dependent Borrelia burgdorferi Genes

    doi: 10.1128/JB.186.21.7390-7402.2004

    Figure Lengend Snippet: The RpoS Ec recognition motif of P ospC lies within a 65-bp core region. The activity levels of P ospC TA (TA), P ospC CG (CG), and P ospC trunc (trunc), and the full-length P ospC , all cloned into pBS( gfp ), were compared after 8 h of growth in either LM5005 (speckled

    Article Snippet: For complementation with either pBAD- rpoS Bb or pBAD- rpoS Ec (see below), the PBb - gfp cassettes, as well as PosmY - gfp , were digested out of the pBS(gfp) intermediate by using ClaI and XbaI and cloned into the corresponding sites of the low-copy-number vector, pACYC184 (New England Biolabs).

    Techniques: Activity Assay, Clone Assay

    (A) The 3′ end of the primer was blocked by the addition of an AZT residue. The ability of wild-type HIV-1 RT and the RT variants to remove the blocking AZT residue (deblocking) and extend the freed end of the primer was tested in the presence of 100.0 μM concentrations of each dNTP, 10.0 μM AZTTP, and varying concentrations of ATP (1.0, 2.0, 5.0, and 10.0 mM) as the pyrophosphate donor. The ratio of dTTP:AZTTP remained at 10:1. The locations of the starting PBS primer and the fully extended primer are marked. (B) The gel in panel A was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of ATP present in the reaction mixture. (C) The 3′ end of the primer was blocked by the addition of a ddT residue. The ability of wild-type HIV-1 RT and the RT variants to remove the blocking ddT residue (deblocking) and extend the freed end of the primer was tested in the presence of 100.0 μM concentrations of each dNTP, 10.0 μM ddTTP, and varying concentrations of ATP (1.0, 2.0, 5.0, and 10.0 mM) as the pyrophosphate donor. The ratio of dTTP:ddTTP remained at 10:1. The locations of the starting PBS primer and the fully extended primer are marked. (D) The gel in panel C was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of ATP present in the reaction mixture. Experiments using D4TTP as the blocking group gave similar results.

    Journal: Journal of Virology

    Article Title: Selective Excision of AZTMP by Drug-Resistant Human Immunodeficiency Virus Reverse Transcriptase

    doi: 10.1128/JVI.75.10.4832-4842.2001

    Figure Lengend Snippet: (A) The 3′ end of the primer was blocked by the addition of an AZT residue. The ability of wild-type HIV-1 RT and the RT variants to remove the blocking AZT residue (deblocking) and extend the freed end of the primer was tested in the presence of 100.0 μM concentrations of each dNTP, 10.0 μM AZTTP, and varying concentrations of ATP (1.0, 2.0, 5.0, and 10.0 mM) as the pyrophosphate donor. The ratio of dTTP:AZTTP remained at 10:1. The locations of the starting PBS primer and the fully extended primer are marked. (B) The gel in panel A was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of ATP present in the reaction mixture. (C) The 3′ end of the primer was blocked by the addition of a ddT residue. The ability of wild-type HIV-1 RT and the RT variants to remove the blocking ddT residue (deblocking) and extend the freed end of the primer was tested in the presence of 100.0 μM concentrations of each dNTP, 10.0 μM ddTTP, and varying concentrations of ATP (1.0, 2.0, 5.0, and 10.0 mM) as the pyrophosphate donor. The ratio of dTTP:ddTTP remained at 10:1. The locations of the starting PBS primer and the fully extended primer are marked. (D) The gel in panel C was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of ATP present in the reaction mixture. Experiments using D4TTP as the blocking group gave similar results.

    Article Snippet: The construct PPT-PBS Litmus 28 ( ) contains the polypurine tract (PPT), a long terminal repeat (U3, R, and U5), and the primer binding site (PBS) of HIV-1 RT cloned into the vector Litmus 28 (New England Biolabs).

    Techniques: Blocking Assay, Radioactivity

    (A) The PBS primer was 5′ end labeled and annealed to the template as described in Materials and Methods. The 3′ end of the primer was blocked by the addition of an AZT residue. The ability of wild-type HIV-1 RT and the RT variants to remove the blocking AZT residue (deblocking) and to extend the freed end of the primer was tested in the presence of 10.0 μM concentrations of each dNTP, 1.0 μM AZTTP, and varying concentrations of ATP (1.0, 2.0, 5.0, and 10.0 mM) as the pyrophosphate donor. In the cell, nucleoside analogs will be present in the triphosphate form, and after a primer is deblocked there is a possibility that HIV-1 RT will add another nucleoside analog back on to the 3′ end of the primer rather than the normal dNTP, which in this case is dTTP. The addition of AZTTP to the reaction mixture is meant to reflect what can occur within the cell. A control lane with no added wild-type RT shows the pattern of the starting template-primer. A control lane to which has been added HIV-1 RT but no ATP indicates the amount of extendable primer. This could result from primer which did not get blocked by an AZT residue or from a low level of deblocking by the enzyme using the dNTPs as the pyrophosphate donor or a combination of both processes. The locations of the starting PBS primer and the fully extended primer are marked. (B) The gel in panel 4A was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of ATP present in the reaction mixture. The percentage of full-length product in the No ATP control lane indicates that the background level is very low (

    Journal: Journal of Virology

    Article Title: Selective Excision of AZTMP by Drug-Resistant Human Immunodeficiency Virus Reverse Transcriptase

    doi: 10.1128/JVI.75.10.4832-4842.2001

    Figure Lengend Snippet: (A) The PBS primer was 5′ end labeled and annealed to the template as described in Materials and Methods. The 3′ end of the primer was blocked by the addition of an AZT residue. The ability of wild-type HIV-1 RT and the RT variants to remove the blocking AZT residue (deblocking) and to extend the freed end of the primer was tested in the presence of 10.0 μM concentrations of each dNTP, 1.0 μM AZTTP, and varying concentrations of ATP (1.0, 2.0, 5.0, and 10.0 mM) as the pyrophosphate donor. In the cell, nucleoside analogs will be present in the triphosphate form, and after a primer is deblocked there is a possibility that HIV-1 RT will add another nucleoside analog back on to the 3′ end of the primer rather than the normal dNTP, which in this case is dTTP. The addition of AZTTP to the reaction mixture is meant to reflect what can occur within the cell. A control lane with no added wild-type RT shows the pattern of the starting template-primer. A control lane to which has been added HIV-1 RT but no ATP indicates the amount of extendable primer. This could result from primer which did not get blocked by an AZT residue or from a low level of deblocking by the enzyme using the dNTPs as the pyrophosphate donor or a combination of both processes. The locations of the starting PBS primer and the fully extended primer are marked. (B) The gel in panel 4A was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of ATP present in the reaction mixture. The percentage of full-length product in the No ATP control lane indicates that the background level is very low (

    Article Snippet: The construct PPT-PBS Litmus 28 ( ) contains the polypurine tract (PPT), a long terminal repeat (U3, R, and U5), and the primer binding site (PBS) of HIV-1 RT cloned into the vector Litmus 28 (New England Biolabs).

    Techniques: Labeling, Blocking Assay, Radioactivity

    (A) The PBS primer was 5′ end labeled and annealed to the template as described in Materials and Methods. The 3′ end of the primer was blocked by the addition of a ddT residue. The ability of wild-type HIV-1 RT and the RT variants to remove the blocking ddT residue (deblocking) and extend the freed end of the primer was tested in the presence of 10.0 μM concentrations of each dNTP, 1.0 μM ddTTP, and varying concentrations of ATP (1.0, 2.0, 5.0, and 10.0 mM) as the pyrophosphate donor. Experiments using D4T as the blocking group gave similar results. (B) The gel in panel A was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of ATP present in the reaction mixture. (C) The 3′ end of the primer was blocked by the addition of a ddT residue, and the ability of wild-type HIV-1 RT and the RT variants to remove the blocking ddT residue and extend the freed end of the primer was tested in the presence of 10.0 μM concentrations of each dNTP, 1.0 μM ddTTP, and varying concentrations of NaPP i (25.0, 50.0, 100.0, and 200.0 μM) as the pyrophosphate donor. The locations of the starting PBS primer and the fully extended primer are marked. (D) The gel in panel C was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of NaPP i present in the reaction mixture.

    Journal: Journal of Virology

    Article Title: Selective Excision of AZTMP by Drug-Resistant Human Immunodeficiency Virus Reverse Transcriptase

    doi: 10.1128/JVI.75.10.4832-4842.2001

    Figure Lengend Snippet: (A) The PBS primer was 5′ end labeled and annealed to the template as described in Materials and Methods. The 3′ end of the primer was blocked by the addition of a ddT residue. The ability of wild-type HIV-1 RT and the RT variants to remove the blocking ddT residue (deblocking) and extend the freed end of the primer was tested in the presence of 10.0 μM concentrations of each dNTP, 1.0 μM ddTTP, and varying concentrations of ATP (1.0, 2.0, 5.0, and 10.0 mM) as the pyrophosphate donor. Experiments using D4T as the blocking group gave similar results. (B) The gel in panel A was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of ATP present in the reaction mixture. (C) The 3′ end of the primer was blocked by the addition of a ddT residue, and the ability of wild-type HIV-1 RT and the RT variants to remove the blocking ddT residue and extend the freed end of the primer was tested in the presence of 10.0 μM concentrations of each dNTP, 1.0 μM ddTTP, and varying concentrations of NaPP i (25.0, 50.0, 100.0, and 200.0 μM) as the pyrophosphate donor. The locations of the starting PBS primer and the fully extended primer are marked. (D) The gel in panel C was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of NaPP i present in the reaction mixture.

    Article Snippet: The construct PPT-PBS Litmus 28 ( ) contains the polypurine tract (PPT), a long terminal repeat (U3, R, and U5), and the primer binding site (PBS) of HIV-1 RT cloned into the vector Litmus 28 (New England Biolabs).

    Techniques: Labeling, Blocking Assay, Radioactivity