akmlc2f promoter  (New England Biolabs)


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    Structured Review

    New England Biolabs akmlc2f promoter
    Analysis of luciferase reporter activity of the <t>Akmlc2f</t> promoter. Cells were seeded in 24-well culture plates and transfected with the indicated reporter vectors, (pcDNA3/C/EBPβ and β-galactosidase expression plasmids). At 24 h after transfection, the cells were lysed and assayed for the luciferase reporter activity. The data are representative of two independent experiments. The values represent means ± SD ( n = 3).
    Akmlc2f Promoter, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 18008 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/akmlc2f promoter/product/New England Biolabs
    Average 85 stars, based on 18008 article reviews
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    akmlc2f promoter - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Molecular and Functional Analyses of the Fast Skeletal Myosin Light Chain2 Gene of the Korean Oily Bitterling, Acheilognathus koreensis"

    Article Title: Molecular and Functional Analyses of the Fast Skeletal Myosin Light Chain2 Gene of the Korean Oily Bitterling, Acheilognathus koreensis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms140816672

    Analysis of luciferase reporter activity of the Akmlc2f promoter. Cells were seeded in 24-well culture plates and transfected with the indicated reporter vectors, (pcDNA3/C/EBPβ and β-galactosidase expression plasmids). At 24 h after transfection, the cells were lysed and assayed for the luciferase reporter activity. The data are representative of two independent experiments. The values represent means ± SD ( n = 3).
    Figure Legend Snippet: Analysis of luciferase reporter activity of the Akmlc2f promoter. Cells were seeded in 24-well culture plates and transfected with the indicated reporter vectors, (pcDNA3/C/EBPβ and β-galactosidase expression plasmids). At 24 h after transfection, the cells were lysed and assayed for the luciferase reporter activity. The data are representative of two independent experiments. The values represent means ± SD ( n = 3).

    Techniques Used: Luciferase, Activity Assay, Transfection, Expressing

    Analysis of DsRed reporter activity of the Akmlc2f promoter. Cells were seeded in 12-well culture plates and transfected with a reporter vector (pDsRed2-1/Akmlc2f P2657) and pcDNA3/C/EBPβ or empty expression plasmid. At 24 h after transfection, the cells were fixed with 4% paraformaldehyde in PBS (pH 7.4), mounted using VECTASHIELD ® with DAPI, and viewed using a confocal laser scanning microscope.
    Figure Legend Snippet: Analysis of DsRed reporter activity of the Akmlc2f promoter. Cells were seeded in 12-well culture plates and transfected with a reporter vector (pDsRed2-1/Akmlc2f P2657) and pcDNA3/C/EBPβ or empty expression plasmid. At 24 h after transfection, the cells were fixed with 4% paraformaldehyde in PBS (pH 7.4), mounted using VECTASHIELD ® with DAPI, and viewed using a confocal laser scanning microscope.

    Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Expressing, Laser-Scanning Microscopy

    Analysis of DsRed reporter activity of the Akmlc2f promoter in the zebrafish embryo. The fluorescent protein reporter of the Akmlc2f promoter, pDsRed2-1/Akmlc2f P2657 was digested with the restriction endonuclease Apa LI and then the DNA solution at concentrations of 25 ng/mL was injected into the one-cell stage embryos using an air-pressure microinjector (WPI). Digital images of embryo (6 days post hatching) were captured using a macro zoom fluorescence microscope (Olympus, Tokyo, Japan). Scale bar = 200 μm
    Figure Legend Snippet: Analysis of DsRed reporter activity of the Akmlc2f promoter in the zebrafish embryo. The fluorescent protein reporter of the Akmlc2f promoter, pDsRed2-1/Akmlc2f P2657 was digested with the restriction endonuclease Apa LI and then the DNA solution at concentrations of 25 ng/mL was injected into the one-cell stage embryos using an air-pressure microinjector (WPI). Digital images of embryo (6 days post hatching) were captured using a macro zoom fluorescence microscope (Olympus, Tokyo, Japan). Scale bar = 200 μm

    Techniques Used: Activity Assay, Injection, Fluorescence, Microscopy

    Tissue distribution of Akmlc2f mRNA. Quantitative real-time polymerase chain reaction was performed on equal amounts of total RNA isolated from the A. koreensis tissues. To determine tissue-specific expression levels, the expression level in each tissue was compared to that in the intestine. Values are means ± SD ( n = 3).
    Figure Legend Snippet: Tissue distribution of Akmlc2f mRNA. Quantitative real-time polymerase chain reaction was performed on equal amounts of total RNA isolated from the A. koreensis tissues. To determine tissue-specific expression levels, the expression level in each tissue was compared to that in the intestine. Values are means ± SD ( n = 3).

    Techniques Used: Real-time Polymerase Chain Reaction, Isolation, Expressing

    Schematic representation of the genomic organization of the Akmlc2f gene. The exon/intron structure is shown and the numbers indicate the length of exon/intron.
    Figure Legend Snippet: Schematic representation of the genomic organization of the Akmlc2f gene. The exon/intron structure is shown and the numbers indicate the length of exon/intron.

    Techniques Used:

    Multiple alignment of AkMLC2f amino acid sequences with MLC2 from other species. Identical residues in all sequences are indicated in blue. Conserved substitutions are indicated in yellow. The sequences were extracted from GenBank: Ictalurus punctafus (NP_001188138), Danio rerio (NP_571263), Sardinops melanostictus (BAA95140), Epinephelus coioides (ACM41847), Sparus aurata (AAD54229), Dicentrarchus labrax (CBN81401), Salmo salar (NP_001117188), Gadus chalcogrammus (BAB18578), Oryzias latipes (XP_004071230), Decapterus maruadsi (BAB69803), Cypselurus agoo (BAA95134), Thunnus thynnus (BAA95125), Siniperca scherzeri (ACT67908), Gallus gallus (NP_001185673), Mus musculus (NP_058034), Rattus norvegicus (NP_036737), Bos Taurus (NP_001069115), and Homo sapiens (AAA91848).
    Figure Legend Snippet: Multiple alignment of AkMLC2f amino acid sequences with MLC2 from other species. Identical residues in all sequences are indicated in blue. Conserved substitutions are indicated in yellow. The sequences were extracted from GenBank: Ictalurus punctafus (NP_001188138), Danio rerio (NP_571263), Sardinops melanostictus (BAA95140), Epinephelus coioides (ACM41847), Sparus aurata (AAD54229), Dicentrarchus labrax (CBN81401), Salmo salar (NP_001117188), Gadus chalcogrammus (BAB18578), Oryzias latipes (XP_004071230), Decapterus maruadsi (BAB69803), Cypselurus agoo (BAA95134), Thunnus thynnus (BAA95125), Siniperca scherzeri (ACT67908), Gallus gallus (NP_001185673), Mus musculus (NP_058034), Rattus norvegicus (NP_036737), Bos Taurus (NP_001069115), and Homo sapiens (AAA91848).

    Techniques Used:

    Nucleotide and deduced amino acid sequences of Akmlc2f cDNA. Start (ATG) and stop (TAA) codons are in bold. The EF-hand calcium-binding motif is in bold and underlined. The polyadenylation signals are underlined.
    Figure Legend Snippet: Nucleotide and deduced amino acid sequences of Akmlc2f cDNA. Start (ATG) and stop (TAA) codons are in bold. The EF-hand calcium-binding motif is in bold and underlined. The polyadenylation signals are underlined.

    Techniques Used: Binding Assay

    2) Product Images from "Molecular and Functional Analyses of the Fast Skeletal Myosin Light Chain2 Gene of the Korean Oily Bitterling, Acheilognathus koreensis"

    Article Title: Molecular and Functional Analyses of the Fast Skeletal Myosin Light Chain2 Gene of the Korean Oily Bitterling, Acheilognathus koreensis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms140816672

    Analysis of DsRed reporter activity of the Akmlc2f promoter in the zebrafish embryo. The fluorescent protein reporter of the Akmlc2f promoter, pDsRed2-1/Akmlc2f P2657 was digested with the restriction endonuclease Apa LI and then the DNA solution at concentrations of 25 ng/mL was injected into the one-cell stage embryos using an air-pressure microinjector (WPI). Digital images of embryo (6 days post hatching) were captured using a macro zoom fluorescence microscope (Olympus, Tokyo, Japan). Scale bar = 200 μm
    Figure Legend Snippet: Analysis of DsRed reporter activity of the Akmlc2f promoter in the zebrafish embryo. The fluorescent protein reporter of the Akmlc2f promoter, pDsRed2-1/Akmlc2f P2657 was digested with the restriction endonuclease Apa LI and then the DNA solution at concentrations of 25 ng/mL was injected into the one-cell stage embryos using an air-pressure microinjector (WPI). Digital images of embryo (6 days post hatching) were captured using a macro zoom fluorescence microscope (Olympus, Tokyo, Japan). Scale bar = 200 μm

    Techniques Used: Activity Assay, Injection, Fluorescence, Microscopy

    3) Product Images from "Correlation between the single nucleotide polymorphisms of the human phosphodiesterase 4D gene and the risk of cerebral infarction in the Uygur and Han ethnic groups of Xinjiang, China"

    Article Title: Correlation between the single nucleotide polymorphisms of the human phosphodiesterase 4D gene and the risk of cerebral infarction in the Uygur and Han ethnic groups of Xinjiang, China

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2013.1370

    Single nucelotide polymorphisms (SNPs) at the rs2910829 locus of the phosphodiesterase 4D (PDE4D) gene. (A) A putative single-base C-to-T alteration at the rs2910829 locus of the PDE4D gene generated an additional Ssp I locus. (B) The single-base C-to-T alteration may occur at the rs2910829 locus of one allele (termed as C–T) or the two alleles (termed as C–T/C–T). (C) SNPs at the rs2910829 locus of the PDE4D gene, as detected using electrophoresis. The experiments were performed ≥3 times. M, DNA marker (50, 100, 150, 200 and 250 bp ladders).
    Figure Legend Snippet: Single nucelotide polymorphisms (SNPs) at the rs2910829 locus of the phosphodiesterase 4D (PDE4D) gene. (A) A putative single-base C-to-T alteration at the rs2910829 locus of the PDE4D gene generated an additional Ssp I locus. (B) The single-base C-to-T alteration may occur at the rs2910829 locus of one allele (termed as C–T) or the two alleles (termed as C–T/C–T). (C) SNPs at the rs2910829 locus of the PDE4D gene, as detected using electrophoresis. The experiments were performed ≥3 times. M, DNA marker (50, 100, 150, 200 and 250 bp ladders).

    Techniques Used: Generated, Electrophoresis, Marker

    4) Product Images from "Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1"

    Article Title: Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0139399

    DIDS inhibits presynaptic filament formation by eh Dmc1. A. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA in the presence and absence of increasing amounts of DIDS at 37°C for 20 min. Products were separated on 12% polyacrylamide gels and analyzed with a phosphorimager. B. eh Dmc1 was incubated with saturating amounts of [ 32 P- γ ]-ATP in the presence and absence of ϕ X174 ssDNA and/or DIDS (66.6 μM). The reactions were stopped at the indicated times, subjected to TLC, and analyzed using a phosphorimager. C. eh Dmc1 was incubated with 32 P-radiolabeled OL90 in the presence and absence of increasing amounts of DIDS followed by exposure to DNase for 15 min at 37°C. The reactions were stopped, separated on 12% polyacrylamide gels, and analyzed with a phosphorimager. D. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA for 2 min prior to the addition of DIDS (2.5 μM, lane 3; 5 μM, lane 4; 7.5 μM, lane 5; and 10 μM, lane 6). After 8 min of incubation, the reaction was initiated by the addition of supercoiled dsDNA. After 12 min, an aliquot was removed and deproteinized. The reaction products were separated by agarose gel electrophoresis, and the gels were analyzed with a phosphorimager. Mean results from three separate experiments were graphed. Error bars represent SEM. DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.
    Figure Legend Snippet: DIDS inhibits presynaptic filament formation by eh Dmc1. A. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA in the presence and absence of increasing amounts of DIDS at 37°C for 20 min. Products were separated on 12% polyacrylamide gels and analyzed with a phosphorimager. B. eh Dmc1 was incubated with saturating amounts of [ 32 P- γ ]-ATP in the presence and absence of ϕ X174 ssDNA and/or DIDS (66.6 μM). The reactions were stopped at the indicated times, subjected to TLC, and analyzed using a phosphorimager. C. eh Dmc1 was incubated with 32 P-radiolabeled OL90 in the presence and absence of increasing amounts of DIDS followed by exposure to DNase for 15 min at 37°C. The reactions were stopped, separated on 12% polyacrylamide gels, and analyzed with a phosphorimager. D. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA for 2 min prior to the addition of DIDS (2.5 μM, lane 3; 5 μM, lane 4; 7.5 μM, lane 5; and 10 μM, lane 6). After 8 min of incubation, the reaction was initiated by the addition of supercoiled dsDNA. After 12 min, an aliquot was removed and deproteinized. The reaction products were separated by agarose gel electrophoresis, and the gels were analyzed with a phosphorimager. Mean results from three separate experiments were graphed. Error bars represent SEM. DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.

    Techniques Used: Incubation, Thin Layer Chromatography, Agarose Gel Electrophoresis

    The eh Dmc1 and eh Rad51 proteins are present in E . histolytica , and purified eh Dmc1 hydrolyzes ATP. A. Purified eh Dmc1 (~1 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. B. Immunoblot of purified recombinant eh Rad51 protein and eh Dmc1 protein (~1 μg, lane 1 and 2, respectively), and E . histolytica partially purified lysate (lane 3) on a 12% SDS-polyacrylamide gel. Anti- sc Rad51 primary antibodies were used. C. Purified eh Dmc1 was incubated with increasing concentrations of [ 32 P- γ ]-ATP. After 60 min, samples were withdrawn and the reaction products were separated using TLC followed by analysis with a phosphorimager. D . Increasing concentrations of ϕ X174 (+) virion single-stranded DNA (ssDNA) or linearized ϕ X174 double-stranded DNA (dsDNA) were incubated with eh Dmc1 and a saturating concentration of [ 32 P- γ ]-ATP. E. Time course analysis of eh Dmc1 ATP hydrolysis activity in the absence or presence of ϕ X174 ssDNA or linearized ϕ X174 dsDNA. Error bars represent SEM, (n = 3).
    Figure Legend Snippet: The eh Dmc1 and eh Rad51 proteins are present in E . histolytica , and purified eh Dmc1 hydrolyzes ATP. A. Purified eh Dmc1 (~1 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. B. Immunoblot of purified recombinant eh Rad51 protein and eh Dmc1 protein (~1 μg, lane 1 and 2, respectively), and E . histolytica partially purified lysate (lane 3) on a 12% SDS-polyacrylamide gel. Anti- sc Rad51 primary antibodies were used. C. Purified eh Dmc1 was incubated with increasing concentrations of [ 32 P- γ ]-ATP. After 60 min, samples were withdrawn and the reaction products were separated using TLC followed by analysis with a phosphorimager. D . Increasing concentrations of ϕ X174 (+) virion single-stranded DNA (ssDNA) or linearized ϕ X174 double-stranded DNA (dsDNA) were incubated with eh Dmc1 and a saturating concentration of [ 32 P- γ ]-ATP. E. Time course analysis of eh Dmc1 ATP hydrolysis activity in the absence or presence of ϕ X174 ssDNA or linearized ϕ X174 dsDNA. Error bars represent SEM, (n = 3).

    Techniques Used: Purification, Staining, Recombinant, Incubation, Thin Layer Chromatography, Concentration Assay, Activity Assay

    5) Product Images from "Correlation between the single nucleotide polymorphisms of the human phosphodiesterase 4D gene and the risk of cerebral infarction in the Uygur and Han ethnic groups of Xinjiang, China"

    Article Title: Correlation between the single nucleotide polymorphisms of the human phosphodiesterase 4D gene and the risk of cerebral infarction in the Uygur and Han ethnic groups of Xinjiang, China

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2013.1370

    Single nucelotide polymorphisms (SNPs) at the rs918592 locus of the phosphodiesterase 4D (PDE4D) gene. (A) A putative single-base G-to-A alteration at the rs918592 locus of the PDE4D gene generated an additional Apa lI locus. (B) The single-base G-to-A alteration may occur at the rs918592 locus of one allele (termed as G–A) or the two alleles (termed as G–A/G–A). (C) SNPs at the rs918592 locus of the PDE4D gene, as detected using electrophoresis. The experiments were performed ≥3 times. M, DNA marker (100, 200, 300, 400, 500 and 600 bp ladders).
    Figure Legend Snippet: Single nucelotide polymorphisms (SNPs) at the rs918592 locus of the phosphodiesterase 4D (PDE4D) gene. (A) A putative single-base G-to-A alteration at the rs918592 locus of the PDE4D gene generated an additional Apa lI locus. (B) The single-base G-to-A alteration may occur at the rs918592 locus of one allele (termed as G–A) or the two alleles (termed as G–A/G–A). (C) SNPs at the rs918592 locus of the PDE4D gene, as detected using electrophoresis. The experiments were performed ≥3 times. M, DNA marker (100, 200, 300, 400, 500 and 600 bp ladders).

    Techniques Used: Generated, Electrophoresis, Marker

    6) Product Images from "Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1"

    Article Title: Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0139399

    eh Dmc1 catalyzes D-loop formation. A. Schematic of D-loop formation assay (ss, single-strand oligonucleotide; sc, supercoiled dsDNA). B. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA (lane 2), dsDNA (lane 3) prior to the addition of dsDNA or ssDNA (lanes 2 and 3, respectively), or both ssDNA and dsDNA (lane 4) simultaneously. Lane 1 is devoid of protein. After a 12 min incubation, an aliquot was removed and deproteinized prior to separation on an agarose gel. The mean percent of six independent experiments was graphed. Error bars represent SEM. C. eh Dmc1 was incubated with 32 P-OL90 ssDNA in the presence of 2 mM nucleotide (ATP, lanes 1–4), ATP- γ -S (lane 5), ADP (lane 6) and AMP-PNP (lane 7). Lane 8 was devoid of nucleotide. At the indicated times, an aliquot was removed and processed as described in B . The mean percent of six independent experiments was graphed. Error bars represent SEM.
    Figure Legend Snippet: eh Dmc1 catalyzes D-loop formation. A. Schematic of D-loop formation assay (ss, single-strand oligonucleotide; sc, supercoiled dsDNA). B. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA (lane 2), dsDNA (lane 3) prior to the addition of dsDNA or ssDNA (lanes 2 and 3, respectively), or both ssDNA and dsDNA (lane 4) simultaneously. Lane 1 is devoid of protein. After a 12 min incubation, an aliquot was removed and deproteinized prior to separation on an agarose gel. The mean percent of six independent experiments was graphed. Error bars represent SEM. C. eh Dmc1 was incubated with 32 P-OL90 ssDNA in the presence of 2 mM nucleotide (ATP, lanes 1–4), ATP- γ -S (lane 5), ADP (lane 6) and AMP-PNP (lane 7). Lane 8 was devoid of nucleotide. At the indicated times, an aliquot was removed and processed as described in B . The mean percent of six independent experiments was graphed. Error bars represent SEM.

    Techniques Used: Tube Formation Assay, Incubation, Agarose Gel Electrophoresis

    eh Dmc1 mediates plasmid length DNA strand exchange. A. Schematic of the 3-strand homologous DNA pairing and strand exchange reaction. Homologous DNA pairing between the circular ssDNA (css) and linear dsDNA (lds) first forms a DNA joint molecule (jm). DNA strand exchange converts the joint molecule into a nicked circular duplex (nc) displacing the linear ssDNA (lss). B. eh Dmc1 (12.5 μM) was incubated with ϕX174 virion ssDNA (css) to allow presynaptic filament formation to occur before the addition of hRPA (3.8 μM) and KCl (150 mM final concentration). The reaction was initiated by the addition of linearized double-strand ϕX174 DNA (lds) and spermidine. At the indicated time points, the reactions were deproteinized, subjected to agarose gel electrophoresis, and stained with ethidium bromide.
    Figure Legend Snippet: eh Dmc1 mediates plasmid length DNA strand exchange. A. Schematic of the 3-strand homologous DNA pairing and strand exchange reaction. Homologous DNA pairing between the circular ssDNA (css) and linear dsDNA (lds) first forms a DNA joint molecule (jm). DNA strand exchange converts the joint molecule into a nicked circular duplex (nc) displacing the linear ssDNA (lss). B. eh Dmc1 (12.5 μM) was incubated with ϕX174 virion ssDNA (css) to allow presynaptic filament formation to occur before the addition of hRPA (3.8 μM) and KCl (150 mM final concentration). The reaction was initiated by the addition of linearized double-strand ϕX174 DNA (lds) and spermidine. At the indicated time points, the reactions were deproteinized, subjected to agarose gel electrophoresis, and stained with ethidium bromide.

    Techniques Used: Plasmid Preparation, Incubation, Concentration Assay, Agarose Gel Electrophoresis, Staining

    DIDS inhibits presynaptic filament formation by eh Dmc1. A. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA in the presence and absence of increasing amounts of DIDS at 37°C for 20 min. Products were separated on 12% polyacrylamide gels and analyzed with a phosphorimager. B. eh Dmc1 was incubated with saturating amounts of [ 32 P- γ ]-ATP in the presence and absence of ϕ X174 ssDNA and/or DIDS (66.6 μM). The reactions were stopped at the indicated times, subjected to TLC, and analyzed using a phosphorimager. C. eh Dmc1 was incubated with 32 P-radiolabeled OL90 in the presence and absence of increasing amounts of DIDS followed by exposure to DNase for 15 min at 37°C. The reactions were stopped, separated on 12% polyacrylamide gels, and analyzed with a phosphorimager. D. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA for 2 min prior to the addition of DIDS (2.5 μM, lane 3; 5 μM, lane 4; 7.5 μM, lane 5; and 10 μM, lane 6). After 8 min of incubation, the reaction was initiated by the addition of supercoiled dsDNA. After 12 min, an aliquot was removed and deproteinized. The reaction products were separated by agarose gel electrophoresis, and the gels were analyzed with a phosphorimager. Mean results from three separate experiments were graphed. Error bars represent SEM. DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.
    Figure Legend Snippet: DIDS inhibits presynaptic filament formation by eh Dmc1. A. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA in the presence and absence of increasing amounts of DIDS at 37°C for 20 min. Products were separated on 12% polyacrylamide gels and analyzed with a phosphorimager. B. eh Dmc1 was incubated with saturating amounts of [ 32 P- γ ]-ATP in the presence and absence of ϕ X174 ssDNA and/or DIDS (66.6 μM). The reactions were stopped at the indicated times, subjected to TLC, and analyzed using a phosphorimager. C. eh Dmc1 was incubated with 32 P-radiolabeled OL90 in the presence and absence of increasing amounts of DIDS followed by exposure to DNase for 15 min at 37°C. The reactions were stopped, separated on 12% polyacrylamide gels, and analyzed with a phosphorimager. D. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA for 2 min prior to the addition of DIDS (2.5 μM, lane 3; 5 μM, lane 4; 7.5 μM, lane 5; and 10 μM, lane 6). After 8 min of incubation, the reaction was initiated by the addition of supercoiled dsDNA. After 12 min, an aliquot was removed and deproteinized. The reaction products were separated by agarose gel electrophoresis, and the gels were analyzed with a phosphorimager. Mean results from three separate experiments were graphed. Error bars represent SEM. DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.

    Techniques Used: Incubation, Thin Layer Chromatography, Agarose Gel Electrophoresis

    The eh Dmc1 and eh Rad51 proteins are present in E . histolytica , and purified eh Dmc1 hydrolyzes ATP. A. Purified eh Dmc1 (~1 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. B. Immunoblot of purified recombinant eh Rad51 protein and eh Dmc1 protein (~1 μg, lane 1 and 2, respectively), and E . histolytica partially purified lysate (lane 3) on a 12% SDS-polyacrylamide gel. Anti- sc Rad51 primary antibodies were used. C. Purified eh Dmc1 was incubated with increasing concentrations of [ 32 P- γ ]-ATP. After 60 min, samples were withdrawn and the reaction products were separated using TLC followed by analysis with a phosphorimager. D . Increasing concentrations of ϕ X174 (+) virion single-stranded DNA (ssDNA) or linearized ϕ X174 double-stranded DNA (dsDNA) were incubated with eh Dmc1 and a saturating concentration of [ 32 P- γ ]-ATP. E. Time course analysis of eh Dmc1 ATP hydrolysis activity in the absence or presence of ϕ X174 ssDNA or linearized ϕ X174 dsDNA. Error bars represent SEM, (n = 3).
    Figure Legend Snippet: The eh Dmc1 and eh Rad51 proteins are present in E . histolytica , and purified eh Dmc1 hydrolyzes ATP. A. Purified eh Dmc1 (~1 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. B. Immunoblot of purified recombinant eh Rad51 protein and eh Dmc1 protein (~1 μg, lane 1 and 2, respectively), and E . histolytica partially purified lysate (lane 3) on a 12% SDS-polyacrylamide gel. Anti- sc Rad51 primary antibodies were used. C. Purified eh Dmc1 was incubated with increasing concentrations of [ 32 P- γ ]-ATP. After 60 min, samples were withdrawn and the reaction products were separated using TLC followed by analysis with a phosphorimager. D . Increasing concentrations of ϕ X174 (+) virion single-stranded DNA (ssDNA) or linearized ϕ X174 double-stranded DNA (dsDNA) were incubated with eh Dmc1 and a saturating concentration of [ 32 P- γ ]-ATP. E. Time course analysis of eh Dmc1 ATP hydrolysis activity in the absence or presence of ϕ X174 ssDNA or linearized ϕ X174 dsDNA. Error bars represent SEM, (n = 3).

    Techniques Used: Purification, Staining, Recombinant, Incubation, Thin Layer Chromatography, Concentration Assay, Activity Assay

    mHop2-Mnd1 and Ca 2+ stimulate eh Dmc1-mediated D-loop formation. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA in the absence (lanes 1–4 and 9–12) or presence of calcium (lanes 5–8 and 13–16) and/or mHop2-Mnd1 (lanes 9–16). The reaction was initiated with the addition of supercoiled dsDNA. Aliquots were removed at the indicated times, deproteinized, and the reaction products were separated by agarose gel electrophoresis. Lanes 1, 5, 9, and 13 were lacking eh Dmc1. Mean values from three individual experiments were graphed. Error bars represent SEM.
    Figure Legend Snippet: mHop2-Mnd1 and Ca 2+ stimulate eh Dmc1-mediated D-loop formation. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA in the absence (lanes 1–4 and 9–12) or presence of calcium (lanes 5–8 and 13–16) and/or mHop2-Mnd1 (lanes 9–16). The reaction was initiated with the addition of supercoiled dsDNA. Aliquots were removed at the indicated times, deproteinized, and the reaction products were separated by agarose gel electrophoresis. Lanes 1, 5, 9, and 13 were lacking eh Dmc1. Mean values from three individual experiments were graphed. Error bars represent SEM.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    eh Dmc1 binds DNA. A. Increasing concentrations of eh Dmc1 (1.3 μM, lane 2; 2.6 μM, lane 3; 3.9 μM, lane 4; and 5.2 μM, lane 5) were incubated with ssDNA ( 32 P-labeled H3 ssDNA). B. The mean binding percentages were graphed for three independent experiments from A . Error bars represent SEM. C. Increasing concentrations of eh Dmc1 (5.2 μM, lane 2; 10.4 μM, lane 3; 20.8 μM, lane 4; and 31.2 μM, lane 5) were incubated with dsDNA ( 32 P-labeled H3 annealed to H3c). D. The mean binding percentages were graphed for three independent experiments from C . Error bars represent SEM. Lane 1 for A and C is devoid of protein, and lane 6 for A and C was SDS/PK (S/P) treated containing the highest concentration of eh Dmc1.
    Figure Legend Snippet: eh Dmc1 binds DNA. A. Increasing concentrations of eh Dmc1 (1.3 μM, lane 2; 2.6 μM, lane 3; 3.9 μM, lane 4; and 5.2 μM, lane 5) were incubated with ssDNA ( 32 P-labeled H3 ssDNA). B. The mean binding percentages were graphed for three independent experiments from A . Error bars represent SEM. C. Increasing concentrations of eh Dmc1 (5.2 μM, lane 2; 10.4 μM, lane 3; 20.8 μM, lane 4; and 31.2 μM, lane 5) were incubated with dsDNA ( 32 P-labeled H3 annealed to H3c). D. The mean binding percentages were graphed for three independent experiments from C . Error bars represent SEM. Lane 1 for A and C is devoid of protein, and lane 6 for A and C was SDS/PK (S/P) treated containing the highest concentration of eh Dmc1.

    Techniques Used: Incubation, Labeling, Binding Assay, Concentration Assay

    7) Product Images from "Comparative analysis of enzymatically produced novel linear DNA constructs with plasmids for use as DNA vaccines"

    Article Title: Comparative analysis of enzymatically produced novel linear DNA constructs with plasmids for use as DNA vaccines

    Journal: Gene therapy

    doi: 10.1038/gt.2014.37

    Characterisation of Doggybone constructs Doggybones were produced using the method outlined by ( 16 ) , a summary of the process is shown (A). Rolling circle replication takes place from a starting plasmid resulting in concatamers which are then resolved through the actions of TelN on the telRL sites included in the sequence. Addition of restriction enzymes and exonuclease removes any contamination from the plasmid backbone sequences to leave the cassette Doggybone only. The end product is a linear dsDNA construct flanked by ssDNA hairpins (Doggybones) (B). To confirm the Doggybone structure constructs were run on a native (C) and a denaturing agarose gel (D). The following were run: Doggybone DB GL (lane 1 created from pGL DOG (Luciferase cassette)), the PCR GL insert only obtained through PCR (lane 2), pUC18 telRL plasmid linearised with TelN (lane 3) or Xmn I (lane 4). The size (E) and charge (F) of Doggybone (●) or plasmid (□) were assessed by dynamic light scattering, points represent mean of n=3 +/− SEM.
    Figure Legend Snippet: Characterisation of Doggybone constructs Doggybones were produced using the method outlined by ( 16 ) , a summary of the process is shown (A). Rolling circle replication takes place from a starting plasmid resulting in concatamers which are then resolved through the actions of TelN on the telRL sites included in the sequence. Addition of restriction enzymes and exonuclease removes any contamination from the plasmid backbone sequences to leave the cassette Doggybone only. The end product is a linear dsDNA construct flanked by ssDNA hairpins (Doggybones) (B). To confirm the Doggybone structure constructs were run on a native (C) and a denaturing agarose gel (D). The following were run: Doggybone DB GL (lane 1 created from pGL DOG (Luciferase cassette)), the PCR GL insert only obtained through PCR (lane 2), pUC18 telRL plasmid linearised with TelN (lane 3) or Xmn I (lane 4). The size (E) and charge (F) of Doggybone (●) or plasmid (□) were assessed by dynamic light scattering, points represent mean of n=3 +/− SEM.

    Techniques Used: Construct, Produced, Plasmid Preparation, Sequencing, Agarose Gel Electrophoresis, Luciferase, Polymerase Chain Reaction

    8) Product Images from "Morphologic, Genetic, and Biochemical Characterization of Helicobacter Magdeburgensis, a Novel Species Isolated from the Intestine of Laboratory Mice"

    Article Title: Morphologic, Genetic, and Biochemical Characterization of Helicobacter Magdeburgensis, a Novel Species Isolated from the Intestine of Laboratory Mice

    Journal: Helicobacter

    doi: 10.1111/j.1523-5378.2010.00770.x

    Analysis of 16S rRNA of different Helicobacter magdeburgensis isolates by PCR and RFLPs. ( A ) DNA isolated from seven individual clones belonging to H. magdeburgensis was investigated by conventional PCR of the 16S rRNA gene. A conserved 1.2-kb DNA fragment
    Figure Legend Snippet: Analysis of 16S rRNA of different Helicobacter magdeburgensis isolates by PCR and RFLPs. ( A ) DNA isolated from seven individual clones belonging to H. magdeburgensis was investigated by conventional PCR of the 16S rRNA gene. A conserved 1.2-kb DNA fragment

    Techniques Used: Polymerase Chain Reaction, Isolation, Clone Assay

    Investigation of 16S rRNA genes of different Helicobacter species by PCR and RFLP analyses. ( A ) DNA isolated from bacteria belonging to the genus Helicobacter ( H. magdeburgensis, H. typhlonicus, H. hepaticus, H. bills, H. mustelae, H. pylori , and the
    Figure Legend Snippet: Investigation of 16S rRNA genes of different Helicobacter species by PCR and RFLP analyses. ( A ) DNA isolated from bacteria belonging to the genus Helicobacter ( H. magdeburgensis, H. typhlonicus, H. hepaticus, H. bills, H. mustelae, H. pylori , and the

    Techniques Used: Polymerase Chain Reaction, Isolation

    9) Product Images from "Functional Regions of Human Telomerase Reverse Transcriptase and Human Telomerase RNA Required for Telomerase Activity and RNA-Protein Interactions"

    Article Title: Functional Regions of Human Telomerase Reverse Transcriptase and Human Telomerase RNA Required for Telomerase Activity and RNA-Protein Interactions

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.5.1888-1897.2001

    Role of the P3 helix of the hTR pseudoknot in telomerase function and two independent hTERT binding sites within hTR. (A) Northern blot of total RNA harvested from RRLs in which hTERT was synthesized in the absence of hTR (lane 1) or presence of wild-type hTR (lane 2) and hTR variants. The Northern blot was probed for hTR-specific sequences. M, DNA markers (sizes, in base pairs, are on the left). RNAs were separated by electrophoresis on a 6% acrylamide–7 M urea gel. (B and C) Equal volumes of RRL reaction products generated in the absence (lanes 11 and 22) or presence of the different hTR variants were subjected to immunoprecipitation (IP) with an antibody to the T7 tag. The washed beads were analyzed for telomerase activity (B) and hTERT-hTR coimmunoprecipitation (C). For panel B, telomerase activity was analyzed by the TRAP assay, and 100 ng of partially purified 293 cell extracts were used as a positive control (lane 21). IC, internal PCR control; WT, wild-type. For panel C, hTERT-hTR coimmunoprecipitation was analyzed by Northern blotting using an hTR-specific probe. The arrowheads indicate the positions at which full-length hTR (FLhTR), hTR164-330, and hTR33-147 migrate.
    Figure Legend Snippet: Role of the P3 helix of the hTR pseudoknot in telomerase function and two independent hTERT binding sites within hTR. (A) Northern blot of total RNA harvested from RRLs in which hTERT was synthesized in the absence of hTR (lane 1) or presence of wild-type hTR (lane 2) and hTR variants. The Northern blot was probed for hTR-specific sequences. M, DNA markers (sizes, in base pairs, are on the left). RNAs were separated by electrophoresis on a 6% acrylamide–7 M urea gel. (B and C) Equal volumes of RRL reaction products generated in the absence (lanes 11 and 22) or presence of the different hTR variants were subjected to immunoprecipitation (IP) with an antibody to the T7 tag. The washed beads were analyzed for telomerase activity (B) and hTERT-hTR coimmunoprecipitation (C). For panel B, telomerase activity was analyzed by the TRAP assay, and 100 ng of partially purified 293 cell extracts were used as a positive control (lane 21). IC, internal PCR control; WT, wild-type. For panel C, hTERT-hTR coimmunoprecipitation was analyzed by Northern blotting using an hTR-specific probe. The arrowheads indicate the positions at which full-length hTR (FLhTR), hTR164-330, and hTR33-147 migrate.

    Techniques Used: Binding Assay, Northern Blot, Synthesized, Electrophoresis, Generated, Immunoprecipitation, Activity Assay, TRAP Assay, Purification, Positive Control, Polymerase Chain Reaction

    10) Product Images from "Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1"

    Article Title: Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0139399

    DIDS inhibits presynaptic filament formation by eh Dmc1. A. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA in the presence and absence of increasing amounts of DIDS at 37°C for 20 min. Products were separated on 12% polyacrylamide gels and analyzed with a phosphorimager. B. eh Dmc1 was incubated with saturating amounts of [ 32 P- γ ]-ATP in the presence and absence of ϕ X174 ssDNA and/or DIDS (66.6 μM). The reactions were stopped at the indicated times, subjected to TLC, and analyzed using a phosphorimager. C. eh Dmc1 was incubated with 32 P-radiolabeled OL90 in the presence and absence of increasing amounts of DIDS followed by exposure to DNase for 15 min at 37°C. The reactions were stopped, separated on 12% polyacrylamide gels, and analyzed with a phosphorimager. D. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA for 2 min prior to the addition of DIDS (2.5 μM, lane 3; 5 μM, lane 4; 7.5 μM, lane 5; and 10 μM, lane 6). After 8 min of incubation, the reaction was initiated by the addition of supercoiled dsDNA. After 12 min, an aliquot was removed and deproteinized. The reaction products were separated by agarose gel electrophoresis, and the gels were analyzed with a phosphorimager. Mean results from three separate experiments were graphed. Error bars represent SEM. DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.
    Figure Legend Snippet: DIDS inhibits presynaptic filament formation by eh Dmc1. A. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA in the presence and absence of increasing amounts of DIDS at 37°C for 20 min. Products were separated on 12% polyacrylamide gels and analyzed with a phosphorimager. B. eh Dmc1 was incubated with saturating amounts of [ 32 P- γ ]-ATP in the presence and absence of ϕ X174 ssDNA and/or DIDS (66.6 μM). The reactions were stopped at the indicated times, subjected to TLC, and analyzed using a phosphorimager. C. eh Dmc1 was incubated with 32 P-radiolabeled OL90 in the presence and absence of increasing amounts of DIDS followed by exposure to DNase for 15 min at 37°C. The reactions were stopped, separated on 12% polyacrylamide gels, and analyzed with a phosphorimager. D. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA for 2 min prior to the addition of DIDS (2.5 μM, lane 3; 5 μM, lane 4; 7.5 μM, lane 5; and 10 μM, lane 6). After 8 min of incubation, the reaction was initiated by the addition of supercoiled dsDNA. After 12 min, an aliquot was removed and deproteinized. The reaction products were separated by agarose gel electrophoresis, and the gels were analyzed with a phosphorimager. Mean results from three separate experiments were graphed. Error bars represent SEM. DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.

    Techniques Used: Incubation, Thin Layer Chromatography, Agarose Gel Electrophoresis

    The eh Dmc1 and eh Rad51 proteins are present in E . histolytica , and purified eh Dmc1 hydrolyzes ATP. A. Purified eh Dmc1 (~1 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. B. Immunoblot of purified recombinant eh Rad51 protein and eh Dmc1 protein (~1 μg, lane 1 and 2, respectively), and E . histolytica partially purified lysate (lane 3) on a 12% SDS-polyacrylamide gel. Anti- sc Rad51 primary antibodies were used. C. Purified eh Dmc1 was incubated with increasing concentrations of [ 32 P- γ ]-ATP. After 60 min, samples were withdrawn and the reaction products were separated using TLC followed by analysis with a phosphorimager. D . Increasing concentrations of ϕ X174 (+) virion single-stranded DNA (ssDNA) or linearized ϕ X174 double-stranded DNA (dsDNA) were incubated with eh Dmc1 and a saturating concentration of [ 32 P- γ ]-ATP. E. Time course analysis of eh Dmc1 ATP hydrolysis activity in the absence or presence of ϕ X174 ssDNA or linearized ϕ X174 dsDNA. Error bars represent SEM, (n = 3).
    Figure Legend Snippet: The eh Dmc1 and eh Rad51 proteins are present in E . histolytica , and purified eh Dmc1 hydrolyzes ATP. A. Purified eh Dmc1 (~1 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. B. Immunoblot of purified recombinant eh Rad51 protein and eh Dmc1 protein (~1 μg, lane 1 and 2, respectively), and E . histolytica partially purified lysate (lane 3) on a 12% SDS-polyacrylamide gel. Anti- sc Rad51 primary antibodies were used. C. Purified eh Dmc1 was incubated with increasing concentrations of [ 32 P- γ ]-ATP. After 60 min, samples were withdrawn and the reaction products were separated using TLC followed by analysis with a phosphorimager. D . Increasing concentrations of ϕ X174 (+) virion single-stranded DNA (ssDNA) or linearized ϕ X174 double-stranded DNA (dsDNA) were incubated with eh Dmc1 and a saturating concentration of [ 32 P- γ ]-ATP. E. Time course analysis of eh Dmc1 ATP hydrolysis activity in the absence or presence of ϕ X174 ssDNA or linearized ϕ X174 dsDNA. Error bars represent SEM, (n = 3).

    Techniques Used: Purification, Staining, Recombinant, Incubation, Thin Layer Chromatography, Concentration Assay, Activity Assay

    11) Product Images from "Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase"

    Article Title: Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase

    Journal: Molecular and biochemical parasitology

    doi: 10.1016/j.molbiopara.2016.09.001

    DIDS disrupts the DNA binding activity of eh Rad51. (A) Time course analysis of eh Rad51 ATPase activity in the presence or absence of DIDS (67 μM), with and without ϕX174 ssDNA or linearized ϕX174 dsDNA. Reactions were stopped with the addition of EDTA at the indicated times prior to separation with thin-layer chromatography and phosphorimager analysis. (B) eh Rad51 (7 μM) incubated with 32 P-radiolabeled ssDNA and increasing amounts of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM; lanes 3–8 respectively). Lane 1 contained no protein or DIDS. Lane 2 contained no DIDS. Lane 9 contained 40 μM DIDS without eh Rad51. (C) eh Rad51 (35 μM) incubated with the 32 P-radiolabled dsDNA and increasing concentrations of DIDS (20 μM, 40 μM, 80 μM, 100 μM, 150 μM, 200 μM; lanes 3–8 respectively). Lane 1 lacked protein and DIDS, lane 2 contained no DIDS and lane 9 contained 200 μM DIDS and no protein. (D) eh Rad51 (7 μM) was incubated with radiolabeled ssDNA in the absence (lane 3) and presence of increasing concentrations of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, and 40 μM; lanes 4–9) prior to the addition of DNase I. The reaction were deproteinized, and the products were separated using non-denaturing PAGE. Lane 1 contained radiolabeled ssDNA alone, lane 2 contained DNase with radiolabeled ssDNA, and lane 10 contained radiolabeled ssDNA in the presence of 40 μM DIDS and DNase I. Error bars represent SEM (n = 3).
    Figure Legend Snippet: DIDS disrupts the DNA binding activity of eh Rad51. (A) Time course analysis of eh Rad51 ATPase activity in the presence or absence of DIDS (67 μM), with and without ϕX174 ssDNA or linearized ϕX174 dsDNA. Reactions were stopped with the addition of EDTA at the indicated times prior to separation with thin-layer chromatography and phosphorimager analysis. (B) eh Rad51 (7 μM) incubated with 32 P-radiolabeled ssDNA and increasing amounts of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM; lanes 3–8 respectively). Lane 1 contained no protein or DIDS. Lane 2 contained no DIDS. Lane 9 contained 40 μM DIDS without eh Rad51. (C) eh Rad51 (35 μM) incubated with the 32 P-radiolabled dsDNA and increasing concentrations of DIDS (20 μM, 40 μM, 80 μM, 100 μM, 150 μM, 200 μM; lanes 3–8 respectively). Lane 1 lacked protein and DIDS, lane 2 contained no DIDS and lane 9 contained 200 μM DIDS and no protein. (D) eh Rad51 (7 μM) was incubated with radiolabeled ssDNA in the absence (lane 3) and presence of increasing concentrations of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, and 40 μM; lanes 4–9) prior to the addition of DNase I. The reaction were deproteinized, and the products were separated using non-denaturing PAGE. Lane 1 contained radiolabeled ssDNA alone, lane 2 contained DNase with radiolabeled ssDNA, and lane 10 contained radiolabeled ssDNA in the presence of 40 μM DIDS and DNase I. Error bars represent SEM (n = 3).

    Techniques Used: Binding Assay, Activity Assay, Thin Layer Chromatography, Incubation, Polyacrylamide Gel Electrophoresis

    eh Rad51 hydrolyzes ATP and binds DNA. (A) Purified recombinant eh Rad51 (0.5 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. (B) Time course analysis of eh Rad51 ATPase activity in the absence and presence of ϕX174 ssDNA or linearized ϕX174 dsDNA. (C) Increasing concentrations of eh Rad51 (lanes 2–6) were incubated with 32 P-labeled ssDNA, and were resolved on a 12% polyacrylamide gel. (D) Increasing concentrations of eh Rad51 (lanes 2–6) were incubated with 32 P-labeled dsDNA. The samples were resolved on a 12% polyacrylamide gel. The results for B, C and D were quantified using a phosphorimager and graphed. Lane 1 for C and D contained no protein, and lane 7 for C and D was treated with SDS/PK (S/P). Error bars represent SEM (n = 3).
    Figure Legend Snippet: eh Rad51 hydrolyzes ATP and binds DNA. (A) Purified recombinant eh Rad51 (0.5 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. (B) Time course analysis of eh Rad51 ATPase activity in the absence and presence of ϕX174 ssDNA or linearized ϕX174 dsDNA. (C) Increasing concentrations of eh Rad51 (lanes 2–6) were incubated with 32 P-labeled ssDNA, and were resolved on a 12% polyacrylamide gel. (D) Increasing concentrations of eh Rad51 (lanes 2–6) were incubated with 32 P-labeled dsDNA. The samples were resolved on a 12% polyacrylamide gel. The results for B, C and D were quantified using a phosphorimager and graphed. Lane 1 for C and D contained no protein, and lane 7 for C and D was treated with SDS/PK (S/P). Error bars represent SEM (n = 3).

    Techniques Used: Purification, Recombinant, Staining, Activity Assay, Incubation, Labeling

    eh Rad51 facilitates plasmid length homologous DNA pairing and DNA strand exchange. (A) Schematic of plasmid length strand exchange assay. Css, circular ssDNA; lds, linearized dsDNA; jm, joint-molecule; nc, nicked-circular; lss, linearized ssDNA. (B) Increasing concentrations of eh Rad51 (lanes 2–8) were incubated with circular ϕX174 ssDNA (css). The reaction was initiated with the introduction of linearized ϕX174 dsDNA (lds) and was deproteinized after 90 min. Reaction products were resolved on an agarose gel and stained with ethidium bromide. Lane 1 contained no protein. (C) The percent product of nicked-circular and total product (jm + nc) were graphed.
    Figure Legend Snippet: eh Rad51 facilitates plasmid length homologous DNA pairing and DNA strand exchange. (A) Schematic of plasmid length strand exchange assay. Css, circular ssDNA; lds, linearized dsDNA; jm, joint-molecule; nc, nicked-circular; lss, linearized ssDNA. (B) Increasing concentrations of eh Rad51 (lanes 2–8) were incubated with circular ϕX174 ssDNA (css). The reaction was initiated with the introduction of linearized ϕX174 dsDNA (lds) and was deproteinized after 90 min. Reaction products were resolved on an agarose gel and stained with ethidium bromide. Lane 1 contained no protein. (C) The percent product of nicked-circular and total product (jm + nc) were graphed.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Staining

    12) Product Images from "Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase"

    Article Title: Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase

    Journal: Molecular and biochemical parasitology

    doi: 10.1016/j.molbiopara.2016.09.001

    DIDS disrupts the DNA binding activity of eh Rad51. (A) Time course analysis of eh Rad51 ATPase activity in the presence or absence of DIDS (67 μM), with and without ϕX174 ssDNA or linearized ϕX174 dsDNA. Reactions were stopped with the addition of EDTA at the indicated times prior to separation with thin-layer chromatography and phosphorimager analysis. (B) eh Rad51 (7 μM) incubated with 32 P-radiolabeled ssDNA and increasing amounts of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM; lanes 3–8 respectively). Lane 1 contained no protein or DIDS. Lane 2 contained no DIDS. Lane 9 contained 40 μM DIDS without eh Rad51. (C) eh Rad51 (35 μM) incubated with the 32 P-radiolabled dsDNA and increasing concentrations of DIDS (20 μM, 40 μM, 80 μM, 100 μM, 150 μM, 200 μM; lanes 3–8 respectively). Lane 1 lacked protein and DIDS, lane 2 contained no DIDS and lane 9 contained 200 μM DIDS and no protein. (D) eh Rad51 (7 μM) was incubated with radiolabeled ssDNA in the absence (lane 3) and presence of increasing concentrations of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, and 40 μM; lanes 4–9) prior to the addition of DNase I. The reaction were deproteinized, and the products were separated using non-denaturing PAGE. Lane 1 contained radiolabeled ssDNA alone, lane 2 contained DNase with radiolabeled ssDNA, and lane 10 contained radiolabeled ssDNA in the presence of 40 μM DIDS and DNase I. Error bars represent SEM (n = 3).
    Figure Legend Snippet: DIDS disrupts the DNA binding activity of eh Rad51. (A) Time course analysis of eh Rad51 ATPase activity in the presence or absence of DIDS (67 μM), with and without ϕX174 ssDNA or linearized ϕX174 dsDNA. Reactions were stopped with the addition of EDTA at the indicated times prior to separation with thin-layer chromatography and phosphorimager analysis. (B) eh Rad51 (7 μM) incubated with 32 P-radiolabeled ssDNA and increasing amounts of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM; lanes 3–8 respectively). Lane 1 contained no protein or DIDS. Lane 2 contained no DIDS. Lane 9 contained 40 μM DIDS without eh Rad51. (C) eh Rad51 (35 μM) incubated with the 32 P-radiolabled dsDNA and increasing concentrations of DIDS (20 μM, 40 μM, 80 μM, 100 μM, 150 μM, 200 μM; lanes 3–8 respectively). Lane 1 lacked protein and DIDS, lane 2 contained no DIDS and lane 9 contained 200 μM DIDS and no protein. (D) eh Rad51 (7 μM) was incubated with radiolabeled ssDNA in the absence (lane 3) and presence of increasing concentrations of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, and 40 μM; lanes 4–9) prior to the addition of DNase I. The reaction were deproteinized, and the products were separated using non-denaturing PAGE. Lane 1 contained radiolabeled ssDNA alone, lane 2 contained DNase with radiolabeled ssDNA, and lane 10 contained radiolabeled ssDNA in the presence of 40 μM DIDS and DNase I. Error bars represent SEM (n = 3).

    Techniques Used: Binding Assay, Activity Assay, Thin Layer Chromatography, Incubation, Polyacrylamide Gel Electrophoresis

    eh Rad51 hydrolyzes ATP and binds DNA. (A) Purified recombinant eh Rad51 (0.5 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. (B) Time course analysis of eh Rad51 ATPase activity in the absence and presence of ϕX174 ssDNA or linearized ϕX174 dsDNA. (C) Increasing concentrations of eh Rad51 (lanes 2–6) were incubated with 32 P-labeled ssDNA, and were resolved on a 12% polyacrylamide gel. (D) Increasing concentrations of eh Rad51 (lanes 2–6) were incubated with 32 P-labeled dsDNA. The samples were resolved on a 12% polyacrylamide gel. The results for B, C and D were quantified using a phosphorimager and graphed. Lane 1 for C and D contained no protein, and lane 7 for C and D was treated with SDS/PK (S/P). Error bars represent SEM (n = 3).
    Figure Legend Snippet: eh Rad51 hydrolyzes ATP and binds DNA. (A) Purified recombinant eh Rad51 (0.5 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. (B) Time course analysis of eh Rad51 ATPase activity in the absence and presence of ϕX174 ssDNA or linearized ϕX174 dsDNA. (C) Increasing concentrations of eh Rad51 (lanes 2–6) were incubated with 32 P-labeled ssDNA, and were resolved on a 12% polyacrylamide gel. (D) Increasing concentrations of eh Rad51 (lanes 2–6) were incubated with 32 P-labeled dsDNA. The samples were resolved on a 12% polyacrylamide gel. The results for B, C and D were quantified using a phosphorimager and graphed. Lane 1 for C and D contained no protein, and lane 7 for C and D was treated with SDS/PK (S/P). Error bars represent SEM (n = 3).

    Techniques Used: Purification, Recombinant, Staining, Activity Assay, Incubation, Labeling

    eh Rad51 facilitates plasmid length homologous DNA pairing and DNA strand exchange. (A) Schematic of plasmid length strand exchange assay. Css, circular ssDNA; lds, linearized dsDNA; jm, joint-molecule; nc, nicked-circular; lss, linearized ssDNA. (B) Increasing concentrations of eh Rad51 (lanes 2–8) were incubated with circular ϕX174 ssDNA (css). The reaction was initiated with the introduction of linearized ϕX174 dsDNA (lds) and was deproteinized after 90 min. Reaction products were resolved on an agarose gel and stained with ethidium bromide. Lane 1 contained no protein. (C) The percent product of nicked-circular and total product (jm + nc) were graphed.
    Figure Legend Snippet: eh Rad51 facilitates plasmid length homologous DNA pairing and DNA strand exchange. (A) Schematic of plasmid length strand exchange assay. Css, circular ssDNA; lds, linearized dsDNA; jm, joint-molecule; nc, nicked-circular; lss, linearized ssDNA. (B) Increasing concentrations of eh Rad51 (lanes 2–8) were incubated with circular ϕX174 ssDNA (css). The reaction was initiated with the introduction of linearized ϕX174 dsDNA (lds) and was deproteinized after 90 min. Reaction products were resolved on an agarose gel and stained with ethidium bromide. Lane 1 contained no protein. (C) The percent product of nicked-circular and total product (jm + nc) were graphed.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Staining

    13) Product Images from "Mediator function of the human Rad51B-Rad51C complex in Rad51/RPA-catalyzed DNA strand exchange"

    Article Title: Mediator function of the human Rad51B-Rad51C complex in Rad51/RPA-catalyzed DNA strand exchange

    Journal: Genes & Development

    doi: 10.1101/gad.935501

    Mediator activity as a function of Rad51B–Rad51C concentration. ( A ) The φX174 ssDNA template (30 μM nucleotides) was incubated with Rad51 (7.5 μM), RPA (1.5 μM), and increasing concentrations of Rad51B–Rad51C (0, 0.6, 0.8, 1.0, and 1.4 μM in lanes 1 – 5 , respectively) for 10 min before the φX174 linear duplex (15 μM base pairs) was incorporated to complete the reaction mixtures. Portions of the reaction mixtures were withdrawn at 30 min (panel I ), 60 min (panel II ), and 80 min (panel III ) and then processed for agarose gel electrophoresis. ( B ) The results from A and from two other independent experiments performed under the same reaction conditions were compiled and graphed. Symbols: results from the 30 min timepoint (squares), the 60 min timepoint (filled triangles), and the 80 min timepoint (circles). Panel I shows the levels of nicked circular duplex formed, and panel II shows the amounts of total reaction products (joint molecules and nicked circular duplex).
    Figure Legend Snippet: Mediator activity as a function of Rad51B–Rad51C concentration. ( A ) The φX174 ssDNA template (30 μM nucleotides) was incubated with Rad51 (7.5 μM), RPA (1.5 μM), and increasing concentrations of Rad51B–Rad51C (0, 0.6, 0.8, 1.0, and 1.4 μM in lanes 1 – 5 , respectively) for 10 min before the φX174 linear duplex (15 μM base pairs) was incorporated to complete the reaction mixtures. Portions of the reaction mixtures were withdrawn at 30 min (panel I ), 60 min (panel II ), and 80 min (panel III ) and then processed for agarose gel electrophoresis. ( B ) The results from A and from two other independent experiments performed under the same reaction conditions were compiled and graphed. Symbols: results from the 30 min timepoint (squares), the 60 min timepoint (filled triangles), and the 80 min timepoint (circles). Panel I shows the levels of nicked circular duplex formed, and panel II shows the amounts of total reaction products (joint molecules and nicked circular duplex).

    Techniques Used: Activity Assay, Concentration Assay, Incubation, Recombinase Polymerase Amplification, Agarose Gel Electrophoresis

    Mediator function of Rad51B–Rad51C. ( A ) Schematic of the homologous DNA pairing and strand exchange reaction using φX174 DNA substrates. Linear duplex is paired with the homologous ssDNA circle to yield a joint molecule. DNA strand exchange, if successful over the length (5.4 kb) of the DNA molecules, results in the formation of the nicked circular duplex. ( B ) Rad51-mediated DNA pairing and strand exchange was carried out with RPA (panel I ) or without it (panel II ). In panel I , the ssDNA was preincubated with Rad51 (R51) before RPA was added. The concentrations of the reaction components were: Rad51, 7.5 μM; RPA, 1.5 μM; ssDNA, 30 μM nucleotides; linear duplex, 15 μM base pairs. ( C ) In the DNA strand exchange reaction in panel I , the ssDNA was incubated with both Rad51 (R51) and RPA simultaneously, and in the reaction in panel II , the ssDNA was incubated with Rad51, RPA and Rad51B–Rad51C ( B – C ) simultaneously. The concentration of Rad51B–Rad51C was 0.8 μM, while the concentrations of the other components were exactly as those in B . In panel III , the amounts of nicked circular duplex in the reactions represented in B panel I (filled squares) and panel II (open circles) and in C panel I (filled circles) and panel II (open squares) are plotted. In panel IV , the amounts of total reaction products (sum of joint molecules and nicked circular duplex) in the reactions represented in B panel I (filled squares) and panel II (open circles) and in C panel I (filled circles) and panel II (open squares) are plotted.
    Figure Legend Snippet: Mediator function of Rad51B–Rad51C. ( A ) Schematic of the homologous DNA pairing and strand exchange reaction using φX174 DNA substrates. Linear duplex is paired with the homologous ssDNA circle to yield a joint molecule. DNA strand exchange, if successful over the length (5.4 kb) of the DNA molecules, results in the formation of the nicked circular duplex. ( B ) Rad51-mediated DNA pairing and strand exchange was carried out with RPA (panel I ) or without it (panel II ). In panel I , the ssDNA was preincubated with Rad51 (R51) before RPA was added. The concentrations of the reaction components were: Rad51, 7.5 μM; RPA, 1.5 μM; ssDNA, 30 μM nucleotides; linear duplex, 15 μM base pairs. ( C ) In the DNA strand exchange reaction in panel I , the ssDNA was incubated with both Rad51 (R51) and RPA simultaneously, and in the reaction in panel II , the ssDNA was incubated with Rad51, RPA and Rad51B–Rad51C ( B – C ) simultaneously. The concentration of Rad51B–Rad51C was 0.8 μM, while the concentrations of the other components were exactly as those in B . In panel III , the amounts of nicked circular duplex in the reactions represented in B panel I (filled squares) and panel II (open circles) and in C panel I (filled circles) and panel II (open squares) are plotted. In panel IV , the amounts of total reaction products (sum of joint molecules and nicked circular duplex) in the reactions represented in B panel I (filled squares) and panel II (open circles) and in C panel I (filled circles) and panel II (open squares) are plotted.

    Techniques Used: Recombinase Polymerase Amplification, Incubation, Concentration Assay

    14) Product Images from "PEX11? Deficiency Is Lethal and Impairs Neuronal Migration but Does Not Abrogate Peroxisome Function"

    Article Title: PEX11? Deficiency Is Lethal and Impairs Neuronal Migration but Does Not Abrogate Peroxisome Function

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.12.4358-4365.2002

    PEX11β −/− mice have a mild, generalized defect in peroxisome metabolism. In all of the graphs, black bars represent average values obtained with PEX11β +/+ , gray bars represents average values obtained with PEX11β −/− , and brackets represent 1 standard deviation. Abundance of VLCFAs (A) and activities of mitochondrial (C 16:0 ) and peroxisomal (C 24:0 ) fatty acid β-oxidation (B) in cultured MEF. (C) Plasmalogens in control and PEX11β −/− mice, expressed as percentages of the total fatty acids. (D) Plasmalogen synthesis activities in cultured MEF, expressed as the ratio of peroxisomal incorporation of [ 14 C]hexadecanol to endoplasmic reticulum incorporation of [ 3 H]hexadecyl-glycerol. (E) Levels of the branched-chain fatty acids phytanic acid and pristanic acid in the liver. (F) Rates of phytanic acid and pristanic acid oxidation in cultured fibroblasts from mutant and control animals.
    Figure Legend Snippet: PEX11β −/− mice have a mild, generalized defect in peroxisome metabolism. In all of the graphs, black bars represent average values obtained with PEX11β +/+ , gray bars represents average values obtained with PEX11β −/− , and brackets represent 1 standard deviation. Abundance of VLCFAs (A) and activities of mitochondrial (C 16:0 ) and peroxisomal (C 24:0 ) fatty acid β-oxidation (B) in cultured MEF. (C) Plasmalogens in control and PEX11β −/− mice, expressed as percentages of the total fatty acids. (D) Plasmalogen synthesis activities in cultured MEF, expressed as the ratio of peroxisomal incorporation of [ 14 C]hexadecanol to endoplasmic reticulum incorporation of [ 3 H]hexadecyl-glycerol. (E) Levels of the branched-chain fatty acids phytanic acid and pristanic acid in the liver. (F) Rates of phytanic acid and pristanic acid oxidation in cultured fibroblasts from mutant and control animals.

    Techniques Used: Mouse Assay, Standard Deviation, Cell Culture, Mutagenesis

    No mitochondrial defects or lipid crystals in PEX11β −/− mouse liver. Ultrastructural analysis of DAB-stained preparations (postfixation and with aqueous osmium) revealed elevated peroxisome clustering (A) and elongation (B) in some hepatocytes of PEX11β −/− mice. Arrowheads indicate the positions of peroxisomes, and arrows in panel B show the position of smooth endoplasmic reticulum (sER). (C) Hepatocytes of PEX11β −/− mice had no mitochondrial structural abnormalities, although in some cells they did appear to be more abundant than in those of controls. MIT, mitochondria; GLY, glycogen; EC, endothelial cell; BC, blood cell. (D) Hepatocytes of PEX11β −/− mice lacked needle-like crystals on the surface of their lipid droplets (D; arrows show where large, needle-like crystals would be seen in Zellweger syndrome patients and mice). (E and F) VLCFA crystals were also absent from phagosomes (PS) of Kupffer cells (KC). NUC, nucleus of a Kupffer cell; EC, endothelial cell; HEP, hepatocyte; PS*, red blood cell in a phagosome. Note the high level of phagocytic degradation of different blood cells in phagosomes (E) and the occasional phagocytosis of hepatocytes (F) in Kupffer cells of PEX11β −/− mice.
    Figure Legend Snippet: No mitochondrial defects or lipid crystals in PEX11β −/− mouse liver. Ultrastructural analysis of DAB-stained preparations (postfixation and with aqueous osmium) revealed elevated peroxisome clustering (A) and elongation (B) in some hepatocytes of PEX11β −/− mice. Arrowheads indicate the positions of peroxisomes, and arrows in panel B show the position of smooth endoplasmic reticulum (sER). (C) Hepatocytes of PEX11β −/− mice had no mitochondrial structural abnormalities, although in some cells they did appear to be more abundant than in those of controls. MIT, mitochondria; GLY, glycogen; EC, endothelial cell; BC, blood cell. (D) Hepatocytes of PEX11β −/− mice lacked needle-like crystals on the surface of their lipid droplets (D; arrows show where large, needle-like crystals would be seen in Zellweger syndrome patients and mice). (E and F) VLCFA crystals were also absent from phagosomes (PS) of Kupffer cells (KC). NUC, nucleus of a Kupffer cell; EC, endothelial cell; HEP, hepatocyte; PS*, red blood cell in a phagosome. Note the high level of phagocytic degradation of different blood cells in phagosomes (E) and the occasional phagocytosis of hepatocytes (F) in Kupffer cells of PEX11β −/− mice.

    Techniques Used: Staining, Mouse Assay

    Neuronal defects in PEX11β −/− mice. Carefully matched coronal (A and B; stained with cresyl fast violet) or sagittal (C to F; stained with PAS and hematoxylin) sections of control (A and C) and PEX11β −/− (B and D to F) mice in medial regions of the neocortex. In panel B, the neuronal migration defect is revealed by higher cell density in the intermediate zone (IZ) and layer V, as well as the altered structure and slightly lesser thickness of the cortical plate (CP). GZ, germinative zone; SP, subplate. Neuronal apoptoses of different stages are indicated by arrows (D to F). Insets 1 and 2 in panel E depict higher magnifications of early stages of chromatin condensation with typical nuclear cap structures of apoptotic cells, indicated by white arrowheads. Black arrowheads indicate macrophages (MP) (slightly more PAS positive) with phagocytosed material. Empty spaces are clearly visible where apoptotic neurons were located (D to F). In contrast, empty spaces in panel C represent endothelium-lined capillaries.
    Figure Legend Snippet: Neuronal defects in PEX11β −/− mice. Carefully matched coronal (A and B; stained with cresyl fast violet) or sagittal (C to F; stained with PAS and hematoxylin) sections of control (A and C) and PEX11β −/− (B and D to F) mice in medial regions of the neocortex. In panel B, the neuronal migration defect is revealed by higher cell density in the intermediate zone (IZ) and layer V, as well as the altered structure and slightly lesser thickness of the cortical plate (CP). GZ, germinative zone; SP, subplate. Neuronal apoptoses of different stages are indicated by arrows (D to F). Insets 1 and 2 in panel E depict higher magnifications of early stages of chromatin condensation with typical nuclear cap structures of apoptotic cells, indicated by white arrowheads. Black arrowheads indicate macrophages (MP) (slightly more PAS positive) with phagocytosed material. Empty spaces are clearly visible where apoptotic neurons were located (D to F). In contrast, empty spaces in panel C represent endothelium-lined capillaries.

    Techniques Used: Mouse Assay, Staining, Migration

    Targeting of PEX11β . (A) Schematic representation of the PEX11β wild-type locus (top), the targeting vector (middle), and the targeted allele (bottom). Two flanking Southern blot probes, A and B, are indicated. (B) Southern blot analyses of the G418 r ES clone DNA with probes A and B and the mouse tail DNA with probe A. Probe A detects an 8.6-kb Eco RI fragment in the wild-type allele and a 5.4-kb fragment in the targeted allele. Probe B detects a 13.4-kb Eco RV/ Sse I fragment in the wild-type allele and a 6.8-kb fragment in the targeted allele. (C) PCR analysis of mouse tail DNA. Positions of the primers are indicated. The wild-type (WT) allele product is 590 bp, and the targeted-allele product is 980 bp. M, molecular size markers. (D) Northern blot analysis of total liver RNA from wild-type (+/+), homozygous (−/−), and heterozygous (+/−) animals. The Northern blot shown was probed with a radioactively labeled murine PEX11β cDNA probe (top), stripped, and probed with labeled β- actin cDNA (bottom). (E and F) PEX11β −/− mice display intrauterine growth retardation and are hypotonic. One litter of newborn mice with heterozygous (+/−) and homozygous (−/−) offspring is depicted either in the conscious state (E) or under ether anesthesia (F).
    Figure Legend Snippet: Targeting of PEX11β . (A) Schematic representation of the PEX11β wild-type locus (top), the targeting vector (middle), and the targeted allele (bottom). Two flanking Southern blot probes, A and B, are indicated. (B) Southern blot analyses of the G418 r ES clone DNA with probes A and B and the mouse tail DNA with probe A. Probe A detects an 8.6-kb Eco RI fragment in the wild-type allele and a 5.4-kb fragment in the targeted allele. Probe B detects a 13.4-kb Eco RV/ Sse I fragment in the wild-type allele and a 6.8-kb fragment in the targeted allele. (C) PCR analysis of mouse tail DNA. Positions of the primers are indicated. The wild-type (WT) allele product is 590 bp, and the targeted-allele product is 980 bp. M, molecular size markers. (D) Northern blot analysis of total liver RNA from wild-type (+/+), homozygous (−/−), and heterozygous (+/−) animals. The Northern blot shown was probed with a radioactively labeled murine PEX11β cDNA probe (top), stripped, and probed with labeled β- actin cDNA (bottom). (E and F) PEX11β −/− mice display intrauterine growth retardation and are hypotonic. One litter of newborn mice with heterozygous (+/−) and homozygous (−/−) offspring is depicted either in the conscious state (E) or under ether anesthesia (F).

    Techniques Used: Plasmid Preparation, Southern Blot, Polymerase Chain Reaction, Northern Blot, Labeling, Mouse Assay

    Developmental delay in kidneys and livers of PEX11β −/− mice. Histological analysis of PAS- and hematoxylin-stained kidney (A to D) and liver (E to I) sections (5 μm) of P0.5 control (A, C, E, and G) and PEX11β −/− (B, D, F, H, and I) mice, depicting the strong delay in the development of both organs in PEX11β −/− animals. Well-developed glomeruli are present in control animals (C, arrows) but absent from PEX11β −/− animals (D). Tubules are also reduced in panel D. Livers of PEX11β −/− mice show regions of underdeveloped hepatocytes (F) with reduced glycogen (reduced PAS staining). Even areas with well-developed PEX11β −/− hepatocytes contained less glycogen (H) than did those of control animals (G). Arrows in panel H depict underdeveloped hepatocytes in PEX11β −/− mouse liver, which were not observed in controls.
    Figure Legend Snippet: Developmental delay in kidneys and livers of PEX11β −/− mice. Histological analysis of PAS- and hematoxylin-stained kidney (A to D) and liver (E to I) sections (5 μm) of P0.5 control (A, C, E, and G) and PEX11β −/− (B, D, F, H, and I) mice, depicting the strong delay in the development of both organs in PEX11β −/− animals. Well-developed glomeruli are present in control animals (C, arrows) but absent from PEX11β −/− animals (D). Tubules are also reduced in panel D. Livers of PEX11β −/− mice show regions of underdeveloped hepatocytes (F) with reduced glycogen (reduced PAS staining). Even areas with well-developed PEX11β −/− hepatocytes contained less glycogen (H) than did those of control animals (G). Arrows in panel H depict underdeveloped hepatocytes in PEX11β −/− mouse liver, which were not observed in controls.

    Techniques Used: Mouse Assay, Staining

    PEX11β deficiency does not affect peroxisomal protein import. Ultrastructural analysis of hepatocytes from P0.5 control (A) and PEX11β −/− mice (B) shows normal import of the peroxisomal enzyme catalase, as determined by DAB staining (post-fixation with aqueous osmium). GLY, glycogen. (C to F) Indirect immunofluorescence analysis of control (C and D) and PEX11β −/− (E and F) MEF with antibodies to catalase (C and E) and PEX14 (D and F), a peroxisomal membrane protein. (G to H) Phytanoyl coenzyme A hydroxylase (PAHX)-myc targets to peroxisomes in PEX11β −/− ), grown for 2 days, and then processed for indirect immunofluorescence assay with a mouse monoclonal antibody to the myc epitope and rabbit anti-PEX14 antibodies.
    Figure Legend Snippet: PEX11β deficiency does not affect peroxisomal protein import. Ultrastructural analysis of hepatocytes from P0.5 control (A) and PEX11β −/− mice (B) shows normal import of the peroxisomal enzyme catalase, as determined by DAB staining (post-fixation with aqueous osmium). GLY, glycogen. (C to F) Indirect immunofluorescence analysis of control (C and D) and PEX11β −/− (E and F) MEF with antibodies to catalase (C and E) and PEX14 (D and F), a peroxisomal membrane protein. (G to H) Phytanoyl coenzyme A hydroxylase (PAHX)-myc targets to peroxisomes in PEX11β −/− ), grown for 2 days, and then processed for indirect immunofluorescence assay with a mouse monoclonal antibody to the myc epitope and rabbit anti-PEX14 antibodies.

    Techniques Used: Mouse Assay, Staining, Immunofluorescence

    15) Product Images from "Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1"

    Article Title: Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0139399

    DIDS inhibits presynaptic filament formation by eh Dmc1. A. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA in the presence and absence of increasing amounts of DIDS at 37°C for 20 min. Products were separated on 12% polyacrylamide gels and analyzed with a phosphorimager. B. eh Dmc1 was incubated with saturating amounts of [ 32 P- γ ]-ATP in the presence and absence of ϕ X174 ssDNA and/or DIDS (66.6 μM). The reactions were stopped at the indicated times, subjected to TLC, and analyzed using a phosphorimager. C. eh Dmc1 was incubated with 32 P-radiolabeled OL90 in the presence and absence of increasing amounts of DIDS followed by exposure to DNase for 15 min at 37°C. The reactions were stopped, separated on 12% polyacrylamide gels, and analyzed with a phosphorimager. D. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA for 2 min prior to the addition of DIDS (2.5 μM, lane 3; 5 μM, lane 4; 7.5 μM, lane 5; and 10 μM, lane 6). After 8 min of incubation, the reaction was initiated by the addition of supercoiled dsDNA. After 12 min, an aliquot was removed and deproteinized. The reaction products were separated by agarose gel electrophoresis, and the gels were analyzed with a phosphorimager. Mean results from three separate experiments were graphed. Error bars represent SEM. DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.
    Figure Legend Snippet: DIDS inhibits presynaptic filament formation by eh Dmc1. A. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA in the presence and absence of increasing amounts of DIDS at 37°C for 20 min. Products were separated on 12% polyacrylamide gels and analyzed with a phosphorimager. B. eh Dmc1 was incubated with saturating amounts of [ 32 P- γ ]-ATP in the presence and absence of ϕ X174 ssDNA and/or DIDS (66.6 μM). The reactions were stopped at the indicated times, subjected to TLC, and analyzed using a phosphorimager. C. eh Dmc1 was incubated with 32 P-radiolabeled OL90 in the presence and absence of increasing amounts of DIDS followed by exposure to DNase for 15 min at 37°C. The reactions were stopped, separated on 12% polyacrylamide gels, and analyzed with a phosphorimager. D. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA for 2 min prior to the addition of DIDS (2.5 μM, lane 3; 5 μM, lane 4; 7.5 μM, lane 5; and 10 μM, lane 6). After 8 min of incubation, the reaction was initiated by the addition of supercoiled dsDNA. After 12 min, an aliquot was removed and deproteinized. The reaction products were separated by agarose gel electrophoresis, and the gels were analyzed with a phosphorimager. Mean results from three separate experiments were graphed. Error bars represent SEM. DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.

    Techniques Used: Incubation, Thin Layer Chromatography, Agarose Gel Electrophoresis

    The eh Dmc1 and eh Rad51 proteins are present in E . histolytica , and purified eh Dmc1 hydrolyzes ATP. A. Purified eh Dmc1 (~1 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. B. Immunoblot of purified recombinant eh Rad51 protein and eh Dmc1 protein (~1 μg, lane 1 and 2, respectively), and E . histolytica partially purified lysate (lane 3) on a 12% SDS-polyacrylamide gel. Anti- sc Rad51 primary antibodies were used. C. Purified eh Dmc1 was incubated with increasing concentrations of [ 32 P- γ ]-ATP. After 60 min, samples were withdrawn and the reaction products were separated using TLC followed by analysis with a phosphorimager. D . Increasing concentrations of ϕ X174 (+) virion single-stranded DNA (ssDNA) or linearized ϕ X174 double-stranded DNA (dsDNA) were incubated with eh Dmc1 and a saturating concentration of [ 32 P- γ ]-ATP. E. Time course analysis of eh Dmc1 ATP hydrolysis activity in the absence or presence of ϕ X174 ssDNA or linearized ϕ X174 dsDNA. Error bars represent SEM, (n = 3).
    Figure Legend Snippet: The eh Dmc1 and eh Rad51 proteins are present in E . histolytica , and purified eh Dmc1 hydrolyzes ATP. A. Purified eh Dmc1 (~1 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. B. Immunoblot of purified recombinant eh Rad51 protein and eh Dmc1 protein (~1 μg, lane 1 and 2, respectively), and E . histolytica partially purified lysate (lane 3) on a 12% SDS-polyacrylamide gel. Anti- sc Rad51 primary antibodies were used. C. Purified eh Dmc1 was incubated with increasing concentrations of [ 32 P- γ ]-ATP. After 60 min, samples were withdrawn and the reaction products were separated using TLC followed by analysis with a phosphorimager. D . Increasing concentrations of ϕ X174 (+) virion single-stranded DNA (ssDNA) or linearized ϕ X174 double-stranded DNA (dsDNA) were incubated with eh Dmc1 and a saturating concentration of [ 32 P- γ ]-ATP. E. Time course analysis of eh Dmc1 ATP hydrolysis activity in the absence or presence of ϕ X174 ssDNA or linearized ϕ X174 dsDNA. Error bars represent SEM, (n = 3).

    Techniques Used: Purification, Staining, Recombinant, Incubation, Thin Layer Chromatography, Concentration Assay, Activity Assay

    16) Product Images from "A Second Allele of eppA in Borrelia burgdorferi Strain B31 Is Located on the Previously Undetected Circular Plasmid cp9-2"

    Article Title: A Second Allele of eppA in Borrelia burgdorferi Strain B31 Is Located on the Previously Undetected Circular Plasmid cp9-2

    Journal: Journal of Bacteriology

    doi:

    (A) Two-dimensional Southern blotting of B31-RML plasmids hybridized with an eppA2 -derived probe. Three forms of cp9-2 are evident and marked as CCC (covalently closed circular), OC (open circular), and Linear. (B) Diagram of cp9-2 indicating the locations of sequences complementary to oligonucleotides EA-G and EA-H, with 5′-to-3′ directions indicated by arrows. (C) Results of long-range PCR of B31-RML and B31-4a. The approximately 9-kb PCR product of cp9-2 is indicated by an arrow. A smaller PCR product was obtained from both cultures and is marked with an asterisk. Positions of DNA size standards (in kilobases) are indicated to the left of panels A and C.
    Figure Legend Snippet: (A) Two-dimensional Southern blotting of B31-RML plasmids hybridized with an eppA2 -derived probe. Three forms of cp9-2 are evident and marked as CCC (covalently closed circular), OC (open circular), and Linear. (B) Diagram of cp9-2 indicating the locations of sequences complementary to oligonucleotides EA-G and EA-H, with 5′-to-3′ directions indicated by arrows. (C) Results of long-range PCR of B31-RML and B31-4a. The approximately 9-kb PCR product of cp9-2 is indicated by an arrow. A smaller PCR product was obtained from both cultures and is marked with an asterisk. Positions of DNA size standards (in kilobases) are indicated to the left of panels A and C.

    Techniques Used: Southern Blot, Derivative Assay, Countercurrent Chromatography, Polymerase Chain Reaction

    17) Product Images from "Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1"

    Article Title: Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0139399

    eh Dmc1 mediates plasmid length DNA strand exchange. A. Schematic of the 3-strand homologous DNA pairing and strand exchange reaction. Homologous DNA pairing between the circular ssDNA (css) and linear dsDNA (lds) first forms a DNA joint molecule (jm). DNA strand exchange converts the joint molecule into a nicked circular duplex (nc) displacing the linear ssDNA (lss). B. eh Dmc1 (12.5 μM) was incubated with ϕX174 virion ssDNA (css) to allow presynaptic filament formation to occur before the addition of hRPA (3.8 μM) and KCl (150 mM final concentration). The reaction was initiated by the addition of linearized double-strand ϕX174 DNA (lds) and spermidine. At the indicated time points, the reactions were deproteinized, subjected to agarose gel electrophoresis, and stained with ethidium bromide.
    Figure Legend Snippet: eh Dmc1 mediates plasmid length DNA strand exchange. A. Schematic of the 3-strand homologous DNA pairing and strand exchange reaction. Homologous DNA pairing between the circular ssDNA (css) and linear dsDNA (lds) first forms a DNA joint molecule (jm). DNA strand exchange converts the joint molecule into a nicked circular duplex (nc) displacing the linear ssDNA (lss). B. eh Dmc1 (12.5 μM) was incubated with ϕX174 virion ssDNA (css) to allow presynaptic filament formation to occur before the addition of hRPA (3.8 μM) and KCl (150 mM final concentration). The reaction was initiated by the addition of linearized double-strand ϕX174 DNA (lds) and spermidine. At the indicated time points, the reactions were deproteinized, subjected to agarose gel electrophoresis, and stained with ethidium bromide.

    Techniques Used: Plasmid Preparation, Incubation, Concentration Assay, Agarose Gel Electrophoresis, Staining

    The eh Dmc1 and eh Rad51 proteins are present in E . histolytica , and purified eh Dmc1 hydrolyzes ATP. A. Purified eh Dmc1 (~1 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. B. Immunoblot of purified recombinant eh Rad51 protein and eh Dmc1 protein (~1 μg, lane 1 and 2, respectively), and E . histolytica partially purified lysate (lane 3) on a 12% SDS-polyacrylamide gel. Anti- sc Rad51 primary antibodies were used. C. Purified eh Dmc1 was incubated with increasing concentrations of [ 32 P- γ ]-ATP. After 60 min, samples were withdrawn and the reaction products were separated using TLC followed by analysis with a phosphorimager. D . Increasing concentrations of ϕ X174 (+) virion single-stranded DNA (ssDNA) or linearized ϕ X174 double-stranded DNA (dsDNA) were incubated with eh Dmc1 and a saturating concentration of [ 32 P- γ ]-ATP. E. Time course analysis of eh Dmc1 ATP hydrolysis activity in the absence or presence of ϕ X174 ssDNA or linearized ϕ X174 dsDNA. Error bars represent SEM, (n = 3).
    Figure Legend Snippet: The eh Dmc1 and eh Rad51 proteins are present in E . histolytica , and purified eh Dmc1 hydrolyzes ATP. A. Purified eh Dmc1 (~1 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. B. Immunoblot of purified recombinant eh Rad51 protein and eh Dmc1 protein (~1 μg, lane 1 and 2, respectively), and E . histolytica partially purified lysate (lane 3) on a 12% SDS-polyacrylamide gel. Anti- sc Rad51 primary antibodies were used. C. Purified eh Dmc1 was incubated with increasing concentrations of [ 32 P- γ ]-ATP. After 60 min, samples were withdrawn and the reaction products were separated using TLC followed by analysis with a phosphorimager. D . Increasing concentrations of ϕ X174 (+) virion single-stranded DNA (ssDNA) or linearized ϕ X174 double-stranded DNA (dsDNA) were incubated with eh Dmc1 and a saturating concentration of [ 32 P- γ ]-ATP. E. Time course analysis of eh Dmc1 ATP hydrolysis activity in the absence or presence of ϕ X174 ssDNA or linearized ϕ X174 dsDNA. Error bars represent SEM, (n = 3).

    Techniques Used: Purification, Staining, Recombinant, Incubation, Thin Layer Chromatography, Concentration Assay, Activity Assay

    eh Dmc1 binds DNA. A. Increasing concentrations of eh Dmc1 (1.3 μM, lane 2; 2.6 μM, lane 3; 3.9 μM, lane 4; and 5.2 μM, lane 5) were incubated with ssDNA ( 32 P-labeled H3 ssDNA). B. The mean binding percentages were graphed for three independent experiments from A . Error bars represent SEM. C. Increasing concentrations of eh Dmc1 (5.2 μM, lane 2; 10.4 μM, lane 3; 20.8 μM, lane 4; and 31.2 μM, lane 5) were incubated with dsDNA ( 32 P-labeled H3 annealed to H3c). D. The mean binding percentages were graphed for three independent experiments from C . Error bars represent SEM. Lane 1 for A and C is devoid of protein, and lane 6 for A and C was SDS/PK (S/P) treated containing the highest concentration of eh Dmc1.
    Figure Legend Snippet: eh Dmc1 binds DNA. A. Increasing concentrations of eh Dmc1 (1.3 μM, lane 2; 2.6 μM, lane 3; 3.9 μM, lane 4; and 5.2 μM, lane 5) were incubated with ssDNA ( 32 P-labeled H3 ssDNA). B. The mean binding percentages were graphed for three independent experiments from A . Error bars represent SEM. C. Increasing concentrations of eh Dmc1 (5.2 μM, lane 2; 10.4 μM, lane 3; 20.8 μM, lane 4; and 31.2 μM, lane 5) were incubated with dsDNA ( 32 P-labeled H3 annealed to H3c). D. The mean binding percentages were graphed for three independent experiments from C . Error bars represent SEM. Lane 1 for A and C is devoid of protein, and lane 6 for A and C was SDS/PK (S/P) treated containing the highest concentration of eh Dmc1.

    Techniques Used: Incubation, Labeling, Binding Assay, Concentration Assay

    18) Product Images from "Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase"

    Article Title: Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase

    Journal: Molecular and biochemical parasitology

    doi: 10.1016/j.molbiopara.2016.09.001

    DIDS disrupts the DNA binding activity of eh Rad51. (A) Time course analysis of eh Rad51 ATPase activity in the presence or absence of DIDS (67 μM), with and without ϕX174 ssDNA or linearized ϕX174 dsDNA. Reactions were stopped with the addition of EDTA at the indicated times prior to separation with thin-layer chromatography and phosphorimager analysis. (B) eh Rad51 (7 μM) incubated with 32 P-radiolabeled ssDNA and increasing amounts of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM; lanes 3–8 respectively). Lane 1 contained no protein or DIDS. Lane 2 contained no DIDS. Lane 9 contained 40 μM DIDS without eh Rad51. (C) eh Rad51 (35 μM) incubated with the 32 P-radiolabled dsDNA and increasing concentrations of DIDS (20 μM, 40 μM, 80 μM, 100 μM, 150 μM, 200 μM; lanes 3–8 respectively). Lane 1 lacked protein and DIDS, lane 2 contained no DIDS and lane 9 contained 200 μM DIDS and no protein. (D) eh Rad51 (7 μM) was incubated with radiolabeled ssDNA in the absence (lane 3) and presence of increasing concentrations of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, and 40 μM; lanes 4–9) prior to the addition of DNase I. The reaction were deproteinized, and the products were separated using non-denaturing PAGE. Lane 1 contained radiolabeled ssDNA alone, lane 2 contained DNase with radiolabeled ssDNA, and lane 10 contained radiolabeled ssDNA in the presence of 40 μM DIDS and DNase I. Error bars represent SEM (n = 3).
    Figure Legend Snippet: DIDS disrupts the DNA binding activity of eh Rad51. (A) Time course analysis of eh Rad51 ATPase activity in the presence or absence of DIDS (67 μM), with and without ϕX174 ssDNA or linearized ϕX174 dsDNA. Reactions were stopped with the addition of EDTA at the indicated times prior to separation with thin-layer chromatography and phosphorimager analysis. (B) eh Rad51 (7 μM) incubated with 32 P-radiolabeled ssDNA and increasing amounts of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM; lanes 3–8 respectively). Lane 1 contained no protein or DIDS. Lane 2 contained no DIDS. Lane 9 contained 40 μM DIDS without eh Rad51. (C) eh Rad51 (35 μM) incubated with the 32 P-radiolabled dsDNA and increasing concentrations of DIDS (20 μM, 40 μM, 80 μM, 100 μM, 150 μM, 200 μM; lanes 3–8 respectively). Lane 1 lacked protein and DIDS, lane 2 contained no DIDS and lane 9 contained 200 μM DIDS and no protein. (D) eh Rad51 (7 μM) was incubated with radiolabeled ssDNA in the absence (lane 3) and presence of increasing concentrations of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, and 40 μM; lanes 4–9) prior to the addition of DNase I. The reaction were deproteinized, and the products were separated using non-denaturing PAGE. Lane 1 contained radiolabeled ssDNA alone, lane 2 contained DNase with radiolabeled ssDNA, and lane 10 contained radiolabeled ssDNA in the presence of 40 μM DIDS and DNase I. Error bars represent SEM (n = 3).

    Techniques Used: Binding Assay, Activity Assay, Thin Layer Chromatography, Incubation, Polyacrylamide Gel Electrophoresis

    eh Rad51 hydrolyzes ATP and binds DNA. (A) Purified recombinant eh Rad51 (0.5 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. (B) Time course analysis of eh Rad51 ATPase activity in the absence and presence of ϕX174 ssDNA or linearized ϕX174 dsDNA. (C) Increasing concentrations of eh Rad51 (lanes 2–6) were incubated with 32 P-labeled ssDNA, and were resolved on a 12% polyacrylamide gel. (D) Increasing concentrations of eh Rad51 (lanes 2–6) were incubated with 32 P-labeled dsDNA. The samples were resolved on a 12% polyacrylamide gel. The results for B, C and D were quantified using a phosphorimager and graphed. Lane 1 for C and D contained no protein, and lane 7 for C and D was treated with SDS/PK (S/P). Error bars represent SEM (n = 3).
    Figure Legend Snippet: eh Rad51 hydrolyzes ATP and binds DNA. (A) Purified recombinant eh Rad51 (0.5 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. (B) Time course analysis of eh Rad51 ATPase activity in the absence and presence of ϕX174 ssDNA or linearized ϕX174 dsDNA. (C) Increasing concentrations of eh Rad51 (lanes 2–6) were incubated with 32 P-labeled ssDNA, and were resolved on a 12% polyacrylamide gel. (D) Increasing concentrations of eh Rad51 (lanes 2–6) were incubated with 32 P-labeled dsDNA. The samples were resolved on a 12% polyacrylamide gel. The results for B, C and D were quantified using a phosphorimager and graphed. Lane 1 for C and D contained no protein, and lane 7 for C and D was treated with SDS/PK (S/P). Error bars represent SEM (n = 3).

    Techniques Used: Purification, Recombinant, Staining, Activity Assay, Incubation, Labeling

    eh Rad51 facilitates plasmid length homologous DNA pairing and DNA strand exchange. (A) Schematic of plasmid length strand exchange assay. Css, circular ssDNA; lds, linearized dsDNA; jm, joint-molecule; nc, nicked-circular; lss, linearized ssDNA. (B) Increasing concentrations of eh Rad51 (lanes 2–8) were incubated with circular ϕX174 ssDNA (css). The reaction was initiated with the introduction of linearized ϕX174 dsDNA (lds) and was deproteinized after 90 min. Reaction products were resolved on an agarose gel and stained with ethidium bromide. Lane 1 contained no protein. (C) The percent product of nicked-circular and total product (jm + nc) were graphed.
    Figure Legend Snippet: eh Rad51 facilitates plasmid length homologous DNA pairing and DNA strand exchange. (A) Schematic of plasmid length strand exchange assay. Css, circular ssDNA; lds, linearized dsDNA; jm, joint-molecule; nc, nicked-circular; lss, linearized ssDNA. (B) Increasing concentrations of eh Rad51 (lanes 2–8) were incubated with circular ϕX174 ssDNA (css). The reaction was initiated with the introduction of linearized ϕX174 dsDNA (lds) and was deproteinized after 90 min. Reaction products were resolved on an agarose gel and stained with ethidium bromide. Lane 1 contained no protein. (C) The percent product of nicked-circular and total product (jm + nc) were graphed.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Staining

    Related Articles

    Clone Assay:

    Article Title: Novel Human Polyomavirus Noncoding Control Regions Differ in Bidirectional Gene Expression according to Host Cell, Large T-Antigen Expression, and Clinically Occurring Rearrangements
    Article Snippet: .. The HPyV NCCRs were chemically synthesized in pUC57 (Eurogentec S.A, Belgium) , excised using the restriction enzymes BssHII and MluI (New England BioLabs, England), and cloned into the corresponding restriction sites of pRG13D12. .. HPyV NCCR constructs were verified by Sanger sequencing for correct NCCR sequences and orientations using the 3130 genetic analyzer (Applied Biosystems, Switzerland).

    Synthesized:

    Article Title: Novel Human Polyomavirus Noncoding Control Regions Differ in Bidirectional Gene Expression according to Host Cell, Large T-Antigen Expression, and Clinically Occurring Rearrangements
    Article Snippet: .. The HPyV NCCRs were chemically synthesized in pUC57 (Eurogentec S.A, Belgium) , excised using the restriction enzymes BssHII and MluI (New England BioLabs, England), and cloned into the corresponding restriction sites of pRG13D12. .. HPyV NCCR constructs were verified by Sanger sequencing for correct NCCR sequences and orientations using the 3130 genetic analyzer (Applied Biosystems, Switzerland).

    Isolation:

    Article Title: Encephalitozoon cuniculi (Microspora) genome: physical map and evidence for telomere-associated rDNA units on all chromosomes
    Article Snippet: .. Probes corresponding to five unique E.cuniculi genes isolated in our laboratory ( , ; C.Biderre, personal communication), were hybridised to Bss HII- and Mlu I-KARD-PFGE gels. ..

    Methylation:

    Article Title: Encephalitozoon cuniculi (Microspora) genome: physical map and evidence for telomere-associated rDNA units on all chromosomes
    Article Snippet: .. This low frequency might also be explained by a DNA modification to which both Bss HII and Mlu I are sensitive, e.g. cytosine methylation as in higher eukaryotes. .. If DNA methylation occurs in E.cuniculi , differences in the methylation pattern of two homologous chromosomes should lead to differentially migrated fragments in KARD-PFGE gels.

    Labeling:

    Article Title: Murine Cytomegalovirus CC Chemokine Homolog MCK-2 (m131-129) Is a Determinant of Dissemination That Increases Inflammation at Initial Sites of Infection
    Article Snippet: .. Virion DNA (0.5 to 1.0 μg) was digested with Hin dIII, Bss HII, Afl II, or Spe I (New England Biolabs, Beverly, Mass.) and end labeled in the presence of 2.5 μCi of [α-32 P]dCTP (Amersham), 125 μM each dATP, dGTP, and dTTP, and 0.5 U of Klenow polymerase (Roche, Indianapolis, Ind.) for 15 min at room temperature in 20 μl of restriction enzyme buffer. .. Restriction fragments were separated on a 0.6% agarose gel, which was fixed in 95% ethanol and vacuum dried at 80°C, followed by autoradiography.

    other:

    Article Title: Encephalitozoon cuniculi (Microspora) genome: physical map and evidence for telomere-associated rDNA units on all chromosomes
    Article Snippet: In order to place the Bss HII and Mlu I restriction sites on each chromosome, we developed a mapping procedure applied to individual chromosomes (DDIC-PFGE) that can be viewed as the counterpart of 2D-PFGE in bacterial genomics ( ).

    Article Title: Genetic relationships between Candida albicans strains isolated from dental plaque, trachea, and bronchoalveolar lavage fluid from mechanically ventilated intensive care unit patients
    Article Snippet: In comparison, restriction endonuclease analysis of the genome (REAG) from the 49 isolates of 22 patients yielded 24 patterns after digestion with Sfi I ( ) and 25 patterns after digestion with Bss HII ( ).

    Modification:

    Article Title: Encephalitozoon cuniculi (Microspora) genome: physical map and evidence for telomere-associated rDNA units on all chromosomes
    Article Snippet: .. This low frequency might also be explained by a DNA modification to which both Bss HII and Mlu I are sensitive, e.g. cytosine methylation as in higher eukaryotes. .. If DNA methylation occurs in E.cuniculi , differences in the methylation pattern of two homologous chromosomes should lead to differentially migrated fragments in KARD-PFGE gels.

    Plasmid Preparation:

    Article Title: Gene cloning, sequence analysis, and expression of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase
    Article Snippet: .. According to the pBluescript map, we would expect to see the DNA fragment next to the pBluescript vector at the T3 promoter site in pBluMH to be about 0.1 kb smaller when double-digested with Bss HII + Pst I than when digested with Bss HII alone. .. The Southern hybridization results show that the 1.1-kb fragment from Bss HII digestion corresponds to a 1-kb fragment in the Bss HII + Pst I double-digestion.

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