dnase i  (New England Biolabs)


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    Name:
    DNase I RNase free
    Description:
    DNase I RNase free 5 000 units
    Catalog Number:
    M0303L
    Price:
    276
    Size:
    5 000 units
    Category:
    Deoxyribonucleases DNase
    Score:
    85
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    New England Biolabs dnase i
    DNase I RNase free
    DNase I RNase free 5 000 units
    https://www.bioz.com/result/dnase i/product/New England Biolabs
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    Images

    1) Product Images from "Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis"

    Article Title: Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01680

    Deoxyribonuclease (DNase) I treatment abolished neutrophil extracellular traps (NETs) formation and ameliorated atherosclerotic burden. WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed on high-fat chow (HFC) for 6 weeks, starting at 3-week HFC, 400 U of DNase I or vehicle control (PBS) was intravenously administered three times weekly until the end of experiments. (A) Representative confocal immunofluorescence microscopy images of aortic root sections stained for DAPI (blue), MPO (green), Ly-6G (red), and Cit-H3 (cyan). Data are representative of five mice in each group. (B) Quantification of NETs from (A) ( n = 5/group). (C) Representative images of aortic root sections stained for lipid (Oil Red O, red) and hematoxylin ( n = 5/group). (D) mRNA levels of IL-1β, TNF-α, CCL2, CXCL1, and CXCL2 in the aorta from WT and PAD4 KO mice placed on HFC for 6 weeks and administered with DNase I or vehicle control (PBS). mRNA levels were normalized to the GAPDH and expressed relative to levels measured in one of the vehicle control-treated WT mice ( n = 5/group). * p
    Figure Legend Snippet: Deoxyribonuclease (DNase) I treatment abolished neutrophil extracellular traps (NETs) formation and ameliorated atherosclerotic burden. WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed on high-fat chow (HFC) for 6 weeks, starting at 3-week HFC, 400 U of DNase I or vehicle control (PBS) was intravenously administered three times weekly until the end of experiments. (A) Representative confocal immunofluorescence microscopy images of aortic root sections stained for DAPI (blue), MPO (green), Ly-6G (red), and Cit-H3 (cyan). Data are representative of five mice in each group. (B) Quantification of NETs from (A) ( n = 5/group). (C) Representative images of aortic root sections stained for lipid (Oil Red O, red) and hematoxylin ( n = 5/group). (D) mRNA levels of IL-1β, TNF-α, CCL2, CXCL1, and CXCL2 in the aorta from WT and PAD4 KO mice placed on HFC for 6 weeks and administered with DNase I or vehicle control (PBS). mRNA levels were normalized to the GAPDH and expressed relative to levels measured in one of the vehicle control-treated WT mice ( n = 5/group). * p

    Techniques Used: Mouse Assay, Immunofluorescence, Microscopy, Staining

    Neutrophil extracellular traps (NETs) present in atherosclerotic lesions stimulate inflammatory responses in arterial macrophages. (A) Bone marrow (BM)-derived neutrophils were stimulated in the absence (UN) or presence (A23187) of A23187 for 4 h. Half the UN-NETs or A23187-NETs were digested by deoxyribonuclease (DNase) I. NETs were quantified by measuring Cit-H3-DNA complexes on ELISA. (B) BM-derived macrophages were stimulated with UN-NETs (BMN-UN), UN-NETs treated with DNase I (BMN-UN-DNase I), A23187-NETs (BMN-A23), or A23187-NETs treated with DNase I (BMN-A23-DNase I) for 4 h. Gene expression levels of IL-1β, CCL2, CXCL1, and CXCL2 were determined. mRNA levels were normalized to GAPDH and expressed relative to levels measured in one of the BMN-UN conditions (C) . WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed high-fat chow (HFC) for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: IL-1β, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. (D) WT and PAD4 KO mice were fed HFC for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: CCL2, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. * p
    Figure Legend Snippet: Neutrophil extracellular traps (NETs) present in atherosclerotic lesions stimulate inflammatory responses in arterial macrophages. (A) Bone marrow (BM)-derived neutrophils were stimulated in the absence (UN) or presence (A23187) of A23187 for 4 h. Half the UN-NETs or A23187-NETs were digested by deoxyribonuclease (DNase) I. NETs were quantified by measuring Cit-H3-DNA complexes on ELISA. (B) BM-derived macrophages were stimulated with UN-NETs (BMN-UN), UN-NETs treated with DNase I (BMN-UN-DNase I), A23187-NETs (BMN-A23), or A23187-NETs treated with DNase I (BMN-A23-DNase I) for 4 h. Gene expression levels of IL-1β, CCL2, CXCL1, and CXCL2 were determined. mRNA levels were normalized to GAPDH and expressed relative to levels measured in one of the BMN-UN conditions (C) . WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed high-fat chow (HFC) for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: IL-1β, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. (D) WT and PAD4 KO mice were fed HFC for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: CCL2, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. * p

    Techniques Used: Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Mouse Assay, Staining, Immunofluorescence, Microscopy

    2) Product Images from "In vitro selection of an XNA aptamer capable of small-molecule recognition"

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky667

    TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.
    Figure Legend Snippet: TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.

    Techniques Used: Binding Assay, Amplification, Modification, Polyacrylamide Gel Electrophoresis, Selection, Incubation, Magnetic Beads, Polymerase Chain Reaction

    Comparison of the biostability of FAM-labeled TNA aptamer A04T.2 and DNA aptamer A08. ( A ) Denaturing PAGE analysis of the TNA (T) and DNA (D) aptamers after incubation in conditions of increasing nuclease stringency: selection buffer (control), 1.5 U DNase I, 50% human blood serum in PBS, and 0.5 mg/mL human liver microsomes. Samples were incubated under these conditions for 3 days at 37°C. ( B ) Bead-binding assay to determine retention of aptamer binding in the presence of nucleases. Each column and error bar represents the average and standard deviation of two trials.
    Figure Legend Snippet: Comparison of the biostability of FAM-labeled TNA aptamer A04T.2 and DNA aptamer A08. ( A ) Denaturing PAGE analysis of the TNA (T) and DNA (D) aptamers after incubation in conditions of increasing nuclease stringency: selection buffer (control), 1.5 U DNase I, 50% human blood serum in PBS, and 0.5 mg/mL human liver microsomes. Samples were incubated under these conditions for 3 days at 37°C. ( B ) Bead-binding assay to determine retention of aptamer binding in the presence of nucleases. Each column and error bar represents the average and standard deviation of two trials.

    Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Incubation, Selection, Binding Assay, Standard Deviation

    3) Product Images from "In-Cell RNA Hydrolysis Assay: A Method for the Determination of the RNase Activity of Potential RNases"

    Article Title: In-Cell RNA Hydrolysis Assay: A Method for the Determination of the RNase Activity of Potential RNases

    Journal: Molecular Biotechnology

    doi: 10.1007/s12033-015-9844-7

    The detection of RNAs released from fixed and permeabilized cells.  a  Experimental procedures for the data presented in ( b ) and ( d ).  b  A549 cells grown in a 48-well plate at a density of 5 × 10 4  cells/well were fixed, permeabilized, and incubated with 100 μl of 10 μM RNase A or 10 μM 3D8 antibody for 2 h at 37 °C. An aliquot of the conditioned medium was added to RiboGreen, and the fluorescence intensity was analyzed.  c  Pure 16S and 23S rRNA from  E. coli  was incubated with RiboGreen in the presence or absence of RNase A prior to fluorescence intensity analysis.  d  A549 cells grown in a 6-well plate at a density of 5 × 10 5  cells/well were fixed, permeabilized, and incubated with 10 μM RNase A or 3D8 antibody for 2 h at 37 °C. Proteins were removed from the conditioned medium by precipitation, and absorbance at 260 nm was measured.  e  Degradation of plasmid DNA (1 μg/ml) by DNase I (2 U) was tested using RiboGreen prepared in DNase I reaction buffer for 2 h at 37 °C.  f  Fixed and permeabilized cells in a 48-well plate were treated with RNase A or 3D8 antibody (10 μM) in the presence or absence of DNase I (2 U) for 2 h at 37 °C. The conditioned medium was mixed with RiboGreen prior to fluorescence intensity analysis.  RFU  relative fluorescence unit. Data represent the mean ± standard error of four independent experiments
    Figure Legend Snippet: The detection of RNAs released from fixed and permeabilized cells. a Experimental procedures for the data presented in ( b ) and ( d ). b A549 cells grown in a 48-well plate at a density of 5 × 10 4  cells/well were fixed, permeabilized, and incubated with 100 μl of 10 μM RNase A or 10 μM 3D8 antibody for 2 h at 37 °C. An aliquot of the conditioned medium was added to RiboGreen, and the fluorescence intensity was analyzed. c Pure 16S and 23S rRNA from E. coli was incubated with RiboGreen in the presence or absence of RNase A prior to fluorescence intensity analysis. d A549 cells grown in a 6-well plate at a density of 5 × 10 5  cells/well were fixed, permeabilized, and incubated with 10 μM RNase A or 3D8 antibody for 2 h at 37 °C. Proteins were removed from the conditioned medium by precipitation, and absorbance at 260 nm was measured. e Degradation of plasmid DNA (1 μg/ml) by DNase I (2 U) was tested using RiboGreen prepared in DNase I reaction buffer for 2 h at 37 °C. f Fixed and permeabilized cells in a 48-well plate were treated with RNase A or 3D8 antibody (10 μM) in the presence or absence of DNase I (2 U) for 2 h at 37 °C. The conditioned medium was mixed with RiboGreen prior to fluorescence intensity analysis. RFU relative fluorescence unit. Data represent the mean ± standard error of four independent experiments

    Techniques Used: Incubation, Fluorescence, Plasmid Preparation

    4) Product Images from "Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis"

    Article Title: Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01680

    Deoxyribonuclease (DNase) I treatment abolished neutrophil extracellular traps (NETs) formation and ameliorated atherosclerotic burden. WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed on high-fat chow (HFC) for 6 weeks, starting at 3-week HFC, 400 U of DNase I or vehicle control (PBS) was intravenously administered three times weekly until the end of experiments. (A) Representative confocal immunofluorescence microscopy images of aortic root sections stained for DAPI (blue), MPO (green), Ly-6G (red), and Cit-H3 (cyan). Data are representative of five mice in each group. (B) Quantification of NETs from (A) ( n = 5/group). (C) Representative images of aortic root sections stained for lipid (Oil Red O, red) and hematoxylin ( n = 5/group). (D) mRNA levels of IL-1β, TNF-α, CCL2, CXCL1, and CXCL2 in the aorta from WT and PAD4 KO mice placed on HFC for 6 weeks and administered with DNase I or vehicle control (PBS). mRNA levels were normalized to the GAPDH and expressed relative to levels measured in one of the vehicle control-treated WT mice ( n = 5/group). * p
    Figure Legend Snippet: Deoxyribonuclease (DNase) I treatment abolished neutrophil extracellular traps (NETs) formation and ameliorated atherosclerotic burden. WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed on high-fat chow (HFC) for 6 weeks, starting at 3-week HFC, 400 U of DNase I or vehicle control (PBS) was intravenously administered three times weekly until the end of experiments. (A) Representative confocal immunofluorescence microscopy images of aortic root sections stained for DAPI (blue), MPO (green), Ly-6G (red), and Cit-H3 (cyan). Data are representative of five mice in each group. (B) Quantification of NETs from (A) ( n = 5/group). (C) Representative images of aortic root sections stained for lipid (Oil Red O, red) and hematoxylin ( n = 5/group). (D) mRNA levels of IL-1β, TNF-α, CCL2, CXCL1, and CXCL2 in the aorta from WT and PAD4 KO mice placed on HFC for 6 weeks and administered with DNase I or vehicle control (PBS). mRNA levels were normalized to the GAPDH and expressed relative to levels measured in one of the vehicle control-treated WT mice ( n = 5/group). * p

    Techniques Used: Mouse Assay, Immunofluorescence, Microscopy, Staining

    Neutrophil extracellular traps (NETs) present in atherosclerotic lesions stimulate inflammatory responses in arterial macrophages. (A) Bone marrow (BM)-derived neutrophils were stimulated in the absence (UN) or presence (A23187) of A23187 for 4 h. Half the UN-NETs or A23187-NETs were digested by deoxyribonuclease (DNase) I. NETs were quantified by measuring Cit-H3-DNA complexes on ELISA. (B) BM-derived macrophages were stimulated with UN-NETs (BMN-UN), UN-NETs treated with DNase I (BMN-UN-DNase I), A23187-NETs (BMN-A23), or A23187-NETs treated with DNase I (BMN-A23-DNase I) for 4 h. Gene expression levels of IL-1β, CCL2, CXCL1, and CXCL2 were determined. mRNA levels were normalized to GAPDH and expressed relative to levels measured in one of the BMN-UN conditions (C) . WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed high-fat chow (HFC) for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: IL-1β, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. (D) WT and PAD4 KO mice were fed HFC for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: CCL2, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. * p
    Figure Legend Snippet: Neutrophil extracellular traps (NETs) present in atherosclerotic lesions stimulate inflammatory responses in arterial macrophages. (A) Bone marrow (BM)-derived neutrophils were stimulated in the absence (UN) or presence (A23187) of A23187 for 4 h. Half the UN-NETs or A23187-NETs were digested by deoxyribonuclease (DNase) I. NETs were quantified by measuring Cit-H3-DNA complexes on ELISA. (B) BM-derived macrophages were stimulated with UN-NETs (BMN-UN), UN-NETs treated with DNase I (BMN-UN-DNase I), A23187-NETs (BMN-A23), or A23187-NETs treated with DNase I (BMN-A23-DNase I) for 4 h. Gene expression levels of IL-1β, CCL2, CXCL1, and CXCL2 were determined. mRNA levels were normalized to GAPDH and expressed relative to levels measured in one of the BMN-UN conditions (C) . WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed high-fat chow (HFC) for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: IL-1β, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. (D) WT and PAD4 KO mice were fed HFC for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: CCL2, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. * p

    Techniques Used: Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Mouse Assay, Staining, Immunofluorescence, Microscopy

    5) Product Images from "Autonomous actions of the human growth hormone long-range enhancer"

    Article Title: Autonomous actions of the human growth hormone long-range enhancer

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv093

    HSI activates noncoding transcription independent of promoter interactions. ( A ) Map of the  hGH  locus and the  hGH BAC  transgene. Each structural gene in the  hGH  locus is indicated along with its transcriptional orientation. The 123 kb  hGH BAC  transgene released from the originating BAC clone (BAC535D15) by NotI digestion was used to generate the  hGH BAC  transgenic mouse lines (  26 ). The vertical arrows labeled with roman numerals indicate the positions of DNase I hypersensitive sites (HS) that form in pituitary chromatin and constitute the  hGH  LCR. The distance (14.5 kb) between the 3′ end of the HSI core and the  hGH-N  gene promoter is indicated with double-headed arrow. Note that this region encompasses the  hCD79b  gene that encodes the B-cell specific Ig receptor component, Igβ. Pit-1 binding sites that constitute core determinants of HSI are indicated by the shaded ovals (expanded inset). The 99 bp deletion that inactivates HSI functions (  18 ) is also shown in expanded view (inset above). ( B ) Map of the wild-type  hGH BAC  and two derivative transgenes. The − 8.0CD79b  transgene (12.5 kb) encompasses HSI and the contiguous  hCD79b  (expanded view below the  hGH  locus map) and isolates this region from the  hGH  gene cluster. The  −8.0CD79bΔ1.6  transgene was derived from the  −8.0CD79b  by deletion of a 1.6-kb internal fragment extending from −0.5 kb of the promoter through intron 2, removing all defined  hCD79b  promoter elements. ( C ) Noncoding transcription across  hCD79b  in the transgenic mouse pituitary is preserved in the absence of defined promoter elements. Transcription across the  hCD79b  region generated by the promoterless − 8.0CD79bΔ1.6  was compared to that of the − 8.0CD79b  transgene (containing the intact  hCD79b  promoter but not the  hGH-N  promoter) and the  hGH BAC  transgene (containing both the  hCD79b  and the  hGH-N  promoters). Pituitary RNA from mice carrying each of these indicated transgenes was assayed for transcription across  hCD79b  by RT-qPCR. The data shown in the histogram (Y-axis) have been normalized to the corresponding transgene copy numbers and to somatotrope-specific levels of the endogenous mouse growth hormone ( mGH1 ) mRNA. The number of genetically distinct lines assessed for each transgene is noted above the corresponding bars in the histogram. Each line was tested in triplicate using three independent mice. Values represent the average ± SD. The level of  hGH BAC  was defined as 1.0. ( D ) Map of the 123 kb  λΔCD/hGH  transgene and the derivative the  −8.0λΔCD  transgene. This transgene was generated by replacing the  hCD79b  gene and promoter (to −500 bp) with a size-matched fragment of bacteriophage λ DNA within the context of an otherwise intact  hGH BAC  (see ‘Materials and Methods' section). The  −8.0λΔCD  transgene was released from the  λΔCD/hGH  transgene with boundaries as indicated. ( E ) Noncoding transcription is activated 3′ of HSI within an inserted (λ) DNA segment. Transcription across the λ-DNA region was compared between the  λΔCD/hGH  and  −8.0λΔCD  transgenes. λ noncoding transcription was measured by RT-qPCR, normalized to transgene copy-number and to  mGH1  mRNA levels. λ noncoding transcription was actively transcribed without  hGH-N  promoter. The number of genetically distinct lines assessed for each transgene is noted above the corresponding bars in the histogram. Each experiment was carried out in triplicate. The study of the single  −8.0λΔCD  line was evaluated in each of three independent mice, each studied in triplicate. Values represent the average ± SD.
    Figure Legend Snippet: HSI activates noncoding transcription independent of promoter interactions. ( A ) Map of the hGH locus and the hGH BAC transgene. Each structural gene in the hGH locus is indicated along with its transcriptional orientation. The 123 kb hGH BAC transgene released from the originating BAC clone (BAC535D15) by NotI digestion was used to generate the hGH BAC transgenic mouse lines ( 26 ). The vertical arrows labeled with roman numerals indicate the positions of DNase I hypersensitive sites (HS) that form in pituitary chromatin and constitute the hGH LCR. The distance (14.5 kb) between the 3′ end of the HSI core and the hGH-N gene promoter is indicated with double-headed arrow. Note that this region encompasses the hCD79b gene that encodes the B-cell specific Ig receptor component, Igβ. Pit-1 binding sites that constitute core determinants of HSI are indicated by the shaded ovals (expanded inset). The 99 bp deletion that inactivates HSI functions ( 18 ) is also shown in expanded view (inset above). ( B ) Map of the wild-type hGH BAC and two derivative transgenes. The − 8.0CD79b transgene (12.5 kb) encompasses HSI and the contiguous hCD79b (expanded view below the hGH locus map) and isolates this region from the hGH gene cluster. The −8.0CD79bΔ1.6 transgene was derived from the −8.0CD79b by deletion of a 1.6-kb internal fragment extending from −0.5 kb of the promoter through intron 2, removing all defined hCD79b promoter elements. ( C ) Noncoding transcription across hCD79b in the transgenic mouse pituitary is preserved in the absence of defined promoter elements. Transcription across the hCD79b region generated by the promoterless − 8.0CD79bΔ1.6 was compared to that of the − 8.0CD79b transgene (containing the intact hCD79b promoter but not the hGH-N promoter) and the hGH BAC transgene (containing both the hCD79b and the hGH-N promoters). Pituitary RNA from mice carrying each of these indicated transgenes was assayed for transcription across hCD79b by RT-qPCR. The data shown in the histogram (Y-axis) have been normalized to the corresponding transgene copy numbers and to somatotrope-specific levels of the endogenous mouse growth hormone ( mGH1 ) mRNA. The number of genetically distinct lines assessed for each transgene is noted above the corresponding bars in the histogram. Each line was tested in triplicate using three independent mice. Values represent the average ± SD. The level of hGH BAC was defined as 1.0. ( D ) Map of the 123 kb λΔCD/hGH transgene and the derivative the −8.0λΔCD transgene. This transgene was generated by replacing the hCD79b gene and promoter (to −500 bp) with a size-matched fragment of bacteriophage λ DNA within the context of an otherwise intact hGH BAC (see ‘Materials and Methods' section). The −8.0λΔCD transgene was released from the λΔCD/hGH transgene with boundaries as indicated. ( E ) Noncoding transcription is activated 3′ of HSI within an inserted (λ) DNA segment. Transcription across the λ-DNA region was compared between the λΔCD/hGH and −8.0λΔCD transgenes. λ noncoding transcription was measured by RT-qPCR, normalized to transgene copy-number and to mGH1 mRNA levels. λ noncoding transcription was actively transcribed without hGH-N promoter. The number of genetically distinct lines assessed for each transgene is noted above the corresponding bars in the histogram. Each experiment was carried out in triplicate. The study of the single −8.0λΔCD line was evaluated in each of three independent mice, each studied in triplicate. Values represent the average ± SD.

    Techniques Used: BAC Assay, Transgenic Assay, Labeling, Binding Assay, Derivative Assay, Generated, Mouse Assay, Quantitative RT-PCR

    6) Product Images from "Molecular Architecture of a Eukaryotic DNA Replication Terminus-Terminator Protein Complex "

    Article Title: Molecular Architecture of a Eukaryotic DNA Replication Terminus-Terminator Protein Complex

    Journal:

    doi: 10.1128/MCB.01102-06

    DNase I footprinting of wild-type and truncated His 6 -Sap1 mutants complexed with Ter1 or SAS1. (A) Sequence comparison of Ter1 and SAS1. Sap1 core motifs a, b, and c are highlighted in light gray, and directions of the repeats are depicted by black arrows
    Figure Legend Snippet: DNase I footprinting of wild-type and truncated His 6 -Sap1 mutants complexed with Ter1 or SAS1. (A) Sequence comparison of Ter1 and SAS1. Sap1 core motifs a, b, and c are highlighted in light gray, and directions of the repeats are depicted by black arrows

    Techniques Used: Footprinting, Sequencing

    Motif a of Ter1 acts as a nucleation center for assembly of the Sap1 dimer. (A) DNase I footprints of His 6 -Sap1 complexed with the top and bottom strands of the Ter1 Δ1 mutant. All lanes contain 30 fmol Ter1 or Ter mutant DNA. Lanes G+A,
    Figure Legend Snippet: Motif a of Ter1 acts as a nucleation center for assembly of the Sap1 dimer. (A) DNase I footprints of His 6 -Sap1 complexed with the top and bottom strands of the Ter1 Δ1 mutant. All lanes contain 30 fmol Ter1 or Ter mutant DNA. Lanes G+A,

    Techniques Used: Mutagenesis

    7) Product Images from "Intronic Cis-Regulatory Modules Mediate Tissue-Specific and Microbial Control of angptl4/fiaf Transcription"

    Article Title: Intronic Cis-Regulatory Modules Mediate Tissue-Specific and Microbial Control of angptl4/fiaf Transcription

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002585

    Non-overlapping regulatory modules within  angptl4  intron 3 confer liver, islet, and enterocyte-specific reporter expression. (A) Depiction of the 6 dpf zebrafish showing liver (li, green), intestine (in, blue), swim bladder (sb, grey), and muscle (mu, grey), with the fish oriented anterior (a) to the left and posterior (p) to the right. The opposite orientation reveals the exocrine pancreas (pa, yellow) and islet (is, orange). (B) Scaled schematic of the zebrafish  angptl4  locus and non-coding DNA assayed for regulatory potential. Modules are color coded according to the tissues in which they confer expression. Ratios of islet or intestine positive fish versus total fish expressing gfp are shown in parentheses next to truncation labels. (C–N) Representative images of GFP reporter expression in mosaic (column 1) and F 1  stable (column 2) animals driven by each non-coding DNA region (rows). Scale bars = 100 µm; li = liver, is = islet, in = intestine, sb = swim bladder. Colored arrowheads indicate tissue with specific reporter expression. (C–D) Full-length intron 3 (in3; 2,136 bp) is sufficient to promote expression of the reporter in the liver, islet (D, inset, scale bar = 50 µm), and intestine. (E–F) Truncation in3.1 (1,219 bp) confers expression in the liver. (G–H) Truncation in3.2 (701 bp) confers expression in both the intestine and islet (H, inset). Inset scale bar = 50 µm. (I–J) Truncation in3.3 (387 bp) confers islet expression. A transverse section (inset, J) reveals islet expression (nuclei stained with DAPI). Inset scale bar = 50 µm. (K–L) Truncation in3.4 (316 bp) confers intestinal expression. Insets in panels K and L contain transverse sections showing expression localized to the intestinal epithelium (nuclei stained with DAPI). Inset scale bar = 25 µm. The dotted lines in panels D, G, H, and I outline the pancreas. The white arrows in panels H, K, and L mark the boundary between the anterior intestine (segment 1) and mid-intestine (segment 2). (M–N) Cells expressing GFP driven by the in3.4 regulatory module colocalize with a marker (4E8, red, white arrow) of the brush border of absorptive enterocytes, but fail to co-localize with marker for secretory cells (2F11, red, asterisk). Nuclei stained with DAPI. Scale bars = 5 µm. (O) Intercross of  Tg(in3.2-Mmu.Fos:tdTomato)  with β-cell specific reporter line ( Tg(ins:CFP-NTR) s892 ) show colocalization of tdTomato and CFP in the islet. Scale bars = 10 µm. (P) Quantitative PCR shows that the in3.4 module and the  angptl4  promoter (TATA box), but not the in3.3 module, are hypersensitive to DNase I cleavage in intestinal epithelial cells isolated from adult zebrafish. Asterisks denote P-value
    Figure Legend Snippet: Non-overlapping regulatory modules within angptl4 intron 3 confer liver, islet, and enterocyte-specific reporter expression. (A) Depiction of the 6 dpf zebrafish showing liver (li, green), intestine (in, blue), swim bladder (sb, grey), and muscle (mu, grey), with the fish oriented anterior (a) to the left and posterior (p) to the right. The opposite orientation reveals the exocrine pancreas (pa, yellow) and islet (is, orange). (B) Scaled schematic of the zebrafish angptl4 locus and non-coding DNA assayed for regulatory potential. Modules are color coded according to the tissues in which they confer expression. Ratios of islet or intestine positive fish versus total fish expressing gfp are shown in parentheses next to truncation labels. (C–N) Representative images of GFP reporter expression in mosaic (column 1) and F 1 stable (column 2) animals driven by each non-coding DNA region (rows). Scale bars = 100 µm; li = liver, is = islet, in = intestine, sb = swim bladder. Colored arrowheads indicate tissue with specific reporter expression. (C–D) Full-length intron 3 (in3; 2,136 bp) is sufficient to promote expression of the reporter in the liver, islet (D, inset, scale bar = 50 µm), and intestine. (E–F) Truncation in3.1 (1,219 bp) confers expression in the liver. (G–H) Truncation in3.2 (701 bp) confers expression in both the intestine and islet (H, inset). Inset scale bar = 50 µm. (I–J) Truncation in3.3 (387 bp) confers islet expression. A transverse section (inset, J) reveals islet expression (nuclei stained with DAPI). Inset scale bar = 50 µm. (K–L) Truncation in3.4 (316 bp) confers intestinal expression. Insets in panels K and L contain transverse sections showing expression localized to the intestinal epithelium (nuclei stained with DAPI). Inset scale bar = 25 µm. The dotted lines in panels D, G, H, and I outline the pancreas. The white arrows in panels H, K, and L mark the boundary between the anterior intestine (segment 1) and mid-intestine (segment 2). (M–N) Cells expressing GFP driven by the in3.4 regulatory module colocalize with a marker (4E8, red, white arrow) of the brush border of absorptive enterocytes, but fail to co-localize with marker for secretory cells (2F11, red, asterisk). Nuclei stained with DAPI. Scale bars = 5 µm. (O) Intercross of Tg(in3.2-Mmu.Fos:tdTomato) with β-cell specific reporter line ( Tg(ins:CFP-NTR) s892 ) show colocalization of tdTomato and CFP in the islet. Scale bars = 10 µm. (P) Quantitative PCR shows that the in3.4 module and the angptl4 promoter (TATA box), but not the in3.3 module, are hypersensitive to DNase I cleavage in intestinal epithelial cells isolated from adult zebrafish. Asterisks denote P-value

    Techniques Used: Expressing, Fluorescence In Situ Hybridization, Staining, Marker, Real-time Polymerase Chain Reaction, Isolation

    8) Product Images from "Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis"

    Article Title: Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01680

    Deoxyribonuclease (DNase) I treatment abolished neutrophil extracellular traps (NETs) formation and ameliorated atherosclerotic burden. WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed on high-fat chow (HFC) for 6 weeks, starting at 3-week HFC, 400 U of DNase I or vehicle control (PBS) was intravenously administered three times weekly until the end of experiments.  (A)  Representative confocal immunofluorescence microscopy images of aortic root sections stained for DAPI (blue), MPO (green), Ly-6G (red), and Cit-H3 (cyan). Data are representative of five mice in each group.  (B)  Quantification of NETs from  (A)  ( n  = 5/group).  (C)  Representative images of aortic root sections stained for lipid (Oil Red O, red) and hematoxylin ( n  = 5/group).  (D)  mRNA levels of IL-1β, TNF-α, CCL2, CXCL1, and CXCL2 in the aorta from WT and PAD4 KO mice placed on HFC for 6 weeks and administered with DNase I or vehicle control (PBS). mRNA levels were normalized to the GAPDH and expressed relative to levels measured in one of the vehicle control-treated WT mice ( n  = 5/group). * p
    Figure Legend Snippet: Deoxyribonuclease (DNase) I treatment abolished neutrophil extracellular traps (NETs) formation and ameliorated atherosclerotic burden. WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed on high-fat chow (HFC) for 6 weeks, starting at 3-week HFC, 400 U of DNase I or vehicle control (PBS) was intravenously administered three times weekly until the end of experiments. (A) Representative confocal immunofluorescence microscopy images of aortic root sections stained for DAPI (blue), MPO (green), Ly-6G (red), and Cit-H3 (cyan). Data are representative of five mice in each group. (B) Quantification of NETs from (A) ( n  = 5/group). (C) Representative images of aortic root sections stained for lipid (Oil Red O, red) and hematoxylin ( n  = 5/group). (D) mRNA levels of IL-1β, TNF-α, CCL2, CXCL1, and CXCL2 in the aorta from WT and PAD4 KO mice placed on HFC for 6 weeks and administered with DNase I or vehicle control (PBS). mRNA levels were normalized to the GAPDH and expressed relative to levels measured in one of the vehicle control-treated WT mice ( n  = 5/group). * p

    Techniques Used: Gene Knockout, Mouse Assay, Immunofluorescence, Microscopy, Staining

    Neutrophil extracellular traps (NETs) present in atherosclerotic lesions stimulate inflammatory responses in arterial macrophages.  (A)  Bone marrow (BM)-derived neutrophils were stimulated in the absence (UN) or presence (A23187) of A23187 for 4 h. Half the UN-NETs or A23187-NETs were digested by deoxyribonuclease (DNase) I. NETs were quantified by measuring Cit-H3-DNA complexes on ELISA.  (B)  BM-derived macrophages were stimulated with UN-NETs (BMN-UN), UN-NETs treated with DNase I (BMN-UN-DNase I), A23187-NETs (BMN-A23), or A23187-NETs treated with DNase I (BMN-A23-DNase I) for 4 h. Gene expression levels of IL-1β, CCL2, CXCL1, and CXCL2 were determined. mRNA levels were normalized to  GAPDH  and expressed relative to levels measured in one of the BMN-UN conditions  (C) . WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed high-fat chow (HFC) for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: IL-1β, and magenta: Cit-H3. Data are representative of four mice in two independent experiments.  (D)  WT and PAD4 KO mice were fed HFC for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: CCL2, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. * p
    Figure Legend Snippet: Neutrophil extracellular traps (NETs) present in atherosclerotic lesions stimulate inflammatory responses in arterial macrophages. (A) Bone marrow (BM)-derived neutrophils were stimulated in the absence (UN) or presence (A23187) of A23187 for 4 h. Half the UN-NETs or A23187-NETs were digested by deoxyribonuclease (DNase) I. NETs were quantified by measuring Cit-H3-DNA complexes on ELISA. (B) BM-derived macrophages were stimulated with UN-NETs (BMN-UN), UN-NETs treated with DNase I (BMN-UN-DNase I), A23187-NETs (BMN-A23), or A23187-NETs treated with DNase I (BMN-A23-DNase I) for 4 h. Gene expression levels of IL-1β, CCL2, CXCL1, and CXCL2 were determined. mRNA levels were normalized to GAPDH and expressed relative to levels measured in one of the BMN-UN conditions (C) . WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed high-fat chow (HFC) for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: IL-1β, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. (D) WT and PAD4 KO mice were fed HFC for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: CCL2, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. * p

    Techniques Used: Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Gene Knockout, Mouse Assay, Staining, Immunofluorescence, Microscopy

    9) Product Images from "Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis"

    Article Title: Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01680

    Deoxyribonuclease (DNase) I treatment abolished neutrophil extracellular traps (NETs) formation and ameliorated atherosclerotic burden. WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed on high-fat chow (HFC) for 6 weeks, starting at 3-week HFC, 400 U of DNase I or vehicle control (PBS) was intravenously administered three times weekly until the end of experiments.  (A)  Representative confocal immunofluorescence microscopy images of aortic root sections stained for DAPI (blue), MPO (green), Ly-6G (red), and Cit-H3 (cyan). Data are representative of five mice in each group.  (B)  Quantification of NETs from  (A)  ( n  = 5/group).  (C)  Representative images of aortic root sections stained for lipid (Oil Red O, red) and hematoxylin ( n  = 5/group).  (D)  mRNA levels of IL-1β, TNF-α, CCL2, CXCL1, and CXCL2 in the aorta from WT and PAD4 KO mice placed on HFC for 6 weeks and administered with DNase I or vehicle control (PBS). mRNA levels were normalized to the GAPDH and expressed relative to levels measured in one of the vehicle control-treated WT mice ( n  = 5/group). * p
    Figure Legend Snippet: Deoxyribonuclease (DNase) I treatment abolished neutrophil extracellular traps (NETs) formation and ameliorated atherosclerotic burden. WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed on high-fat chow (HFC) for 6 weeks, starting at 3-week HFC, 400 U of DNase I or vehicle control (PBS) was intravenously administered three times weekly until the end of experiments. (A) Representative confocal immunofluorescence microscopy images of aortic root sections stained for DAPI (blue), MPO (green), Ly-6G (red), and Cit-H3 (cyan). Data are representative of five mice in each group. (B) Quantification of NETs from (A) ( n  = 5/group). (C) Representative images of aortic root sections stained for lipid (Oil Red O, red) and hematoxylin ( n  = 5/group). (D) mRNA levels of IL-1β, TNF-α, CCL2, CXCL1, and CXCL2 in the aorta from WT and PAD4 KO mice placed on HFC for 6 weeks and administered with DNase I or vehicle control (PBS). mRNA levels were normalized to the GAPDH and expressed relative to levels measured in one of the vehicle control-treated WT mice ( n  = 5/group). * p

    Techniques Used: Gene Knockout, Mouse Assay, Immunofluorescence, Microscopy, Staining

    Neutrophil extracellular traps (NETs) present in atherosclerotic lesions stimulate inflammatory responses in arterial macrophages.  (A)  Bone marrow (BM)-derived neutrophils were stimulated in the absence (UN) or presence (A23187) of A23187 for 4 h. Half the UN-NETs or A23187-NETs were digested by deoxyribonuclease (DNase) I. NETs were quantified by measuring Cit-H3-DNA complexes on ELISA.  (B)  BM-derived macrophages were stimulated with UN-NETs (BMN-UN), UN-NETs treated with DNase I (BMN-UN-DNase I), A23187-NETs (BMN-A23), or A23187-NETs treated with DNase I (BMN-A23-DNase I) for 4 h. Gene expression levels of IL-1β, CCL2, CXCL1, and CXCL2 were determined. mRNA levels were normalized to  GAPDH  and expressed relative to levels measured in one of the BMN-UN conditions  (C) . WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed high-fat chow (HFC) for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: IL-1β, and magenta: Cit-H3. Data are representative of four mice in two independent experiments.  (D)  WT and PAD4 KO mice were fed HFC for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: CCL2, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. * p
    Figure Legend Snippet: Neutrophil extracellular traps (NETs) present in atherosclerotic lesions stimulate inflammatory responses in arterial macrophages. (A) Bone marrow (BM)-derived neutrophils were stimulated in the absence (UN) or presence (A23187) of A23187 for 4 h. Half the UN-NETs or A23187-NETs were digested by deoxyribonuclease (DNase) I. NETs were quantified by measuring Cit-H3-DNA complexes on ELISA. (B) BM-derived macrophages were stimulated with UN-NETs (BMN-UN), UN-NETs treated with DNase I (BMN-UN-DNase I), A23187-NETs (BMN-A23), or A23187-NETs treated with DNase I (BMN-A23-DNase I) for 4 h. Gene expression levels of IL-1β, CCL2, CXCL1, and CXCL2 were determined. mRNA levels were normalized to GAPDH and expressed relative to levels measured in one of the BMN-UN conditions (C) . WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed high-fat chow (HFC) for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: IL-1β, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. (D) WT and PAD4 KO mice were fed HFC for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: CCL2, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. * p

    Techniques Used: Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Gene Knockout, Mouse Assay, Staining, Immunofluorescence, Microscopy

    10) Product Images from "Chemical perturbation of an intrinsically disordered region of TFIID distinguishes two modes of transcription initiation"

    Article Title: Chemical perturbation of an intrinsically disordered region of TFIID distinguishes two modes of transcription initiation

    Journal: eLife

    doi: 10.7554/eLife.07777

    DNase I footprinting assays monitoring PIC assembly and structural isomerization. ( A ) Early steps of PIC assembly. Specified GTFs were incubated with the end-labeled DNA template, followed by DNase I digestion. Blue bracket highlights the TFIID footprint. The numbers are relative to the transcription start site (+1). ( B ) Arresting of the conformational isomerization. Top, the scheme. The inhibitor used here was SnOCl 2 ·pyridine. Lanes 1 and 9 are 10 bp DNA ladder. Lanes 2 and 10 are digestion of naked DNA. The lower case ‘d’ and ‘f’ in lanes #11–14 reflect the use of less TFIID and TFIIF (together with the omission of spermidine and carrier nucleic acid in the reaction—see ‘Material and methods’ for detail). Letter abbreviations are explained in   Figure 6  legend. DOI: http://dx.doi.org/10.7554/eLife.07777.016
    Figure Legend Snippet: DNase I footprinting assays monitoring PIC assembly and structural isomerization. ( A ) Early steps of PIC assembly. Specified GTFs were incubated with the end-labeled DNA template, followed by DNase I digestion. Blue bracket highlights the TFIID footprint. The numbers are relative to the transcription start site (+1). ( B ) Arresting of the conformational isomerization. Top, the scheme. The inhibitor used here was SnOCl 2 ·pyridine. Lanes 1 and 9 are 10 bp DNA ladder. Lanes 2 and 10 are digestion of naked DNA. The lower case ‘d’ and ‘f’ in lanes #11–14 reflect the use of less TFIID and TFIIF (together with the omission of spermidine and carrier nucleic acid in the reaction—see ‘Material and methods’ for detail). Letter abbreviations are explained in Figure 6 legend. DOI: http://dx.doi.org/10.7554/eLife.07777.016

    Techniques Used: Footprinting, Incubation, Labeling

    ( A ) DNase I footprinting assay of TFIID-promoter binding and its enhancement by the specific inhibitor at different salt concentrations. ( B ) A non-specific inhibitor (Maybridge BTB08574): its structure (top) and inhibition of both TFIID- and TBP-directed transcription (bottom). The lead compound used here was ChemDiv 7241-4207. DOI: http://dx.doi.org/10.7554/eLife.07777.013
    Figure Legend Snippet: ( A ) DNase I footprinting assay of TFIID-promoter binding and its enhancement by the specific inhibitor at different salt concentrations. ( B ) A non-specific inhibitor (Maybridge BTB08574): its structure (top) and inhibition of both TFIID- and TBP-directed transcription (bottom). The lead compound used here was ChemDiv 7241-4207. DOI: http://dx.doi.org/10.7554/eLife.07777.013

    Techniques Used: Footprinting, Binding Assay, Inhibition

    A model of inhibition that mechanistically distinguishes the two modes of transcription initiation. ( A ) Initially, TFIID forms multiple contacts with an extended promoter DNA region that is stabilized by TFIIA and TFIIB. TFIIA doesn't affect transcription at this promoter with purified factors, but it does facilitate the TATA box protection by TFIID alone (  Cianfrocco et al., 2013 ) or by TFIID together with TFIIB (ZZ and RT unpublished). We propose a critical isomerization step during de novo PIC assembly involving a TFIID conformational change (i.e., release of at least part of the promoter DNA, illustrated by the change in the shape of TFIID) to allow entry and engagement of Pol II. Once Pol II becomes engaged and further stabilized by other factors (TFIIE, TFIIF, etc) transcription can proceed. ( B ) The inhibitor, by binding and interfering with the TAF2 IDR, arrests TFIID isomerization and Pol II engagement, thus, blocking the assembly of a functional PIC. DNase I footprint assay reveals that Pol II molecules can still partially interact with the downstream portion of promoter DNA in the presence of the inhibitor. ( C ) Once the first round of Pol II engagement is accomplished and isomerization has occurred, the PIC intermediate establishes a state resistant to inhibition. After Pol II enters the elongation phase, TFIID remains at the isomerized state as part of a reinitiation scaffold. This reinitiation complex bypasses the initial stages of de novo PIC assembly where TFIID contacts an extended DNA region and thus is resistant to the inhibition by the tin(IV) oxochloride cluster. In addition, this shortcut may be a mechanism for the reinitiation scaffold to facilitate reloading of more Pol II molecules. DOI: http://dx.doi.org/10.7554/eLife.07777.019
    Figure Legend Snippet: A model of inhibition that mechanistically distinguishes the two modes of transcription initiation. ( A ) Initially, TFIID forms multiple contacts with an extended promoter DNA region that is stabilized by TFIIA and TFIIB. TFIIA doesn't affect transcription at this promoter with purified factors, but it does facilitate the TATA box protection by TFIID alone ( Cianfrocco et al., 2013 ) or by TFIID together with TFIIB (ZZ and RT unpublished). We propose a critical isomerization step during de novo PIC assembly involving a TFIID conformational change (i.e., release of at least part of the promoter DNA, illustrated by the change in the shape of TFIID) to allow entry and engagement of Pol II. Once Pol II becomes engaged and further stabilized by other factors (TFIIE, TFIIF, etc) transcription can proceed. ( B ) The inhibitor, by binding and interfering with the TAF2 IDR, arrests TFIID isomerization and Pol II engagement, thus, blocking the assembly of a functional PIC. DNase I footprint assay reveals that Pol II molecules can still partially interact with the downstream portion of promoter DNA in the presence of the inhibitor. ( C ) Once the first round of Pol II engagement is accomplished and isomerization has occurred, the PIC intermediate establishes a state resistant to inhibition. After Pol II enters the elongation phase, TFIID remains at the isomerized state as part of a reinitiation scaffold. This reinitiation complex bypasses the initial stages of de novo PIC assembly where TFIID contacts an extended DNA region and thus is resistant to the inhibition by the tin(IV) oxochloride cluster. In addition, this shortcut may be a mechanism for the reinitiation scaffold to facilitate reloading of more Pol II molecules. DOI: http://dx.doi.org/10.7554/eLife.07777.019

    Techniques Used: Inhibition, Purification, Binding Assay, Blocking Assay, Functional Assay

    11) Product Images from "Chemical perturbation of an intrinsically disordered region of TFIID distinguishes two modes of transcription initiation"

    Article Title: Chemical perturbation of an intrinsically disordered region of TFIID distinguishes two modes of transcription initiation

    Journal: eLife

    doi: 10.7554/eLife.07777

    DNase I footprinting assays monitoring PIC assembly and structural isomerization. ( A ) Early steps of PIC assembly. Specified GTFs were incubated with the end-labeled DNA template, followed by DNase I digestion. Blue bracket highlights the TFIID footprint. The numbers are relative to the transcription start site (+1). ( B ) Arresting of the conformational isomerization. Top, the scheme. The inhibitor used here was SnOCl 2 ·pyridine. Lanes 1 and 9 are 10 bp DNA ladder. Lanes 2 and 10 are digestion of naked DNA. The lower case ‘d’ and ‘f’ in lanes #11–14 reflect the use of less TFIID and TFIIF (together with the omission of spermidine and carrier nucleic acid in the reaction—see ‘Material and methods’ for detail). Letter abbreviations are explained in   Figure 6  legend. DOI: http://dx.doi.org/10.7554/eLife.07777.016
    Figure Legend Snippet: DNase I footprinting assays monitoring PIC assembly and structural isomerization. ( A ) Early steps of PIC assembly. Specified GTFs were incubated with the end-labeled DNA template, followed by DNase I digestion. Blue bracket highlights the TFIID footprint. The numbers are relative to the transcription start site (+1). ( B ) Arresting of the conformational isomerization. Top, the scheme. The inhibitor used here was SnOCl 2 ·pyridine. Lanes 1 and 9 are 10 bp DNA ladder. Lanes 2 and 10 are digestion of naked DNA. The lower case ‘d’ and ‘f’ in lanes #11–14 reflect the use of less TFIID and TFIIF (together with the omission of spermidine and carrier nucleic acid in the reaction—see ‘Material and methods’ for detail). Letter abbreviations are explained in Figure 6 legend. DOI: http://dx.doi.org/10.7554/eLife.07777.016

    Techniques Used: Footprinting, Incubation, Labeling

    ( A ) DNase I footprinting assay of TFIID-promoter binding and its enhancement by the specific inhibitor at different salt concentrations. ( B ) A non-specific inhibitor (Maybridge BTB08574): its structure (top) and inhibition of both TFIID- and TBP-directed transcription (bottom). The lead compound used here was ChemDiv 7241-4207. DOI: http://dx.doi.org/10.7554/eLife.07777.013
    Figure Legend Snippet: ( A ) DNase I footprinting assay of TFIID-promoter binding and its enhancement by the specific inhibitor at different salt concentrations. ( B ) A non-specific inhibitor (Maybridge BTB08574): its structure (top) and inhibition of both TFIID- and TBP-directed transcription (bottom). The lead compound used here was ChemDiv 7241-4207. DOI: http://dx.doi.org/10.7554/eLife.07777.013

    Techniques Used: Footprinting, Binding Assay, Inhibition

    A model of inhibition that mechanistically distinguishes the two modes of transcription initiation. ( A ) Initially, TFIID forms multiple contacts with an extended promoter DNA region that is stabilized by TFIIA and TFIIB. TFIIA doesn't affect transcription at this promoter with purified factors, but it does facilitate the TATA box protection by TFIID alone (  Cianfrocco et al., 2013 ) or by TFIID together with TFIIB (ZZ and RT unpublished). We propose a critical isomerization step during de novo PIC assembly involving a TFIID conformational change (i.e., release of at least part of the promoter DNA, illustrated by the change in the shape of TFIID) to allow entry and engagement of Pol II. Once Pol II becomes engaged and further stabilized by other factors (TFIIE, TFIIF, etc) transcription can proceed. ( B ) The inhibitor, by binding and interfering with the TAF2 IDR, arrests TFIID isomerization and Pol II engagement, thus, blocking the assembly of a functional PIC. DNase I footprint assay reveals that Pol II molecules can still partially interact with the downstream portion of promoter DNA in the presence of the inhibitor. ( C ) Once the first round of Pol II engagement is accomplished and isomerization has occurred, the PIC intermediate establishes a state resistant to inhibition. After Pol II enters the elongation phase, TFIID remains at the isomerized state as part of a reinitiation scaffold. This reinitiation complex bypasses the initial stages of de novo PIC assembly where TFIID contacts an extended DNA region and thus is resistant to the inhibition by the tin(IV) oxochloride cluster. In addition, this shortcut may be a mechanism for the reinitiation scaffold to facilitate reloading of more Pol II molecules. DOI: http://dx.doi.org/10.7554/eLife.07777.019
    Figure Legend Snippet: A model of inhibition that mechanistically distinguishes the two modes of transcription initiation. ( A ) Initially, TFIID forms multiple contacts with an extended promoter DNA region that is stabilized by TFIIA and TFIIB. TFIIA doesn't affect transcription at this promoter with purified factors, but it does facilitate the TATA box protection by TFIID alone ( Cianfrocco et al., 2013 ) or by TFIID together with TFIIB (ZZ and RT unpublished). We propose a critical isomerization step during de novo PIC assembly involving a TFIID conformational change (i.e., release of at least part of the promoter DNA, illustrated by the change in the shape of TFIID) to allow entry and engagement of Pol II. Once Pol II becomes engaged and further stabilized by other factors (TFIIE, TFIIF, etc) transcription can proceed. ( B ) The inhibitor, by binding and interfering with the TAF2 IDR, arrests TFIID isomerization and Pol II engagement, thus, blocking the assembly of a functional PIC. DNase I footprint assay reveals that Pol II molecules can still partially interact with the downstream portion of promoter DNA in the presence of the inhibitor. ( C ) Once the first round of Pol II engagement is accomplished and isomerization has occurred, the PIC intermediate establishes a state resistant to inhibition. After Pol II enters the elongation phase, TFIID remains at the isomerized state as part of a reinitiation scaffold. This reinitiation complex bypasses the initial stages of de novo PIC assembly where TFIID contacts an extended DNA region and thus is resistant to the inhibition by the tin(IV) oxochloride cluster. In addition, this shortcut may be a mechanism for the reinitiation scaffold to facilitate reloading of more Pol II molecules. DOI: http://dx.doi.org/10.7554/eLife.07777.019

    Techniques Used: Inhibition, Purification, Binding Assay, Blocking Assay, Functional Assay

    12) Product Images from "Chemical perturbation of an intrinsically disordered region of TFIID distinguishes two modes of transcription initiation"

    Article Title: Chemical perturbation of an intrinsically disordered region of TFIID distinguishes two modes of transcription initiation

    Journal: eLife

    doi: 10.7554/eLife.07777

    DNase I footprinting assays monitoring PIC assembly and structural isomerization. ( A ) Early steps of PIC assembly. Specified GTFs were incubated with the end-labeled DNA template, followed by DNase I digestion. Blue bracket highlights the TFIID footprint. The numbers are relative to the transcription start site (+1). ( B ) Arresting of the conformational isomerization. Top, the scheme. The inhibitor used here was SnOCl 2 ·pyridine. Lanes 1 and 9 are 10 bp DNA ladder. Lanes 2 and 10 are digestion of naked DNA. The lower case ‘d’ and ‘f’ in lanes #11–14 reflect the use of less TFIID and TFIIF (together with the omission of spermidine and carrier nucleic acid in the reaction—see ‘Material and methods’ for detail). Letter abbreviations are explained in   Figure 6  legend. DOI: http://dx.doi.org/10.7554/eLife.07777.016
    Figure Legend Snippet: DNase I footprinting assays monitoring PIC assembly and structural isomerization. ( A ) Early steps of PIC assembly. Specified GTFs were incubated with the end-labeled DNA template, followed by DNase I digestion. Blue bracket highlights the TFIID footprint. The numbers are relative to the transcription start site (+1). ( B ) Arresting of the conformational isomerization. Top, the scheme. The inhibitor used here was SnOCl 2 ·pyridine. Lanes 1 and 9 are 10 bp DNA ladder. Lanes 2 and 10 are digestion of naked DNA. The lower case ‘d’ and ‘f’ in lanes #11–14 reflect the use of less TFIID and TFIIF (together with the omission of spermidine and carrier nucleic acid in the reaction—see ‘Material and methods’ for detail). Letter abbreviations are explained in Figure 6 legend. DOI: http://dx.doi.org/10.7554/eLife.07777.016

    Techniques Used: Footprinting, Incubation, Labeling

    ( A ) DNase I footprinting assay of TFIID-promoter binding and its enhancement by the specific inhibitor at different salt concentrations. ( B ) A non-specific inhibitor (Maybridge BTB08574): its structure (top) and inhibition of both TFIID- and TBP-directed transcription (bottom). The lead compound used here was ChemDiv 7241-4207. DOI: http://dx.doi.org/10.7554/eLife.07777.013
    Figure Legend Snippet: ( A ) DNase I footprinting assay of TFIID-promoter binding and its enhancement by the specific inhibitor at different salt concentrations. ( B ) A non-specific inhibitor (Maybridge BTB08574): its structure (top) and inhibition of both TFIID- and TBP-directed transcription (bottom). The lead compound used here was ChemDiv 7241-4207. DOI: http://dx.doi.org/10.7554/eLife.07777.013

    Techniques Used: Footprinting, Binding Assay, Inhibition

    A model of inhibition that mechanistically distinguishes the two modes of transcription initiation. ( A ) Initially, TFIID forms multiple contacts with an extended promoter DNA region that is stabilized by TFIIA and TFIIB. TFIIA doesn't affect transcription at this promoter with purified factors, but it does facilitate the TATA box protection by TFIID alone (  Cianfrocco et al., 2013 ) or by TFIID together with TFIIB (ZZ and RT unpublished). We propose a critical isomerization step during de novo PIC assembly involving a TFIID conformational change (i.e., release of at least part of the promoter DNA, illustrated by the change in the shape of TFIID) to allow entry and engagement of Pol II. Once Pol II becomes engaged and further stabilized by other factors (TFIIE, TFIIF, etc) transcription can proceed. ( B ) The inhibitor, by binding and interfering with the TAF2 IDR, arrests TFIID isomerization and Pol II engagement, thus, blocking the assembly of a functional PIC. DNase I footprint assay reveals that Pol II molecules can still partially interact with the downstream portion of promoter DNA in the presence of the inhibitor. ( C ) Once the first round of Pol II engagement is accomplished and isomerization has occurred, the PIC intermediate establishes a state resistant to inhibition. After Pol II enters the elongation phase, TFIID remains at the isomerized state as part of a reinitiation scaffold. This reinitiation complex bypasses the initial stages of de novo PIC assembly where TFIID contacts an extended DNA region and thus is resistant to the inhibition by the tin(IV) oxochloride cluster. In addition, this shortcut may be a mechanism for the reinitiation scaffold to facilitate reloading of more Pol II molecules. DOI: http://dx.doi.org/10.7554/eLife.07777.019
    Figure Legend Snippet: A model of inhibition that mechanistically distinguishes the two modes of transcription initiation. ( A ) Initially, TFIID forms multiple contacts with an extended promoter DNA region that is stabilized by TFIIA and TFIIB. TFIIA doesn't affect transcription at this promoter with purified factors, but it does facilitate the TATA box protection by TFIID alone ( Cianfrocco et al., 2013 ) or by TFIID together with TFIIB (ZZ and RT unpublished). We propose a critical isomerization step during de novo PIC assembly involving a TFIID conformational change (i.e., release of at least part of the promoter DNA, illustrated by the change in the shape of TFIID) to allow entry and engagement of Pol II. Once Pol II becomes engaged and further stabilized by other factors (TFIIE, TFIIF, etc) transcription can proceed. ( B ) The inhibitor, by binding and interfering with the TAF2 IDR, arrests TFIID isomerization and Pol II engagement, thus, blocking the assembly of a functional PIC. DNase I footprint assay reveals that Pol II molecules can still partially interact with the downstream portion of promoter DNA in the presence of the inhibitor. ( C ) Once the first round of Pol II engagement is accomplished and isomerization has occurred, the PIC intermediate establishes a state resistant to inhibition. After Pol II enters the elongation phase, TFIID remains at the isomerized state as part of a reinitiation scaffold. This reinitiation complex bypasses the initial stages of de novo PIC assembly where TFIID contacts an extended DNA region and thus is resistant to the inhibition by the tin(IV) oxochloride cluster. In addition, this shortcut may be a mechanism for the reinitiation scaffold to facilitate reloading of more Pol II molecules. DOI: http://dx.doi.org/10.7554/eLife.07777.019

    Techniques Used: Inhibition, Purification, Binding Assay, Blocking Assay, Functional Assay

    13) Product Images from "Silencing of toxic gene expression by Fis"

    Article Title: Silencing of toxic gene expression by Fis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks037

    Model showing Fis-mediated silencing of  mom.  The rightmost gene in the Mu genome is  mom . P mom , the regulatory region of  mom  has been enlarged. The −35 and −10 promoter elements of P mom  are boxed. The dashed arrows indicate binding of Fis (small oval) to multiple sites in the promoter (as shown by DNase I footprinting). RNAP (large oval) cannot bind to P mom  because Fis is occluding the RNAP binding region. The Fis binding sites implicated in interfering with RNAP binding and thus with transcription are indicated by thickened arrows. Effect of Fis on other regions of the genome (see ‘Discussion’ section) is not shown.
    Figure Legend Snippet: Model showing Fis-mediated silencing of mom. The rightmost gene in the Mu genome is mom . P mom , the regulatory region of mom has been enlarged. The −35 and −10 promoter elements of P mom are boxed. The dashed arrows indicate binding of Fis (small oval) to multiple sites in the promoter (as shown by DNase I footprinting). RNAP (large oval) cannot bind to P mom because Fis is occluding the RNAP binding region. The Fis binding sites implicated in interfering with RNAP binding and thus with transcription are indicated by thickened arrows. Effect of Fis on other regions of the genome (see ‘Discussion’ section) is not shown.

    Techniques Used: Binding Assay, Footprinting

    Footprinting analysis of Fis binding at P mom . ( A ) DNase I footprinting analysis of Fis binding sites in P mom .  Plasmid pUW4 was incubated with Fis (288 and 384 nM in lanes 2 and 3, respectively) prior to digestion with DNase I. Primer extension was carried out with Klenow polymerase using  mom  reverse primer. The vertical bars on the left side indicate regions protected from DNase I in the presence of Fis. G, A, T and C refer to Sanger's dideoxy sequencing ladders of P mom  obtained using  mom  reverse primer. Numbers on the right denote nucleotide positions with respect to the +1 start site of P mom . DNase I hypersensitive sites are depicted by arrowheads. ( B ) Sequence of  mom  promoter. Regions protected by Fis are underlined and sequences matching with the consensus Fis binding site are highlighted with the central nucleotide emboldened and its position indicated. The −35 and −10 promoter elements are boxed.
    Figure Legend Snippet: Footprinting analysis of Fis binding at P mom . ( A ) DNase I footprinting analysis of Fis binding sites in P mom . Plasmid pUW4 was incubated with Fis (288 and 384 nM in lanes 2 and 3, respectively) prior to digestion with DNase I. Primer extension was carried out with Klenow polymerase using mom reverse primer. The vertical bars on the left side indicate regions protected from DNase I in the presence of Fis. G, A, T and C refer to Sanger's dideoxy sequencing ladders of P mom obtained using mom reverse primer. Numbers on the right denote nucleotide positions with respect to the +1 start site of P mom . DNase I hypersensitive sites are depicted by arrowheads. ( B ) Sequence of mom promoter. Regions protected by Fis are underlined and sequences matching with the consensus Fis binding site are highlighted with the central nucleotide emboldened and its position indicated. The −35 and −10 promoter elements are boxed.

    Techniques Used: Footprinting, Binding Assay, Plasmid Preparation, Incubation, Sequencing

    14) Product Images from "In vitro selection of an XNA aptamer capable of small-molecule recognition"

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky667

    TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.
    Figure Legend Snippet: TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.

    Techniques Used: Binding Assay, Amplification, Modification, Polyacrylamide Gel Electrophoresis, Selection, Incubation, Magnetic Beads, Polymerase Chain Reaction

    Comparison of the biostability of FAM-labeled TNA aptamer A04T.2 and DNA aptamer A08. ( A ) Denaturing PAGE analysis of the TNA (T) and DNA (D) aptamers after incubation in conditions of increasing nuclease stringency: selection buffer (control), 1.5 U DNase I, 50% human blood serum in PBS, and 0.5 mg/mL human liver microsomes. Samples were incubated under these conditions for 3 days at 37°C. ( B ) Bead-binding assay to determine retention of aptamer binding in the presence of nucleases. Each column and error bar represents the average and standard deviation of two trials.
    Figure Legend Snippet: Comparison of the biostability of FAM-labeled TNA aptamer A04T.2 and DNA aptamer A08. ( A ) Denaturing PAGE analysis of the TNA (T) and DNA (D) aptamers after incubation in conditions of increasing nuclease stringency: selection buffer (control), 1.5 U DNase I, 50% human blood serum in PBS, and 0.5 mg/mL human liver microsomes. Samples were incubated under these conditions for 3 days at 37°C. ( B ) Bead-binding assay to determine retention of aptamer binding in the presence of nucleases. Each column and error bar represents the average and standard deviation of two trials.

    Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Incubation, Selection, Binding Assay, Standard Deviation

    15) Product Images from "In vitro selection of an XNA aptamer capable of small-molecule recognition"

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky667

    TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.
    Figure Legend Snippet: TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.

    Techniques Used: Binding Assay, Amplification, Modification, Polyacrylamide Gel Electrophoresis, Selection, Incubation, Magnetic Beads, Polymerase Chain Reaction

    Comparison of the biostability of FAM-labeled TNA aptamer A04T.2 and DNA aptamer A08. ( A ) Denaturing PAGE analysis of the TNA (T) and DNA (D) aptamers after incubation in conditions of increasing nuclease stringency: selection buffer (control), 1.5 U DNase I, 50% human blood serum in PBS, and 0.5 mg/mL human liver microsomes. Samples were incubated under these conditions for 3 days at 37°C. ( B ) Bead-binding assay to determine retention of aptamer binding in the presence of nucleases. Each column and error bar represents the average and standard deviation of two trials.
    Figure Legend Snippet: Comparison of the biostability of FAM-labeled TNA aptamer A04T.2 and DNA aptamer A08. ( A ) Denaturing PAGE analysis of the TNA (T) and DNA (D) aptamers after incubation in conditions of increasing nuclease stringency: selection buffer (control), 1.5 U DNase I, 50% human blood serum in PBS, and 0.5 mg/mL human liver microsomes. Samples were incubated under these conditions for 3 days at 37°C. ( B ) Bead-binding assay to determine retention of aptamer binding in the presence of nucleases. Each column and error bar represents the average and standard deviation of two trials.

    Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Incubation, Selection, Binding Assay, Standard Deviation

    16) Product Images from "In the Staphylococcus aureus Two-Component System sae, the Response Regulator SaeR Binds to a Direct Repeat Sequence and DNA Binding Requires Phosphorylation by the Sensor Kinase SaeS"

    Article Title: In the Staphylococcus aureus Two-Component System sae, the Response Regulator SaeR Binds to a Direct Repeat Sequence and DNA Binding Requires Phosphorylation by the Sensor Kinase SaeS

    Journal:

    doi: 10.1128/JB.01524-09

    Identification of SaeR binding sequences. (A) DNase I footprinting analysis of the P1 promoter with SaeR C and P-SaeR. Sequencing of the DNA probe was carried out by the Maxam-Gilbert method. The nucleotide positions are indicated to the left of the footprinting
    Figure Legend Snippet: Identification of SaeR binding sequences. (A) DNase I footprinting analysis of the P1 promoter with SaeR C and P-SaeR. Sequencing of the DNA probe was carried out by the Maxam-Gilbert method. The nucleotide positions are indicated to the left of the footprinting

    Techniques Used: Binding Assay, Footprinting, Sequencing

    DNase I footprinting assays identify the SaeR binding sequence.
    Figure Legend Snippet: DNase I footprinting assays identify the SaeR binding sequence.

    Techniques Used: Footprinting, Binding Assay, Sequencing

    DNase I footprinting assays identify the SaeR binding sequence.
    Figure Legend Snippet: DNase I footprinting assays identify the SaeR binding sequence.

    Techniques Used: Footprinting, Binding Assay, Sequencing

    17) Product Images from "Transcriptional Analysis of the grlRA Virulence Operon from Citrobacter rodentium "

    Article Title: Transcriptional Analysis of the grlRA Virulence Operon from Citrobacter rodentium

    Journal:

    doi: 10.1128/JB.01540-09

    Binding of Ler to the grlRA regulatory region. (A) A DNase I footprinting assay was performed on a 234-bp DNA fragment spanning positions −398 to −165 relative to the transcription start site of grlRA (Fig. ). Samples
    Figure Legend Snippet: Binding of Ler to the grlRA regulatory region. (A) A DNase I footprinting assay was performed on a 234-bp DNA fragment spanning positions −398 to −165 relative to the transcription start site of grlRA (Fig. ). Samples

    Techniques Used: Binding Assay, Footprinting

    Binding of Ler to the grlA regulatory region. DNase I footprinting assays were performed on the bottom (A) and top (B) strands of a DNA fragment spanning 294 nucleotides between positions −364 and −69 relative to the transcription start
    Figure Legend Snippet: Binding of Ler to the grlA regulatory region. DNase I footprinting assays were performed on the bottom (A) and top (B) strands of a DNA fragment spanning 294 nucleotides between positions −364 and −69 relative to the transcription start

    Techniques Used: Binding Assay, Footprinting

    18) Product Images from "Identification and Characterization of Novel Helicobacter pylori apo-Fur-Regulated Target Genes"

    Article Title: Identification and Characterization of Novel Helicobacter pylori apo-Fur-Regulated Target Genes

    Journal:

    doi: 10.1128/JB.01026-13

    DNase I footprinting of the hydA promoter. A fragment of the hydA promoter fluorescently labeled at the 5′ end was subjected to DNase I digestion in the absence and presence of apo -Fur (A). Protected regions are those with reduced peak height
    Figure Legend Snippet: DNase I footprinting of the hydA promoter. A fragment of the hydA promoter fluorescently labeled at the 5′ end was subjected to DNase I digestion in the absence and presence of apo -Fur (A). Protected regions are those with reduced peak height

    Techniques Used: Footprinting, Labeling

    DNase I footprinting of the pfr promoter. A fragment of the pfr promoter fluorescently labeled at the 3′ end was subjected to DNase I digestion in the absence and presence of apo -Fur (A). A fragment of the pfr promoter fluorescently labeled at
    Figure Legend Snippet: DNase I footprinting of the pfr promoter. A fragment of the pfr promoter fluorescently labeled at the 3′ end was subjected to DNase I digestion in the absence and presence of apo -Fur (A). A fragment of the pfr promoter fluorescently labeled at

    Techniques Used: Footprinting, Labeling

    19) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.105411

    Effect of phosphomimetic RPA-32D8 on WRN-E84A-mediated fork regression. A , RPA-32D8 (0.04–8.0 fmol) was incubated with the fork substrate (1 fmol) for 10 min at 25 °C, and DNA binding of RPA-32D8 was analyzed by EMSA as described. The positions of the RPA-32D8-fork complexes and the fork substrate are indicated at the  left. B , for experiments as presented in  A , the amount of fork substrate bound was determined by comparing the amount of bound DNA to the total DNA for each reaction and plotted  versus  RPA-32D8 concentration. Each data point is the average of two independent experiments.  C , binding of RPA-32D8 (1.0–32 fmol) to the fork substrate (5 fmol) is analyzed by DNase I footprinting as described under “Experimental Procedures.” Position of the markers, the boundary of the leading arm gap ( dashed bracket ), and the area of protection by RPA-32D8 ( solid bracket ) is denoted on the  right. D , in fork regression assays, shown is the fork substrate (1 fmol) with or without RPA-32D8 (0.2–8.0 fmol) for 5 min at 4 °C followed by the addition of WRN-E84A (3.5 fmol), except where indicated, and further incubation at 37 °C for 15 min. The position of individual DNA species is noted on the  left  and for parental ( PD ) and daughter duplexes ( DD ) by  arrowheads  also on the  right. E , for WRN-mediated fork regression reactions as depicted in  D , amounts of daughter duplexes and products of forward unwinding were quantitated as described, and the data are plotted as a  bar graph  showing the amounts of these products with respect to RPA-32D8 concentration.
    Figure Legend Snippet: Effect of phosphomimetic RPA-32D8 on WRN-E84A-mediated fork regression. A , RPA-32D8 (0.04–8.0 fmol) was incubated with the fork substrate (1 fmol) for 10 min at 25 °C, and DNA binding of RPA-32D8 was analyzed by EMSA as described. The positions of the RPA-32D8-fork complexes and the fork substrate are indicated at the left. B , for experiments as presented in A , the amount of fork substrate bound was determined by comparing the amount of bound DNA to the total DNA for each reaction and plotted versus RPA-32D8 concentration. Each data point is the average of two independent experiments. C , binding of RPA-32D8 (1.0–32 fmol) to the fork substrate (5 fmol) is analyzed by DNase I footprinting as described under “Experimental Procedures.” Position of the markers, the boundary of the leading arm gap ( dashed bracket ), and the area of protection by RPA-32D8 ( solid bracket ) is denoted on the right. D , in fork regression assays, shown is the fork substrate (1 fmol) with or without RPA-32D8 (0.2–8.0 fmol) for 5 min at 4 °C followed by the addition of WRN-E84A (3.5 fmol), except where indicated, and further incubation at 37 °C for 15 min. The position of individual DNA species is noted on the left and for parental ( PD ) and daughter duplexes ( DD ) by arrowheads also on the right. E , for WRN-mediated fork regression reactions as depicted in D , amounts of daughter duplexes and products of forward unwinding were quantitated as described, and the data are plotted as a bar graph showing the amounts of these products with respect to RPA-32D8 concentration.

    Techniques Used: Recombinase Polymerase Amplification, Incubation, Binding Assay, Concentration Assay, Footprinting

    RPA-wt binding to a replication fork substrate containing a single-stranded gap on the leading arm. A , the model replication fork substrate generated by two-step annealing of leadP122, lagP122, leadD52, and lagD82 oligonucleotides as indicated that contains a 32-nt single-stranded gap on the leading arm is shown, with parental and daughter strands depicted in  black  and  light gray , respectively (for construction details and oligo sequences, see “Experimental Procedures” and  supplemental Table 1 , respectively). The parental-daughter arms were completely homologous except for a 5-nt non-complementary region ( dark gray ) at the fork junction to prevent spontaneous branch migration. The relevant lengths of the duplex and single-stranded regions are indicated.  B , RPA-wt (0–5.0 fmol) was incubated with the fork substrate (1 fmol) for 10 min at 25 °C, and binding of RPA to the substrate was analyzed by EMSA as described under “Experimental Procedures.” The positions of the RPA-fork complexes and the fork substrate are indicated at the  left. C , graphic representation of RPA binding to the fork substrate, derived from experiments performed as in  B , calculated as the percentage of Fork-RPA complex compared with the total DNA for each reaction is shown. Data points are the mean of three independent experiments, except for values at 0.5 and 1 fmol of RPA-wt (four independent experiments).  D , shown is RPA-wt (0.1–5 fmol) binding to the fork substrate (5 fmol) analyzed by DNase I footprinting as described under “Experimental Procedures.” The sizes of the markers ( lane 10 ) and the boundaries of the leading arm gap ( dashed bracket ) and of the area of protection by RPA-wt ( solid bracket ) are denoted at the  right .
    Figure Legend Snippet: RPA-wt binding to a replication fork substrate containing a single-stranded gap on the leading arm. A , the model replication fork substrate generated by two-step annealing of leadP122, lagP122, leadD52, and lagD82 oligonucleotides as indicated that contains a 32-nt single-stranded gap on the leading arm is shown, with parental and daughter strands depicted in black and light gray , respectively (for construction details and oligo sequences, see “Experimental Procedures” and supplemental Table 1 , respectively). The parental-daughter arms were completely homologous except for a 5-nt non-complementary region ( dark gray ) at the fork junction to prevent spontaneous branch migration. The relevant lengths of the duplex and single-stranded regions are indicated. B , RPA-wt (0–5.0 fmol) was incubated with the fork substrate (1 fmol) for 10 min at 25 °C, and binding of RPA to the substrate was analyzed by EMSA as described under “Experimental Procedures.” The positions of the RPA-fork complexes and the fork substrate are indicated at the left. C , graphic representation of RPA binding to the fork substrate, derived from experiments performed as in B , calculated as the percentage of Fork-RPA complex compared with the total DNA for each reaction is shown. Data points are the mean of three independent experiments, except for values at 0.5 and 1 fmol of RPA-wt (four independent experiments). D , shown is RPA-wt (0.1–5 fmol) binding to the fork substrate (5 fmol) analyzed by DNase I footprinting as described under “Experimental Procedures.” The sizes of the markers ( lane 10 ) and the boundaries of the leading arm gap ( dashed bracket ) and of the area of protection by RPA-wt ( solid bracket ) are denoted at the right .

    Techniques Used: Recombinase Polymerase Amplification, Binding Assay, Generated, Migration, Incubation, Derivative Assay, Footprinting

    20) Product Images from "Synthesizing topological structures containing RNA"

    Article Title: Synthesizing topological structures containing RNA

    Journal: Nature Communications

    doi: 10.1038/ncomms14936

    Design and construction of a ds DNA–RNA hybrid knot. ( a – c ) Three views of 3D helical model of the ds DNA–RNA hybrid knot: along the threefold rotation axis ( a ), the axis of the outer ( b ) and inner ( c ) helices. The template DNA strand is shown in red and the complementary RNA strand in blue. Also see  Supplementary Fig. 3  for stereo views. ( d , e ) AFM images for the ds hybrid circle ( d ) and knot ( e ). Scale bar, 50 nm. ( f ) Nuclease digestion confirming the formation of the hybrid knot. Lanes 1–3, purified ds hybrid knot undigested (lane 1), digested by DNase I (lane 2) or by RNase H (lane 3). Lanes 4–6, purified ds hybrid circle undigested (lane 4), digested by DNase I (lane 5) or by RNase H (lane 6). Lanes 7 and 8, ssDNA references of the ssDNA template knot and circle. Lanes 9 and 10, ssRNA references ( TK j ( + ) and  C j ), which are of the same size with the RNA strand in the ds hybrid structures but a different sequence.
    Figure Legend Snippet: Design and construction of a ds DNA–RNA hybrid knot. ( a – c ) Three views of 3D helical model of the ds DNA–RNA hybrid knot: along the threefold rotation axis ( a ), the axis of the outer ( b ) and inner ( c ) helices. The template DNA strand is shown in red and the complementary RNA strand in blue. Also see Supplementary Fig. 3 for stereo views. ( d , e ) AFM images for the ds hybrid circle ( d ) and knot ( e ). Scale bar, 50 nm. ( f ) Nuclease digestion confirming the formation of the hybrid knot. Lanes 1–3, purified ds hybrid knot undigested (lane 1), digested by DNase I (lane 2) or by RNase H (lane 3). Lanes 4–6, purified ds hybrid circle undigested (lane 4), digested by DNase I (lane 5) or by RNase H (lane 6). Lanes 7 and 8, ssDNA references of the ssDNA template knot and circle. Lanes 9 and 10, ssRNA references ( TK j ( + ) and C j ), which are of the same size with the RNA strand in the ds hybrid structures but a different sequence.

    Techniques Used: Purification, Sequencing

    Constructing ssRNA topological structures with the junction-based method. ( a ) Preparation of the ssRNA strand with uniform ends and proper end groups for the DNA-splinted RNA ligation. ( b ) Both the positive ( TK j ( + )) and negative ( TK j ( − )) RNA trefoil knots of the same sequence are constructed by configuring tensegrity triangles with different handedness. The same scaffolds (blue) are threaded by different staple sets (grey or purple) to form a 17-bp-edged right-handed tensegrity triangle for the positive trefoil knot, or a 14-bp-edged left-handed tensegrity triangle for the negative one, respectively. Each topology is designated by the Alexander–Briggs notation  n i  or  , where  n  is the minimal number of nodes,  C  is the number of components (for links), and  i  distinguishes different topologies with the same  n  and  C . ( c ) dPAGE analysis of  TK j ( + ) (lanes 1 and 2),  TK j ( − ) (lanes 3 and 4), and their circular ( C j , lanes 5 and 6) and linear ( L j , lanes 7 and 8) counterparts. Lanes 2, 4, 6 and 8 contain samples digested by RNase R. ( d ) The assembly complex for the hybrid BR contains tensegrity triangles of both handedness to generate three positive nodes plus three negative nodes. ( e ) Topological analyses of the hybrid BR. Lane 1, gel-purified BR; lanes 2–6, BR treated by RNase H, Nt.AlwI (for cleaving the red ring), Nt.BspQI (for cleaving the green ring), DNase I, and  E. coli  DNA Topo I; lanes 7 and 8, DNA and RNA references of the three individual components. During the purification of BR, the breaking down of the 95-nt circular RNA component is unavoidable, and a portion of BR falls apart as a result. The treatment of BR by RNase H, Nt.AlwI and Nt.BspQI is conducted in the presence of an assisting DNA strand complementary to the corresponding ssRNA or ssDNA ring. LX and CX represent X-nt linear and circular species, respectively. In all the gels, lane M contains the DNA size markers.
    Figure Legend Snippet: Constructing ssRNA topological structures with the junction-based method. ( a ) Preparation of the ssRNA strand with uniform ends and proper end groups for the DNA-splinted RNA ligation. ( b ) Both the positive ( TK j ( + )) and negative ( TK j ( − )) RNA trefoil knots of the same sequence are constructed by configuring tensegrity triangles with different handedness. The same scaffolds (blue) are threaded by different staple sets (grey or purple) to form a 17-bp-edged right-handed tensegrity triangle for the positive trefoil knot, or a 14-bp-edged left-handed tensegrity triangle for the negative one, respectively. Each topology is designated by the Alexander–Briggs notation n i or , where n is the minimal number of nodes, C is the number of components (for links), and i distinguishes different topologies with the same n and C . ( c ) dPAGE analysis of TK j ( + ) (lanes 1 and 2), TK j ( − ) (lanes 3 and 4), and their circular ( C j , lanes 5 and 6) and linear ( L j , lanes 7 and 8) counterparts. Lanes 2, 4, 6 and 8 contain samples digested by RNase R. ( d ) The assembly complex for the hybrid BR contains tensegrity triangles of both handedness to generate three positive nodes plus three negative nodes. ( e ) Topological analyses of the hybrid BR. Lane 1, gel-purified BR; lanes 2–6, BR treated by RNase H, Nt.AlwI (for cleaving the red ring), Nt.BspQI (for cleaving the green ring), DNase I, and E. coli DNA Topo I; lanes 7 and 8, DNA and RNA references of the three individual components. During the purification of BR, the breaking down of the 95-nt circular RNA component is unavoidable, and a portion of BR falls apart as a result. The treatment of BR by RNase H, Nt.AlwI and Nt.BspQI is conducted in the presence of an assisting DNA strand complementary to the corresponding ssRNA or ssDNA ring. LX and CX represent X-nt linear and circular species, respectively. In all the gels, lane M contains the DNA size markers.

    Techniques Used: Ligation, Sequencing, Construct, Purification

    Strategies of constructing ssRNA topological structures. ( a , b ) Seeman's method of using A-form RNA helix to generate ssRNA topological structures 21 . In  a , one turn of an A-form RNA helix is shown with the helical and schematic representations. The two component strands form two negative nodes within this one-turn helix. In  b , a strand of ssRNA (orange) is designed to contain alternating complementary pairing segments to form two one-turn A-form helices, and a trefoil knot is formed after enzymatic ligation aided by a DNA splint (green). However, topological structures constructed in this way contain very strong intrastrand base pairings. ( c , d ) Junction-based method to generate ssRNA topological structures. In  c , a 4WJ is formed with two RNA strands (blue) as the helical strands and two DNA strands (grey) as the crossover strands, and is shown with the helical and schematic representations. The two RNA helical strands generate a node. In  d , the assembly complex for the trefoil knot is formed, where the RNA scaffolds (blue) are threaded into the targeted topology by DNA staples (grey) and linked end to end by DNA splints (green). After ligation and subsequent removal of the DNA staples and splints, a ssRNA knot free of strong intrastrand base pairings is generated. ( e ) The ssDNA trefoil knot can be used as a template for the construction of ssRNA trefoil knot. The ssDNA knot template (red) is pre-prepared and annealed with the complementary RNA strands (blue). After ligation, the DNA–RNA hybrid knot is formed. The ds DNA–RNA hybrid is more rigid than single-stranded structure, the careful design of curvature (by adding bulges) and torsion (by adjusting the length of hybrid helix) is necessary. The hybrid knot can then be subjected to DNase I digestion to obtain the ssRNA knot.
    Figure Legend Snippet: Strategies of constructing ssRNA topological structures. ( a , b ) Seeman's method of using A-form RNA helix to generate ssRNA topological structures 21 . In a , one turn of an A-form RNA helix is shown with the helical and schematic representations. The two component strands form two negative nodes within this one-turn helix. In b , a strand of ssRNA (orange) is designed to contain alternating complementary pairing segments to form two one-turn A-form helices, and a trefoil knot is formed after enzymatic ligation aided by a DNA splint (green). However, topological structures constructed in this way contain very strong intrastrand base pairings. ( c , d ) Junction-based method to generate ssRNA topological structures. In c , a 4WJ is formed with two RNA strands (blue) as the helical strands and two DNA strands (grey) as the crossover strands, and is shown with the helical and schematic representations. The two RNA helical strands generate a node. In d , the assembly complex for the trefoil knot is formed, where the RNA scaffolds (blue) are threaded into the targeted topology by DNA staples (grey) and linked end to end by DNA splints (green). After ligation and subsequent removal of the DNA staples and splints, a ssRNA knot free of strong intrastrand base pairings is generated. ( e ) The ssDNA trefoil knot can be used as a template for the construction of ssRNA trefoil knot. The ssDNA knot template (red) is pre-prepared and annealed with the complementary RNA strands (blue). After ligation, the DNA–RNA hybrid knot is formed. The ds DNA–RNA hybrid is more rigid than single-stranded structure, the careful design of curvature (by adding bulges) and torsion (by adjusting the length of hybrid helix) is necessary. The hybrid knot can then be subjected to DNase I digestion to obtain the ssRNA knot.

    Techniques Used: Ligation, Construct, Generated

    Related Articles

    Centrifugation:

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    Amplification:

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    Synthesized:

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    Quantitative RT-PCR:

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    Real-time Polymerase Chain Reaction:

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    Microarray:

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    Incubation:

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    Activity Assay:

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    Cell Culture:

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    Expressing:

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    Recombinase Polymerase Amplification:

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    Hybridization:

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    Transfection:

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    Protease Inhibitor:

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    Northern Blot:

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    Infection:

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    Reverse Transcription Polymerase Chain Reaction:

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    Polymerase Chain Reaction:

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    Sonication:

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    Nucleic Acid Electrophoresis:

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    Mutagenesis:

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    Isolation:

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    Article Snippet: Total RNA from four tissues was extracted separately using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). .. Total RNA was isolated by following the manufacturer's protocol and then treated with RNase-free DNase I (New England Biolabs) at 37°C for 30 min to remove the potential DNA. .. After that, RNA was suspended in RNase-free water for cDNA synthesis. cDNA was synthesized using 2 µg of total RNA using the SMART cDNA synthesis kit (Clontech Laboratories, Inc., Mountain View, CA, USA).

    Article Title: Genome-wide identification, classification and expression profiling of nicotianamine synthase (NAS) gene family in maize
    Article Snippet: The proteins and their accession numbers used for alignment and phylogenetic tree construction are as follows: ZmNAS1;1 [MaizeSequence:GRMZM2G385200], ZmNAS1;2 [MaizeSequence:GRMZM2G312481], ZmNAS2;1 [MaizeSequence:GRMZM2G030036], ZmNAS2;2 [MaizeSequence:GRMZM2G124785], ZmNAS3 [MaizeSequence:GRMZM2G478568], ZmNAS4 [MaizeSequence:GRMZM2G439195], ZmNAS5 [MaizeSequence:GRMZM2G050108], ZmNAS6;1 [MaizeSequence:GRMZM2G704488], and ZmNAS6;2 [MaizeSequence:AC233955.1_FGT003] from Maize (Zea mays ); NASHOR1 [GenBank:AF136941], NASHOR2 [GenBank:AF136942], HvNAS1 [GenBank:AB010086], HvNAS2 [GenBank:AB011265], HvNAS3 [GenBank:AB011264], HvNAS4 [GenBank:AB011266], HvNAS5 [GenBank:AB011268], HvNAS6 [GenBank:AB011269] and HvNAS7 [GenBank:AB019525] from barley (Hordeum vulgare ), OsNAS1 [GenBank:AB021746], OsNAS2 [GenBank:AB023818] and OsNAS3 [GenBank:AB023819] from rice (Oryza sativa ); AtNAS1 [GenBank:NM_120577], AtNAS2 [GenBank:NM_124990], AtNAS3 [GenBank:NM_100794] and AtNAS4 [GenBank:NM_104521] from Arabidopsis thaliana ; chlN [GenBank:AJ242045] from Lycopersicon esculen-tum . .. Total RNA was isolated using TRIzol reagent according to the manufacturer’s instructions (Invitrogen) Genomic DNA contaminants were removed from RNA samples using DNaseI (NEB). .. The amount and quality of the total RNA was confirmed by electrophoresis in 1% formamide agarose gel.

    Article Title: Energy, ageing, fidelity and sex: oocyte mitochondrial DNA as a protected genetic template
    Article Snippet: A.2. .. Total RNA from dissected A. aurita tissues was isolated using TRI reagent (Ambion), treated with DNase I (New England Biolabs) for 10 min at 37 °C and re-purified using Pure LinkTM RNA Mini Kit (Ambion), all according to the manufacturers’ instructions. .. A Chromo4 real-time detector (Bio-Rad), and Brilliant III Ultra-Fast SYBR Green qRT-PCR (Agilent Technologies) were used for the reactions.

    Article Title: Iron-Regulated Expression of Alginate Production, Mucoid Phenotype, and Biofilm Formation by Pseudomonas aeruginosa
    Article Snippet: The samples were then per-O -trimethylsilylated by treatment with Tri-Sil (Pierce) at 80°C for 0.5 h. GC-MS analysis of the TMS methylglycosides was performed on an Agilent 6890 N GC interfaced to a 5975B MSD, using an Agilent DB-1 fused silica capillary column (30 m BY 0.25 mm inner diameter [ID]). .. Total RNA was isolated using Qiagen RNeasy minicolumns and DNase treated with RNase-free DNase I (NEB). .. ImPromII reverse transcription system (Promega) was used for cDNA synthesis.

    Article Title: Overexpression of piRNA Pathway Genes in Epithelial Ovarian Cancer
    Article Snippet: Paragraph title: RNA isolation and cDNA synthesis ... RNA was treated with DNase I (New England Biolabs) before reverse transcription.

    Article Title: New Genomic Structure for Prostate Cancer Specific Gene PCA3 within BMCC1: Implications for Prostate Cancer Detection and Progression
    Article Snippet: Paragraph title: RNA isolation and cDNA synthesis ... Subsequent DNase treatment was performed with DNase I (NEB Biolabs: Cat No. M0303S), ethanol precipitated, resuspended in DEPC-treated water and quality controlled via spectrophotometry and gel electrophoresis.

    RNA Extraction:

    Article Title: Transcriptome-wide selection of a reliable set of reference genes for gene expression studies in potato cyst nematodes (Globodera spp.)
    Article Snippet: Paragraph title: RNA extraction and cDNA synthesis ... All samples were treated with DNase (DNase I, New England Biolabs, Ipswich, Massachusetts, United States).

    Article Title: Activation of Nucleases, PCD, and Mobilization of Reserves in the Araucaria angustifolia Megagametophyte During Germination
    Article Snippet: Paragraph title: RNA Extraction and Semi-Quantitative PCR (RT-PCR) ... Total RNA was treated with DNase I (New England Biolabs).

    Article Title: Identification of aberrant microRNA expression pattern in pediatric gliomas by microarray
    Article Snippet: Paragraph title: Total RNA extraction and amplification ... DNase I (New England Biolabs) was then added to digest the residual RNA.

    Article Title: Replication and Transcription Activities of Ribonucleoprotein Complexes Reconstituted from Avian H5N1, H1N1pdm09 and H3N2 Influenza A Viruses
    Article Snippet: Paragraph title: RNA Extraction and cDNA Synthesis ... Total RNA was extracted from transfected cells using TRIzol plus RNA purification kit (Invitrogen) and followed by DNaseI treatment (New England Biolabs, Ipswich, MA, USA).

    Labeling:

    Article Title: Identification of aberrant microRNA expression pattern in pediatric gliomas by microarray
    Article Snippet: DNase I (New England Biolabs) was then added to digest the residual RNA. .. T7 Enzyme Mix (Life technology) was then used to perform transcription of cDNAs to aRNAs in vitro.

    Article Title:
    Article Snippet: Although EMSA analysis identified stable complexes between RPA-wt and the fork DNA substrate and it would be expected that RPA would bind to the single-stranded gap, we used DNase I footprinting to determine the precise site of RPA binding. .. DNase I digestion of the fork DNA substrate (labeled on the leading parental strand, leadP122) without RPA-wt is shown in D (lanes 2 and 9); the digestion pattern is weak in the region of the 32-nt gap ( D , denoted by a dashed bracket ) because of the known weaker activity of DNase I on ssDNA. .. However, increasing concentrations of RPA further inhibited DNase I incision in the 32-nt gap region near the fork junction ( D , lanes 3–8 ).

    Purification:

    Article Title: Silencing efficiency of dsRNA fragments targeting Fusarium graminearum TRI6 and patterns of small interfering RNA associated with reduced virulence and mycotoxin production
    Article Snippet: RNA was extracted from 100 mg of frozen powder using the GeneJET Plant Purification Minikit (Thermo Fisher Scientific, Waltham, MA, USA). .. Samples were then treated with DNase I and DNase 5x buffer (New England Biolabs).

    Article Title: Identification of aberrant microRNA expression pattern in pediatric gliomas by microarray
    Article Snippet: DNase I (New England Biolabs) was then added to digest the residual RNA. .. DNase I (New England Biolabs) was then added to digest the residual RNA.

    Article Title: Mammalian RNase H2 removes ribonucleotides from DNA to maintain genome integrity
    Article Snippet: Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions. .. For qRT-PCR, RNA was digested with DNaseI (New England Biolabs, Inc.) and purified using the RNeasy Mini kit (QIAGEN). .. 200 ng–1 µg RNA was reverse transcribed using a poly(dT) primer of the RevertAid H Minus First Strand cDNA Synthesis kit (Thermo Fisher Scientific) according to the manufacturer’s recommendations.

    Article Title: Replication and Transcription Activities of Ribonucleoprotein Complexes Reconstituted from Avian H5N1, H1N1pdm09 and H3N2 Influenza A Viruses
    Article Snippet: Polymerase activity was normalized with the expression of a reporter plasmid pGL4.73 [hRluc/SV40] (Promega), encoding a Renilla luciferase gene. .. Total RNA was extracted from transfected cells using TRIzol plus RNA purification kit (Invitrogen) and followed by DNaseI treatment (New England Biolabs, Ipswich, MA, USA). .. A previously described approach was used to achieve specific quantification for each RNA species , .

    Sequencing:

    Article Title: Caveolin-1 Associated Adenovirus Entry into Human Corneal Cells
    Article Snippet: To quantify the presence of viral DNA in endosomal compartments, the E1A gene was amplified from endosomal fractions after sucrose gradient with primers (IDT, ) designed based on the HAdV-D37 genomic E1A nucleotide sequence [ ]. .. Total RNA was then treated with DNase I (1 unit) (New England BioLabs, Ipswich, MA) at 37°C for 1hr.

    Article Title: Transcriptome Analysis of Crucian Carp (Carassius auratus), an Important Aquaculture and Hypoxia-Tolerant Species
    Article Snippet: Paragraph title: RNA Isolation, cDNA Libraries Construction and 454 Sequencing ... Total RNA was isolated by following the manufacturer's protocol and then treated with RNase-free DNase I (New England Biolabs) at 37°C for 30 min to remove the potential DNA.

    Construct:

    Article Title: Transcriptome Analysis of Crucian Carp (Carassius auratus), an Important Aquaculture and Hypoxia-Tolerant Species
    Article Snippet: Total RNA was isolated by following the manufacturer's protocol and then treated with RNase-free DNase I (New England Biolabs) at 37°C for 30 min to remove the potential DNA. .. Before cDNA synthesis, the RNA quantity and quality was checked using gel electrophoresis and Bioanalyzer (Agilent).

    SDS Page:

    Article Title:
    Article Snippet: For co-immunoprecipitation experiments, cells were lysed by sonication in immunoprecipitation buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, and 1 mm EDTA) supplemented with protease inhibitor mixture, 1 mm PMSF, and 10 units/ml of DNase I (New England Biolabs). .. After the final wash, equal portions of immunoprecipitation buffer and 2× SDS sample buffer were added to the beads, and immunoprecipitated proteins were released by heating at 95 °C for 5 min.

    Software:

    Article Title: Silencing efficiency of dsRNA fragments targeting Fusarium graminearum TRI6 and patterns of small interfering RNA associated with reduced virulence and mycotoxin production
    Article Snippet: Samples were then treated with DNase I and DNase 5x buffer (New England Biolabs). .. Estimation of TRI5 expression was determined for PH1 and mutant strains in three experiments, each with three technical replicates for the target gene and the reference gene.

    Article Title: Caveolin-1 Associated Adenovirus Entry into Human Corneal Cells
    Article Snippet: Total RNA was then treated with DNase I (1 unit) (New England BioLabs, Ipswich, MA) at 37°C for 1hr. .. Total RNA was then treated with DNase I (1 unit) (New England BioLabs, Ipswich, MA) at 37°C for 1hr.

    Article Title: Mammalian RNase H2 removes ribonucleotides from DNA to maintain genome integrity
    Article Snippet: For qRT-PCR, RNA was digested with DNaseI (New England Biolabs, Inc.) and purified using the RNeasy Mini kit (QIAGEN). .. 1 µl of the cDNA product served as template in the subsequent qRT-PCR using the Maxima SYBR green/ROX qPCR Master Mix (Thermo Fisher Scientific) using 0.6 µM target specific primers, initial denaturation at 95°C for 15 min, 40 cycles of denaturation (15 s at 95°C), annealing (30 s at 58°C), and elongation (30 s at 72°C).

    SYBR Green Assay:

    Article Title: A new cell culture model to genetically dissect the complete human papillomavirus life cycle
    Article Snippet: Isolated RNA samples were treated with DNase I (NEB; M0303L) prior to reverse transcription. .. 1 or 0.5 μg total RNA was used to reverse-transcribe into cDNA using ImProm-II Reverse Transcriptase kit (Promega).

    Article Title: Caveolin-1 Associated Adenovirus Entry into Human Corneal Cells
    Article Snippet: Real-time PCR was performed with Fast SYBR Green master mix (Applied Biosystems, Foster City, CA) with the following conditions, 40 cycles at 95°C (10 s), 55°C (20 s), and a final extension at 72°C for 10 min. To determine mRNA expression for caveolin-1 and IL-8, total RNA was extracted from caveolin-1 or scrambled siRNA transfected, HAdV-D37 infected cells, at 2 hr post-infection using TRIzol (Invitrogen) according to the manufacturer’s instructions. .. Total RNA was then treated with DNase I (1 unit) (New England BioLabs, Ipswich, MA) at 37°C for 1hr.

    Article Title: Mammalian RNase H2 removes ribonucleotides from DNA to maintain genome integrity
    Article Snippet: For qRT-PCR, RNA was digested with DNaseI (New England Biolabs, Inc.) and purified using the RNeasy Mini kit (QIAGEN). .. 1 µl of the cDNA product served as template in the subsequent qRT-PCR using the Maxima SYBR green/ROX qPCR Master Mix (Thermo Fisher Scientific) using 0.6 µM target specific primers, initial denaturation at 95°C for 15 min, 40 cycles of denaturation (15 s at 95°C), annealing (30 s at 58°C), and elongation (30 s at 72°C).

    Article Title: Analysis of TET Expression/Activity and 5mC Oxidation during Normal and Malignant Germ Cell Development
    Article Snippet: For first strand synthesis 500 ng of DNAse (NEB, Frankfurt, Germany) digested RNA template was used. .. For first strand synthesis 500 ng of DNAse (NEB, Frankfurt, Germany) digested RNA template was used.

    Negative Control:

    Article Title: A new cell culture model to genetically dissect the complete human papillomavirus life cycle
    Article Snippet: Isolated RNA samples were treated with DNase I (NEB; M0303L) prior to reverse transcription. .. Equal amounts of cDNA were quantified by RT-qPCR using the IQ SYBR Green Supermix (BIO-RAD) and a CFX96 Real-Time System (BIO-RAD).

    Agarose Gel Electrophoresis:

    Article Title: Activation of Nucleases, PCD, and Mobilization of Reserves in the Araucaria angustifolia Megagametophyte During Germination
    Article Snippet: Total RNA was treated with DNase I (New England Biolabs). .. Total RNA was treated with DNase I (New England Biolabs).

    In Vitro:

    Article Title: Identification of aberrant microRNA expression pattern in pediatric gliomas by microarray
    Article Snippet: DNase I (New England Biolabs) was then added to digest the residual RNA. .. After purification, the concentration was measured using Nanodrop2000, all total RNA was reversely transcribed to cDNA using a MessageAmp™ II aRNA Amplification Kit (Ambion).

    Electrophoresis:

    Article Title: Activation of Nucleases, PCD, and Mobilization of Reserves in the Araucaria angustifolia Megagametophyte During Germination
    Article Snippet: Total RNA was treated with DNase I (New England Biolabs). .. Total RNA was treated with DNase I (New England Biolabs).

    Spectrophotometry:

    Article Title: Activation of Nucleases, PCD, and Mobilization of Reserves in the Araucaria angustifolia Megagametophyte During Germination
    Article Snippet: The quantity and purity of the RNA samples were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States); samples with 260/280 nm and 260/230 nm ratios between 1.8–2.2 and 1.6–2.2, respectively, were considered pure enough. .. Total RNA was treated with DNase I (New England Biolabs).

    Article Title: New Genomic Structure for Prostate Cancer Specific Gene PCA3 within BMCC1: Implications for Prostate Cancer Detection and Progression
    Article Snippet: Total RNA was extracted from prostate tissues using Trizol (Invitrogen) following manufacture's protocol. .. Subsequent DNase treatment was performed with DNase I (NEB Biolabs: Cat No. M0303S), ethanol precipitated, resuspended in DEPC-treated water and quality controlled via spectrophotometry and gel electrophoresis. .. All RNA was confirmed to be of good quality and thus suitable for subsequent experiments if the A260/280 ratio was > 1.7 and little RNA degradation was evident by gel electrophoresis.

    Immunoprecipitation:

    Article Title:
    Article Snippet: For DNA damaging treatments, cells were incubated in medium containing 1 mm MMS for 4 h or 2 mm HU for 10 h before harvesting. .. For co-immunoprecipitation experiments, cells were lysed by sonication in immunoprecipitation buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, and 1 mm EDTA) supplemented with protease inhibitor mixture, 1 mm PMSF, and 10 units/ml of DNase I (New England Biolabs). .. Samples (800 μg of protein each) were precleared with Protein G Plus/Protein A-agarose beads (Calbiochem) and 1 μg of normal mouse IgG (Santa Cruz) for 30 min, then incubated with mouse monoclonal anti-RPA32 antibody (Calbiochem) for 15 h at 4 °C.

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    New England Biolabs t7 endonuclease 1
    Gene disruption efficiencies of paired D10A Cas9 nickases vs. Cas9 nucleases using sgRNA (5′GGX20) and control sgRNA in human and mouse cells. HEK293T were analyzed 3 days after transfection with plasmids encoding paired Cas9 nickases or Cas9 nuclease, sgRNAs targeting AAVS1-S1 (T1 and B1; A ), AAVS1-S2 (T2 and B1; B ), MET (T1 and B1; C ), EMX1-S1 (T1 and B1; D ), VEGFA-S1 (T1 and B1; E ), VEGFA-S2 (T1 and B1; F ). NIH3T3 were analyzed 3 days after transfection with plasmids encoding paired Cas9 nickases or Cas9 nuclease, sgRNAs targeting Tsc1-S1 (T1 and B1; G ), Tsc2-S1 (T1 and B1; H ), Mtor-S1 (T1 and B1; I ), Mtor-S2 (T1 and B1; J ), Mtor-S3 (T1 and B1; K ) and fluorescent proteins (i.e., eGFP or mCherry). A sgRNA that does not target the respective genes was used as a control and is designated as Con. The frequency of Cas9 nuclease- or paired nickases-driven mutations as determined by the <t>T7E1</t> assay are shown using bar graph. Error bars were derived from three independent experiments ( n = 3). For brevity, statistical significance was shown only for comparison between a group-of-interest and the paired nickase group, except for the negative control group; * P
    T7 Endonuclease 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t7 endonuclease 1/product/New England Biolabs
    Average 99 stars, based on 13 article reviews
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    t7 endonuclease 1 - by Bioz Stars, 2019-10
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    79
    New England Biolabs promastigotes
    Leishmania MCM4 interacts with PCNA and colocalizes with it in S phase. A . MCM4 - PCNA interaction in pull-down experiments. Lanes 1–3 – without ATPγS; lanes 4–6 – with ATPγS. Lanes 1 4 – dummy metal affinity beads exposed to Leishmania whole cell extracts and elution carried out with imidazole. Lanes 2 5 – recombinant immobilized His-PCNA eluted with imidazole. Lanes 3 6 – recombinant immobilized His-PCNA exposed to Leishmania whole cell extracts, and eluted with imidazole. WCE- whole cell extracts. The PCNA band detected in lanes 1 and 4 is probably due to background PCNA from the Leishmania whole cell extracts poured on the beads. B . Immunofluorescence analysis of MCM4-GFP and PCNA expression in synchronized cells. MCM4-GFP expression was analyzed by direct fluorescence. PCNA expression was analyzed by indirect fluorescence using anti-PCNA antibodies. Magnification bar represents 5 µm. C . MCM4 colocalizes with PCNA in S phase cells. MCM4-GFP transfectant Leishmania <t>promastigotes</t> synchronized with hydroxyurea and harvested three hours after release were labeled for PCNA immunofluorescence as described. Cells were analyzed by collecting Z stack images using a confocal microscope. Magnification bar represents 2 µm.
    Promastigotes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    promastigotes - by Bioz Stars, 2019-10
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    Image Search Results


    Gene disruption efficiencies of paired D10A Cas9 nickases vs. Cas9 nucleases using sgRNA (5′GGX20) and control sgRNA in human and mouse cells. HEK293T were analyzed 3 days after transfection with plasmids encoding paired Cas9 nickases or Cas9 nuclease, sgRNAs targeting AAVS1-S1 (T1 and B1; A ), AAVS1-S2 (T2 and B1; B ), MET (T1 and B1; C ), EMX1-S1 (T1 and B1; D ), VEGFA-S1 (T1 and B1; E ), VEGFA-S2 (T1 and B1; F ). NIH3T3 were analyzed 3 days after transfection with plasmids encoding paired Cas9 nickases or Cas9 nuclease, sgRNAs targeting Tsc1-S1 (T1 and B1; G ), Tsc2-S1 (T1 and B1; H ), Mtor-S1 (T1 and B1; I ), Mtor-S2 (T1 and B1; J ), Mtor-S3 (T1 and B1; K ) and fluorescent proteins (i.e., eGFP or mCherry). A sgRNA that does not target the respective genes was used as a control and is designated as Con. The frequency of Cas9 nuclease- or paired nickases-driven mutations as determined by the T7E1 assay are shown using bar graph. Error bars were derived from three independent experiments ( n = 3). For brevity, statistical significance was shown only for comparison between a group-of-interest and the paired nickase group, except for the negative control group; * P

    Journal: Nucleic Acids Research

    Article Title: Paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption

    doi: 10.1093/nar/gky222

    Figure Lengend Snippet: Gene disruption efficiencies of paired D10A Cas9 nickases vs. Cas9 nucleases using sgRNA (5′GGX20) and control sgRNA in human and mouse cells. HEK293T were analyzed 3 days after transfection with plasmids encoding paired Cas9 nickases or Cas9 nuclease, sgRNAs targeting AAVS1-S1 (T1 and B1; A ), AAVS1-S2 (T2 and B1; B ), MET (T1 and B1; C ), EMX1-S1 (T1 and B1; D ), VEGFA-S1 (T1 and B1; E ), VEGFA-S2 (T1 and B1; F ). NIH3T3 were analyzed 3 days after transfection with plasmids encoding paired Cas9 nickases or Cas9 nuclease, sgRNAs targeting Tsc1-S1 (T1 and B1; G ), Tsc2-S1 (T1 and B1; H ), Mtor-S1 (T1 and B1; I ), Mtor-S2 (T1 and B1; J ), Mtor-S3 (T1 and B1; K ) and fluorescent proteins (i.e., eGFP or mCherry). A sgRNA that does not target the respective genes was used as a control and is designated as Con. The frequency of Cas9 nuclease- or paired nickases-driven mutations as determined by the T7E1 assay are shown using bar graph. Error bars were derived from three independent experiments ( n = 3). For brevity, statistical significance was shown only for comparison between a group-of-interest and the paired nickase group, except for the negative control group; * P

    Article Snippet: The amplicons were denatured by heating and annealed to allow formation of heteroduplex DNA, which was treated with 5 units of T7 endonuclease 1 (New England Biolabs) for 20 min at 37°C followed by analysis using 2% agarose gel electrophoresis.

    Techniques: Transfection, Derivative Assay, Negative Control

    Comparison of gene disruption efficiencies of paired Cas9 nickases versus Cas9 nucleases in human and mouse cells. Comparison of indel frequencies associated with nickase pairs and corresponding nucleases or double the amount of nucleases as measured using the T7E1 assay in human and mouse cells. Sixteen pairs of sgRNAs targeting human genes and twelve pairs of sgRNAs targeting mouse genes were used in HEK293T cells and Neuro-2a cells, respectively. The average values from three independent experiments are shown. Representative bar graphs from each experiment are shown in detail in Figures 2 and 3 . Error bars represent s.e.m. *** P

    Journal: Nucleic Acids Research

    Article Title: Paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption

    doi: 10.1093/nar/gky222

    Figure Lengend Snippet: Comparison of gene disruption efficiencies of paired Cas9 nickases versus Cas9 nucleases in human and mouse cells. Comparison of indel frequencies associated with nickase pairs and corresponding nucleases or double the amount of nucleases as measured using the T7E1 assay in human and mouse cells. Sixteen pairs of sgRNAs targeting human genes and twelve pairs of sgRNAs targeting mouse genes were used in HEK293T cells and Neuro-2a cells, respectively. The average values from three independent experiments are shown. Representative bar graphs from each experiment are shown in detail in Figures 2 and 3 . Error bars represent s.e.m. *** P

    Article Snippet: The amplicons were denatured by heating and annealed to allow formation of heteroduplex DNA, which was treated with 5 units of T7 endonuclease 1 (New England Biolabs) for 20 min at 37°C followed by analysis using 2% agarose gel electrophoresis.

    Techniques:

    Comparison of indel generation efficiencies of paired Cas9 D10A nickases versus paired Cas9 H840A nickases using reverse-identical 5′GX19 sgRNAs without control sgRNA. ( A ) Sequence of the human EMX1-S2 locus with designed sgRNAs (T1, B2, T3 and B4). Reverse-identical sgRNAs (T1 and T3; B2 and B4) are shown in blue letters. sgRNA target sites (T1, B2, T3 and B4) are indicated by red letters and PAM sequences are marked by bold underlined letters. The cleavage by D10A Cas9 nickase and the sgRNA pair of T1 and B2 leads to a double-strand break with a 5′ overhang. Similarly, the cleavage by H840A Cas9 nickase and the sgRNA pair of T3 and B4 also leads to a double strand break with a 5′ overhang. Red arrowheads indicate the cleavage site. ( B ) The frequency of double the amount of Cas9 nuclease- or paired nickase-driven mutations as determined by the T7E1 assay are shown using bar graph. Error bars were derived from three independent experiments ( n = 3). ‘+’ and ‘++’ denote 1 and 2 μg concentrations of Cas9 nucleases or paired Cas9 nickases using top or bottom sgRNAs, respectively.

    Journal: Nucleic Acids Research

    Article Title: Paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption

    doi: 10.1093/nar/gky222

    Figure Lengend Snippet: Comparison of indel generation efficiencies of paired Cas9 D10A nickases versus paired Cas9 H840A nickases using reverse-identical 5′GX19 sgRNAs without control sgRNA. ( A ) Sequence of the human EMX1-S2 locus with designed sgRNAs (T1, B2, T3 and B4). Reverse-identical sgRNAs (T1 and T3; B2 and B4) are shown in blue letters. sgRNA target sites (T1, B2, T3 and B4) are indicated by red letters and PAM sequences are marked by bold underlined letters. The cleavage by D10A Cas9 nickase and the sgRNA pair of T1 and B2 leads to a double-strand break with a 5′ overhang. Similarly, the cleavage by H840A Cas9 nickase and the sgRNA pair of T3 and B4 also leads to a double strand break with a 5′ overhang. Red arrowheads indicate the cleavage site. ( B ) The frequency of double the amount of Cas9 nuclease- or paired nickase-driven mutations as determined by the T7E1 assay are shown using bar graph. Error bars were derived from three independent experiments ( n = 3). ‘+’ and ‘++’ denote 1 and 2 μg concentrations of Cas9 nucleases or paired Cas9 nickases using top or bottom sgRNAs, respectively.

    Article Snippet: The amplicons were denatured by heating and annealed to allow formation of heteroduplex DNA, which was treated with 5 units of T7 endonuclease 1 (New England Biolabs) for 20 min at 37°C followed by analysis using 2% agarose gel electrophoresis.

    Techniques: Sequencing, Derivative Assay

    Gene disruption efficiencies of paired Cas9 nickases versus double the amount of Cas9 nucleases using sgRNA (5′GX20 or 5′GX19) without control sgRNA in mouse cells. (A–L) paired Cas9 nickase- or nuclease-driven mutations in the mouse genes detected by the T7E1 assay. Neuro-2a were analyzed 3 days after transfection with plasmids encoding paired Cas9 nickases or Cas9 nuclease, 5′GX20 sgRNAs targeting Tsc1-S2 (T1 and B1; A ), Tsc2-S1 (T1 and B1; B ), Tsc2-S2 (T1 and B1; C ), Mtor-S1 (T1 and B1; D ), Mtor-S2 (T1 and B1; E ), Mtor-S3 (T1 and B1; F ), Usp3 (T1 and B1; G ), Usp7-S1 (T1 and B1; H ) and Usp7-S2 (T1 and B2; I ). Neuro-2a were analyzed 3 days after transfection with plasmids encoding paired Cas9 nickases or Cas9 nuclease, 5′GX19 sgRNAs targeting Tsc2-S3 (T1 and B1; J ), Tsc2-S4 (T1 and B1; K ) and mUSP7-S3 (T1 and B1; L ). The frequency of Cas9 nuclease- or paired nickases-driven mutations as determined by the T7E1 assay are shown using bar graph. Error bars were derived from three independent experiments ( n = 3). For brevity, statistical significance was shown only for comparison between a group-of-interest and the paired nickase group, except for the negative control group; * P

    Journal: Nucleic Acids Research

    Article Title: Paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption

    doi: 10.1093/nar/gky222

    Figure Lengend Snippet: Gene disruption efficiencies of paired Cas9 nickases versus double the amount of Cas9 nucleases using sgRNA (5′GX20 or 5′GX19) without control sgRNA in mouse cells. (A–L) paired Cas9 nickase- or nuclease-driven mutations in the mouse genes detected by the T7E1 assay. Neuro-2a were analyzed 3 days after transfection with plasmids encoding paired Cas9 nickases or Cas9 nuclease, 5′GX20 sgRNAs targeting Tsc1-S2 (T1 and B1; A ), Tsc2-S1 (T1 and B1; B ), Tsc2-S2 (T1 and B1; C ), Mtor-S1 (T1 and B1; D ), Mtor-S2 (T1 and B1; E ), Mtor-S3 (T1 and B1; F ), Usp3 (T1 and B1; G ), Usp7-S1 (T1 and B1; H ) and Usp7-S2 (T1 and B2; I ). Neuro-2a were analyzed 3 days after transfection with plasmids encoding paired Cas9 nickases or Cas9 nuclease, 5′GX19 sgRNAs targeting Tsc2-S3 (T1 and B1; J ), Tsc2-S4 (T1 and B1; K ) and mUSP7-S3 (T1 and B1; L ). The frequency of Cas9 nuclease- or paired nickases-driven mutations as determined by the T7E1 assay are shown using bar graph. Error bars were derived from three independent experiments ( n = 3). For brevity, statistical significance was shown only for comparison between a group-of-interest and the paired nickase group, except for the negative control group; * P

    Article Snippet: The amplicons were denatured by heating and annealed to allow formation of heteroduplex DNA, which was treated with 5 units of T7 endonuclease 1 (New England Biolabs) for 20 min at 37°C followed by analysis using 2% agarose gel electrophoresis.

    Techniques: Transfection, Derivative Assay, Negative Control

    Gene disruption efficiencies of paired Cas9 nickases vs. double the amount of Cas9 nucleases using sgRNA (5′GX20 or 5′GX19) without control sgRNA in human cells. (A–P) Cas9 paired nickase- or nuclease-driven mutations in the human genes detected by the T7E1 assay. HEK293T were analyzed 3 days after transfection with plasmids encoding paired Cas9 nickases or Cas9 nuclease, 5′GX20 sgRNAs targeting AAVS1-S1 (T1 and B1; A ), MET (T1 and B1; B ), EMX1-S1 (T1 and B1; C ), EMX1-S2 (T1 and B1; D ), USP1 (T1 and B1; E ), USP3 (T1 and B1; F ), USP16 (T1 and B1; G ), USP31 (T1 and B1; H ), USP33 (T1 and B1; I ), USP47 (T1 and B1; J ), VEGFA-S1 (T1 and B1; K ), and VEGFA-S2 (T1 and B1; L ). HEK293T were analyzed 3 days after transfection with plasmids encoding paired Cas9 nickases or Cas9 nuclease, 5′GX19 sgRNAs targeting EMX1-S3 (T1 and B1; M ), EMX1-S4 (T1 and B1; N ), EMX1-S5 (T2 and B1; O ) and MET-S2 (T1 and B1; P ). The frequency of Cas9 nuclease- or paired nickases-driven mutations as determined by the T7E1 assay are shown using bar graph. Error bars were derived from three independent experiments ( n = 3). For brevity, statistical significance was shown only for comparison between a group-of-interest and the paired nickase group, except for the negative control group; * P

    Journal: Nucleic Acids Research

    Article Title: Paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption

    doi: 10.1093/nar/gky222

    Figure Lengend Snippet: Gene disruption efficiencies of paired Cas9 nickases vs. double the amount of Cas9 nucleases using sgRNA (5′GX20 or 5′GX19) without control sgRNA in human cells. (A–P) Cas9 paired nickase- or nuclease-driven mutations in the human genes detected by the T7E1 assay. HEK293T were analyzed 3 days after transfection with plasmids encoding paired Cas9 nickases or Cas9 nuclease, 5′GX20 sgRNAs targeting AAVS1-S1 (T1 and B1; A ), MET (T1 and B1; B ), EMX1-S1 (T1 and B1; C ), EMX1-S2 (T1 and B1; D ), USP1 (T1 and B1; E ), USP3 (T1 and B1; F ), USP16 (T1 and B1; G ), USP31 (T1 and B1; H ), USP33 (T1 and B1; I ), USP47 (T1 and B1; J ), VEGFA-S1 (T1 and B1; K ), and VEGFA-S2 (T1 and B1; L ). HEK293T were analyzed 3 days after transfection with plasmids encoding paired Cas9 nickases or Cas9 nuclease, 5′GX19 sgRNAs targeting EMX1-S3 (T1 and B1; M ), EMX1-S4 (T1 and B1; N ), EMX1-S5 (T2 and B1; O ) and MET-S2 (T1 and B1; P ). The frequency of Cas9 nuclease- or paired nickases-driven mutations as determined by the T7E1 assay are shown using bar graph. Error bars were derived from three independent experiments ( n = 3). For brevity, statistical significance was shown only for comparison between a group-of-interest and the paired nickase group, except for the negative control group; * P

    Article Snippet: The amplicons were denatured by heating and annealed to allow formation of heteroduplex DNA, which was treated with 5 units of T7 endonuclease 1 (New England Biolabs) for 20 min at 37°C followed by analysis using 2% agarose gel electrophoresis.

    Techniques: Transfection, Derivative Assay, Negative Control

    Comparison of indel generation efficiencies of paired Cas9 D10A nickases vs. paired Cas9 H840A nickases using identical 5′GX20 sgRNAs without control sgRNA. (A–C) Three surrogate reporters containing sequences that can be targeted with a fixed sgRNA pair (T1 and B2; shown in blue letters) were constructed. sgRNA target sites (T1 and B2) are indicated by red letters and PAM sequences are marked by bold underlined letters. ( A ) A sequence that can be targeted by D10A Cas9n and the sgRNA pair with a +6 bp offset. The cleavage is expected to lead to a 40 bp 5′ overhang. ( B ) A sequence that can be targeted by H840A Cas9n and the sgRNA pair with a +6 bp offset. The cleavage is expected to lead to a 12 bp 5′ overhang. ( C ) A sequence that can be targeted by H840A Cas9n and the sgRNA pair with a +28 bp offset. The cleavage is expected to lead to a 40 bp 5′ overhang. (D–E) HEK293T cells were analyzed 3 days after transfection with a plasmid encoding Cas9 nickase (D10A or H840A) or double the amount of Cas9 nuclease, plasmids encoding sgRNAs (T1 and B2), and a reporter plasmid containing the target sequence shown above (A, B or C). ( D ) Representative flow cytometry. The percentages of GFP + cells in the total RFP + cell population (G/R) are shown (e.g. G/R = 77%). ( E ) The mutation frequencies in the target sequence of the transfected reporter plasmids detected by the T7E1 assay.

    Journal: Nucleic Acids Research

    Article Title: Paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption

    doi: 10.1093/nar/gky222

    Figure Lengend Snippet: Comparison of indel generation efficiencies of paired Cas9 D10A nickases vs. paired Cas9 H840A nickases using identical 5′GX20 sgRNAs without control sgRNA. (A–C) Three surrogate reporters containing sequences that can be targeted with a fixed sgRNA pair (T1 and B2; shown in blue letters) were constructed. sgRNA target sites (T1 and B2) are indicated by red letters and PAM sequences are marked by bold underlined letters. ( A ) A sequence that can be targeted by D10A Cas9n and the sgRNA pair with a +6 bp offset. The cleavage is expected to lead to a 40 bp 5′ overhang. ( B ) A sequence that can be targeted by H840A Cas9n and the sgRNA pair with a +6 bp offset. The cleavage is expected to lead to a 12 bp 5′ overhang. ( C ) A sequence that can be targeted by H840A Cas9n and the sgRNA pair with a +28 bp offset. The cleavage is expected to lead to a 40 bp 5′ overhang. (D–E) HEK293T cells were analyzed 3 days after transfection with a plasmid encoding Cas9 nickase (D10A or H840A) or double the amount of Cas9 nuclease, plasmids encoding sgRNAs (T1 and B2), and a reporter plasmid containing the target sequence shown above (A, B or C). ( D ) Representative flow cytometry. The percentages of GFP + cells in the total RFP + cell population (G/R) are shown (e.g. G/R = 77%). ( E ) The mutation frequencies in the target sequence of the transfected reporter plasmids detected by the T7E1 assay.

    Article Snippet: The amplicons were denatured by heating and annealed to allow formation of heteroduplex DNA, which was treated with 5 units of T7 endonuclease 1 (New England Biolabs) for 20 min at 37°C followed by analysis using 2% agarose gel electrophoresis.

    Techniques: Construct, Sequencing, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry, Mutagenesis

    lenti-X4R5-Cas9 modified primary CD4 + T cell resists HIV challenge. a T7E1 analysis of CXCR4 and CCR5 disruption. b Deep sequencing analysis of typical NHEJ (indels) of related targets. c lenti-X4R5-Cas9 modified CD4 + T cell challenged with HIV-1 NL4-3 or HIV-1 YU-2 . d lenti-X4R5-Cas9 modified CD4 + T cell exposed to dual-tropic HIV-1 variants (NL4-3 YU-2, 1:1). The CCR5, CXCR4-1, CXCR4-2 represent single disruption of CCR5 or CXCR4, which use the same corresponding gRNAs used in lenti-X4R5-Cas9-#1 or #2. The data shown were the mean ± SD of three independent experiments. *P

    Journal: Cell & Bioscience

    Article Title: Genome editing of the HIV co-receptors CCR5 and CXCR4 by CRISPR-Cas9 protects CD4+ T cells from HIV-1 infection

    doi: 10.1186/s13578-017-0174-2

    Figure Lengend Snippet: lenti-X4R5-Cas9 modified primary CD4 + T cell resists HIV challenge. a T7E1 analysis of CXCR4 and CCR5 disruption. b Deep sequencing analysis of typical NHEJ (indels) of related targets. c lenti-X4R5-Cas9 modified CD4 + T cell challenged with HIV-1 NL4-3 or HIV-1 YU-2 . d lenti-X4R5-Cas9 modified CD4 + T cell exposed to dual-tropic HIV-1 variants (NL4-3 YU-2, 1:1). The CCR5, CXCR4-1, CXCR4-2 represent single disruption of CCR5 or CXCR4, which use the same corresponding gRNAs used in lenti-X4R5-Cas9-#1 or #2. The data shown were the mean ± SD of three independent experiments. *P

    Article Snippet: After PCR amplification of CXCR4 or CCR5 fragment, T7 endonuclease 1 assay was performed according to manufacturer’s instruction with 400 ng PCR product annealed and digested with T7 endonuclease 1 (10 units/µl, NEB) and analyzed with ethidium bromide (EB) stained 1.5% agarose gel.

    Techniques: Modification, Sequencing, Non-Homologous End Joining

    off-target analysis of CCR5. a Mutation frequency analysis at predicted off-target sites of CCR5. The off-target sites were predicted and aligned with the human genome. The sites were amplified and cloned into T-vector and subjected to sequencing. b T7E1 analysis of all predicted off-target sites

    Journal: Cell & Bioscience

    Article Title: Genome editing of the HIV co-receptors CCR5 and CXCR4 by CRISPR-Cas9 protects CD4+ T cells from HIV-1 infection

    doi: 10.1186/s13578-017-0174-2

    Figure Lengend Snippet: off-target analysis of CCR5. a Mutation frequency analysis at predicted off-target sites of CCR5. The off-target sites were predicted and aligned with the human genome. The sites were amplified and cloned into T-vector and subjected to sequencing. b T7E1 analysis of all predicted off-target sites

    Article Snippet: After PCR amplification of CXCR4 or CCR5 fragment, T7 endonuclease 1 assay was performed according to manufacturer’s instruction with 400 ng PCR product annealed and digested with T7 endonuclease 1 (10 units/µl, NEB) and analyzed with ethidium bromide (EB) stained 1.5% agarose gel.

    Techniques: Mutagenesis, Amplification, Clone Assay, Plasmid Preparation, Sequencing

    Disruption of CXCR4 and CCR5 protects TZM-bl cells from HIV-1 infection. a T7E1 assay for genome level cleavage efficacy by lenti-X4R5-Cas9-#1,#2 in TZM-bl. b Expression of CXCR4 or CCR5 in TZM-bl cell line transfected with lenti-X4R5-Cas9 by lipo2000 transfection reagent were analyzed with flow cytometry. c On-target analysis of the cleavage on target sites. d lenti-X4R5-Cas9 transfected TZM-bl cell line challenged with HIV-1 NL4-3 or HIV-1 YU-2 (3 days post infection). The data shown were the mean ± SD of three independent experiments. *P

    Journal: Cell & Bioscience

    Article Title: Genome editing of the HIV co-receptors CCR5 and CXCR4 by CRISPR-Cas9 protects CD4+ T cells from HIV-1 infection

    doi: 10.1186/s13578-017-0174-2

    Figure Lengend Snippet: Disruption of CXCR4 and CCR5 protects TZM-bl cells from HIV-1 infection. a T7E1 assay for genome level cleavage efficacy by lenti-X4R5-Cas9-#1,#2 in TZM-bl. b Expression of CXCR4 or CCR5 in TZM-bl cell line transfected with lenti-X4R5-Cas9 by lipo2000 transfection reagent were analyzed with flow cytometry. c On-target analysis of the cleavage on target sites. d lenti-X4R5-Cas9 transfected TZM-bl cell line challenged with HIV-1 NL4-3 or HIV-1 YU-2 (3 days post infection). The data shown were the mean ± SD of three independent experiments. *P

    Article Snippet: After PCR amplification of CXCR4 or CCR5 fragment, T7 endonuclease 1 assay was performed according to manufacturer’s instruction with 400 ng PCR product annealed and digested with T7 endonuclease 1 (10 units/µl, NEB) and analyzed with ethidium bromide (EB) stained 1.5% agarose gel.

    Techniques: Infection, Expressing, Transfection, Flow Cytometry, Cytometry

    X4R5-Cas9 lentivirus modified Jurkat T cells were enriched after CXCR4-tropic (NL4-3) and CCR5-tropic (YU-2) HIV-1 challenge.  a  HIV replication in X4R5-Cas9 lentivirus modified as well as mock and control Jurkat T cells infected with X4-tropic and R5-tropic HIV-1 concurrently. Values represent the mean of duplicate infections.  b  cleavage analysis of CXCR4 and CCR5 by T7 endonuclease 1 in mock, control, lenti-X4R5-Cas9-#1 and lenti-X4R5-Cas9-#2 group at 0, 9 and 18 days after HIV-1 challenge. The lower migrating bands (indicated by  arrows ) in  each lane  indicate the disrupted CXCR4 and CCR5 alleles.  DPI  days post infection

    Journal: Cell & Bioscience

    Article Title: Genome editing of the HIV co-receptors CCR5 and CXCR4 by CRISPR-Cas9 protects CD4+ T cells from HIV-1 infection

    doi: 10.1186/s13578-017-0174-2

    Figure Lengend Snippet: X4R5-Cas9 lentivirus modified Jurkat T cells were enriched after CXCR4-tropic (NL4-3) and CCR5-tropic (YU-2) HIV-1 challenge. a HIV replication in X4R5-Cas9 lentivirus modified as well as mock and control Jurkat T cells infected with X4-tropic and R5-tropic HIV-1 concurrently. Values represent the mean of duplicate infections. b cleavage analysis of CXCR4 and CCR5 by T7 endonuclease 1 in mock, control, lenti-X4R5-Cas9-#1 and lenti-X4R5-Cas9-#2 group at 0, 9 and 18 days after HIV-1 challenge. The lower migrating bands (indicated by arrows ) in each lane indicate the disrupted CXCR4 and CCR5 alleles. DPI days post infection

    Article Snippet: After PCR amplification of CXCR4 or CCR5 fragment, T7 endonuclease 1 assay was performed according to manufacturer’s instruction with 400 ng PCR product annealed and digested with T7 endonuclease 1 (10 units/µl, NEB) and analyzed with ethidium bromide (EB) stained 1.5% agarose gel.

    Techniques: Modification, Infection

    Jurkat T cell line modified by X4R5-Cas9 lentivirus antagonized HIV infection. a T7E1 assay identification of packaged X4R5-Cas9 lentivirus mediated cleavage at the genome level. b On-target analysis of each target in Jurkat T cells. c Flow cytometry analysis of CXCR4 expression on the cell surface. Jurkat T cell line was transduced with X4R5-Cas9 lentivirus at MOI = 40. CCR5 surface expression detection was excluded because of its low expression on Jurkat T cells. d Detection of protein level of CXCR4 and CCR5 after Jurkat cells were transduced with X4R5-Cas9 lentivirus. e HIV-1 titer change detected by p24 gag ELISA from day 1 to day 5 post-infection. The data shown were the mean ± SD of three independent experiments. *P

    Journal: Cell & Bioscience

    Article Title: Genome editing of the HIV co-receptors CCR5 and CXCR4 by CRISPR-Cas9 protects CD4+ T cells from HIV-1 infection

    doi: 10.1186/s13578-017-0174-2

    Figure Lengend Snippet: Jurkat T cell line modified by X4R5-Cas9 lentivirus antagonized HIV infection. a T7E1 assay identification of packaged X4R5-Cas9 lentivirus mediated cleavage at the genome level. b On-target analysis of each target in Jurkat T cells. c Flow cytometry analysis of CXCR4 expression on the cell surface. Jurkat T cell line was transduced with X4R5-Cas9 lentivirus at MOI = 40. CCR5 surface expression detection was excluded because of its low expression on Jurkat T cells. d Detection of protein level of CXCR4 and CCR5 after Jurkat cells were transduced with X4R5-Cas9 lentivirus. e HIV-1 titer change detected by p24 gag ELISA from day 1 to day 5 post-infection. The data shown were the mean ± SD of three independent experiments. *P

    Article Snippet: After PCR amplification of CXCR4 or CCR5 fragment, T7 endonuclease 1 assay was performed according to manufacturer’s instruction with 400 ng PCR product annealed and digested with T7 endonuclease 1 (10 units/µl, NEB) and analyzed with ethidium bromide (EB) stained 1.5% agarose gel.

    Techniques: Modification, Infection, Flow Cytometry, Cytometry, Expressing, Transduction, Enzyme-linked Immunosorbent Assay

    Leishmania MCM4 interacts with PCNA and colocalizes with it in S phase. A . MCM4 - PCNA interaction in pull-down experiments. Lanes 1–3 – without ATPγS; lanes 4–6 – with ATPγS. Lanes 1 4 – dummy metal affinity beads exposed to Leishmania whole cell extracts and elution carried out with imidazole. Lanes 2 5 – recombinant immobilized His-PCNA eluted with imidazole. Lanes 3 6 – recombinant immobilized His-PCNA exposed to Leishmania whole cell extracts, and eluted with imidazole. WCE- whole cell extracts. The PCNA band detected in lanes 1 and 4 is probably due to background PCNA from the Leishmania whole cell extracts poured on the beads. B . Immunofluorescence analysis of MCM4-GFP and PCNA expression in synchronized cells. MCM4-GFP expression was analyzed by direct fluorescence. PCNA expression was analyzed by indirect fluorescence using anti-PCNA antibodies. Magnification bar represents 5 µm. C . MCM4 colocalizes with PCNA in S phase cells. MCM4-GFP transfectant Leishmania promastigotes synchronized with hydroxyurea and harvested three hours after release were labeled for PCNA immunofluorescence as described. Cells were analyzed by collecting Z stack images using a confocal microscope. Magnification bar represents 2 µm.

    Journal: PLoS ONE

    Article Title: Characterization of Leishmania donovani MCM4: Expression Patterns and Interaction with PCNA

    doi: 10.1371/journal.pone.0023107

    Figure Lengend Snippet: Leishmania MCM4 interacts with PCNA and colocalizes with it in S phase. A . MCM4 - PCNA interaction in pull-down experiments. Lanes 1–3 – without ATPγS; lanes 4–6 – with ATPγS. Lanes 1 4 – dummy metal affinity beads exposed to Leishmania whole cell extracts and elution carried out with imidazole. Lanes 2 5 – recombinant immobilized His-PCNA eluted with imidazole. Lanes 3 6 – recombinant immobilized His-PCNA exposed to Leishmania whole cell extracts, and eluted with imidazole. WCE- whole cell extracts. The PCNA band detected in lanes 1 and 4 is probably due to background PCNA from the Leishmania whole cell extracts poured on the beads. B . Immunofluorescence analysis of MCM4-GFP and PCNA expression in synchronized cells. MCM4-GFP expression was analyzed by direct fluorescence. PCNA expression was analyzed by indirect fluorescence using anti-PCNA antibodies. Magnification bar represents 5 µm. C . MCM4 colocalizes with PCNA in S phase cells. MCM4-GFP transfectant Leishmania promastigotes synchronized with hydroxyurea and harvested three hours after release were labeled for PCNA immunofluorescence as described. Cells were analyzed by collecting Z stack images using a confocal microscope. Magnification bar represents 2 µm.

    Article Snippet: 30–40 µg His-PCNA (overexpressed and purified from pASK-PCNA by Strep-Tactin chromatography) was incubated with Talon metal affinity resin beads (BD Biosciences) equilibrated with 50 mM Tris.Cl (pH 8.0) containing 300 mM NaCl, using a nutator mixer, at 4°C for 1 h. After removing unbound PCNA by two successive washes, whole cell extract from 1×109 exponentially growing promastigotes [that had been treated with 20 U of DNase I (New England Biolabs, USA) for 15 min with mixing using a nutator mixer during isolation of extract], was added in the presence or absence of 500 µM ATPγS.

    Techniques: Recombinant, Immunofluorescence, Expressing, Fluorescence, Transfection, Labeling, Microscopy

    Analysis of MCM4-GFP expression in Leishmania promastigotes. A . Western blot analysis of extracts from 5×10 7 cell equivalents (8% SDS-PAGE), analyzed using anti-GFP (1∶1000 dilution; Invitrogen) or anti-MCM4 antibody (1∶1000 dilution). Filled arrowheads – MCM4-GFP; open arrowheads – endogenous MCM4. B . Immunofluorescence analysis. G1/early S phase cells – one nucleus, one short kinetoplast. Late S phase/early G2/M cells – one nucleus, one elongated kinetoplast. Late G2/M phase cells - two nuclei, one kinetoplast or one nucleus, two kinetoplasts. Post-mitosis – two nuclei, two kinetoplasts. Cells were analyzed by collecting Z stack images using a confocal microscope. Magnification bar represents 2 µm. N-nucleus; K- kinetoplast. C . Immunofluorescence analysis of MCM4-GFP expression in logarithmically growing and stationary phase promastigotes. Magnification bar represents 5 µm.

    Journal: PLoS ONE

    Article Title: Characterization of Leishmania donovani MCM4: Expression Patterns and Interaction with PCNA

    doi: 10.1371/journal.pone.0023107

    Figure Lengend Snippet: Analysis of MCM4-GFP expression in Leishmania promastigotes. A . Western blot analysis of extracts from 5×10 7 cell equivalents (8% SDS-PAGE), analyzed using anti-GFP (1∶1000 dilution; Invitrogen) or anti-MCM4 antibody (1∶1000 dilution). Filled arrowheads – MCM4-GFP; open arrowheads – endogenous MCM4. B . Immunofluorescence analysis. G1/early S phase cells – one nucleus, one short kinetoplast. Late S phase/early G2/M cells – one nucleus, one elongated kinetoplast. Late G2/M phase cells - two nuclei, one kinetoplast or one nucleus, two kinetoplasts. Post-mitosis – two nuclei, two kinetoplasts. Cells were analyzed by collecting Z stack images using a confocal microscope. Magnification bar represents 2 µm. N-nucleus; K- kinetoplast. C . Immunofluorescence analysis of MCM4-GFP expression in logarithmically growing and stationary phase promastigotes. Magnification bar represents 5 µm.

    Article Snippet: 30–40 µg His-PCNA (overexpressed and purified from pASK-PCNA by Strep-Tactin chromatography) was incubated with Talon metal affinity resin beads (BD Biosciences) equilibrated with 50 mM Tris.Cl (pH 8.0) containing 300 mM NaCl, using a nutator mixer, at 4°C for 1 h. After removing unbound PCNA by two successive washes, whole cell extract from 1×109 exponentially growing promastigotes [that had been treated with 20 U of DNase I (New England Biolabs, USA) for 15 min with mixing using a nutator mixer during isolation of extract], was added in the presence or absence of 500 µM ATPγS.

    Techniques: Expressing, Western Blot, SDS Page, Immunofluorescence, Microscopy

    Analysis of cell cycle progression in Leishmania promastigotes. A . Flow cytometry analysis of Ld1S and Ld1S/MCM4-GFP. Data from 25000 events was collected for each time-point. In overlay histogram panels, solidly filled histograms - Ld1S/MCM4-GFP; green line - Ld1S histogram. B . The percent of cells at 4 h, 5 h and 6 h after release, that display the different stages of cell cycle (no significant differences were seen at earlier time-points), determined using the CellQuest Pro software. C . Flow cytometry analysis of Ld1S and Ld1S/GFP – overlay histograms. Solidly filled histograms - Ld1S/GFP; green line - histogram of Ld1S. G1, S and G2/M phases are indicated on the histograms by the gates M1, M2 and M3 respectively.

    Journal: PLoS ONE

    Article Title: Characterization of Leishmania donovani MCM4: Expression Patterns and Interaction with PCNA

    doi: 10.1371/journal.pone.0023107

    Figure Lengend Snippet: Analysis of cell cycle progression in Leishmania promastigotes. A . Flow cytometry analysis of Ld1S and Ld1S/MCM4-GFP. Data from 25000 events was collected for each time-point. In overlay histogram panels, solidly filled histograms - Ld1S/MCM4-GFP; green line - Ld1S histogram. B . The percent of cells at 4 h, 5 h and 6 h after release, that display the different stages of cell cycle (no significant differences were seen at earlier time-points), determined using the CellQuest Pro software. C . Flow cytometry analysis of Ld1S and Ld1S/GFP – overlay histograms. Solidly filled histograms - Ld1S/GFP; green line - histogram of Ld1S. G1, S and G2/M phases are indicated on the histograms by the gates M1, M2 and M3 respectively.

    Article Snippet: 30–40 µg His-PCNA (overexpressed and purified from pASK-PCNA by Strep-Tactin chromatography) was incubated with Talon metal affinity resin beads (BD Biosciences) equilibrated with 50 mM Tris.Cl (pH 8.0) containing 300 mM NaCl, using a nutator mixer, at 4°C for 1 h. After removing unbound PCNA by two successive washes, whole cell extract from 1×109 exponentially growing promastigotes [that had been treated with 20 U of DNase I (New England Biolabs, USA) for 15 min with mixing using a nutator mixer during isolation of extract], was added in the presence or absence of 500 µM ATPγS.

    Techniques: Flow Cytometry, Cytometry, Software

    Analysis of endogenous MCM4 expression in Leishmania promastigotes. A . Western blot analysis of extracts from proliferating promastigotes. Extracts (10% SDS-PAGE) probed with anti-MCM4 antibody (1∶1000 dilution), anti-PCNA antibody (1∶5000 dilution; loading control for nuclear extracts), anti-tubulin antibody (1∶2000 dilution; loading control for cytosolic extracts). CE- cytosolic extract; NE- nuclear extract. B . MCM4 expression at different stages of the cell cycle. Upper panel – Flow cytometry profiles of cells harvested at different timepoints. Lower panel - Western blot analysis of nuclear extracts (4×10 7 cell equivalents) using anti-MCM4 antibody (1∶1000 dilution) and anti-PCNA antibody (1∶5000 dilution; loading control). 1- log; 2- HU block; 3- S phase; 4- late S phase; 5- G2/M phase. Bar chart represents expression of MCM4 relative to PCNA. Arrowheads indicate MCM4.

    Journal: PLoS ONE

    Article Title: Characterization of Leishmania donovani MCM4: Expression Patterns and Interaction with PCNA

    doi: 10.1371/journal.pone.0023107

    Figure Lengend Snippet: Analysis of endogenous MCM4 expression in Leishmania promastigotes. A . Western blot analysis of extracts from proliferating promastigotes. Extracts (10% SDS-PAGE) probed with anti-MCM4 antibody (1∶1000 dilution), anti-PCNA antibody (1∶5000 dilution; loading control for nuclear extracts), anti-tubulin antibody (1∶2000 dilution; loading control for cytosolic extracts). CE- cytosolic extract; NE- nuclear extract. B . MCM4 expression at different stages of the cell cycle. Upper panel – Flow cytometry profiles of cells harvested at different timepoints. Lower panel - Western blot analysis of nuclear extracts (4×10 7 cell equivalents) using anti-MCM4 antibody (1∶1000 dilution) and anti-PCNA antibody (1∶5000 dilution; loading control). 1- log; 2- HU block; 3- S phase; 4- late S phase; 5- G2/M phase. Bar chart represents expression of MCM4 relative to PCNA. Arrowheads indicate MCM4.

    Article Snippet: 30–40 µg His-PCNA (overexpressed and purified from pASK-PCNA by Strep-Tactin chromatography) was incubated with Talon metal affinity resin beads (BD Biosciences) equilibrated with 50 mM Tris.Cl (pH 8.0) containing 300 mM NaCl, using a nutator mixer, at 4°C for 1 h. After removing unbound PCNA by two successive washes, whole cell extract from 1×109 exponentially growing promastigotes [that had been treated with 20 U of DNase I (New England Biolabs, USA) for 15 min with mixing using a nutator mixer during isolation of extract], was added in the presence or absence of 500 µM ATPγS.

    Techniques: Expressing, Western Blot, SDS Page, Flow Cytometry, Cytometry, Blocking Assay

    MCM4 expression in Leishmania . A . Schematic representation of the conserved domains in LdMCM4. PIP box - PCNA interacting protein motif; A – Walker A motif; B – Walker B motif; R – arginine finger; Z – zinc finger; S1 – sensor 1; S2 – sensor 2. B . SDS-PAGE (10% PAGE) analysis of overexpressed and purified recombinant LdMCM4 (2 ug; ∼97.2 kDa). C . Western blot analysis of recombinant LdMCM4 with mouse anti-MCM4 antibody (1∶5000 dilution). D . Western blot analysis (10% SDS-PAGE) using anti-MCM4 antibodies (dilution of 1∶1000). First panel - lane 1, recombinant MCM4; lane 2, L. donovani whole cell extracts. Second panel - Whole cell extracts from Leishmania promastigotes and amastigotes (4×10 7 cell equivalents) probed with anti-MCM4 antibodies and anti-tubulin antibodies (Zymed Laboratories; 1∶2000 dilution; loading control). E . Western blot analysis (10% SDS-PAGE) of whole cell extracts from Leishmania procyclics and metacyclics (4×10 7 cell equivalents) probed with anti-MCM4 antibodies and anti-tubulin antibodies (loading control).

    Journal: PLoS ONE

    Article Title: Characterization of Leishmania donovani MCM4: Expression Patterns and Interaction with PCNA

    doi: 10.1371/journal.pone.0023107

    Figure Lengend Snippet: MCM4 expression in Leishmania . A . Schematic representation of the conserved domains in LdMCM4. PIP box - PCNA interacting protein motif; A – Walker A motif; B – Walker B motif; R – arginine finger; Z – zinc finger; S1 – sensor 1; S2 – sensor 2. B . SDS-PAGE (10% PAGE) analysis of overexpressed and purified recombinant LdMCM4 (2 ug; ∼97.2 kDa). C . Western blot analysis of recombinant LdMCM4 with mouse anti-MCM4 antibody (1∶5000 dilution). D . Western blot analysis (10% SDS-PAGE) using anti-MCM4 antibodies (dilution of 1∶1000). First panel - lane 1, recombinant MCM4; lane 2, L. donovani whole cell extracts. Second panel - Whole cell extracts from Leishmania promastigotes and amastigotes (4×10 7 cell equivalents) probed with anti-MCM4 antibodies and anti-tubulin antibodies (Zymed Laboratories; 1∶2000 dilution; loading control). E . Western blot analysis (10% SDS-PAGE) of whole cell extracts from Leishmania procyclics and metacyclics (4×10 7 cell equivalents) probed with anti-MCM4 antibodies and anti-tubulin antibodies (loading control).

    Article Snippet: 30–40 µg His-PCNA (overexpressed and purified from pASK-PCNA by Strep-Tactin chromatography) was incubated with Talon metal affinity resin beads (BD Biosciences) equilibrated with 50 mM Tris.Cl (pH 8.0) containing 300 mM NaCl, using a nutator mixer, at 4°C for 1 h. After removing unbound PCNA by two successive washes, whole cell extract from 1×109 exponentially growing promastigotes [that had been treated with 20 U of DNase I (New England Biolabs, USA) for 15 min with mixing using a nutator mixer during isolation of extract], was added in the presence or absence of 500 µM ATPγS.

    Techniques: Expressing, SDS Page, Polyacrylamide Gel Electrophoresis, Purification, Recombinant, Western Blot

    Analysis of the importance of the PIP box domain. A . Creation of MCM4 PIP-box mutant. The mutated residues are indicated in red. B . Growth analysis of strains expressing MCM4-GFP and MCM4/PIP-GFP. Closed circles – MCM4-GFP; closed squares – MCM4/PIP-GFP. Cells were counted every 24 hours. Error bars indicate standard deviation. C . Western blot analysis of whole cell extracts made from MCM4/PIP-GFP transfectant cultures. Extracts were analyzed on 8% SDS-PAGE by probing with anti-GFP (1∶1000; Invitrogen) or anti-MCM4 (1∶1000) antibody. 1- MCM4-GFP (wild type); 2- MCM4/PIP-GFP mutant. Filled arrowheads - MCM4-GFP, open arrowheads - endogenous MCM4. D . Immunofluorescence analysis of promastigotes overexpressing MCM4-GFP and MCM4/PIP-GFP. Cells were examined for direct fluorescence of the protein, 12-14 days after drug-induced selection pressure. Magnification bar represents 5 µm.

    Journal: PLoS ONE

    Article Title: Characterization of Leishmania donovani MCM4: Expression Patterns and Interaction with PCNA

    doi: 10.1371/journal.pone.0023107

    Figure Lengend Snippet: Analysis of the importance of the PIP box domain. A . Creation of MCM4 PIP-box mutant. The mutated residues are indicated in red. B . Growth analysis of strains expressing MCM4-GFP and MCM4/PIP-GFP. Closed circles – MCM4-GFP; closed squares – MCM4/PIP-GFP. Cells were counted every 24 hours. Error bars indicate standard deviation. C . Western blot analysis of whole cell extracts made from MCM4/PIP-GFP transfectant cultures. Extracts were analyzed on 8% SDS-PAGE by probing with anti-GFP (1∶1000; Invitrogen) or anti-MCM4 (1∶1000) antibody. 1- MCM4-GFP (wild type); 2- MCM4/PIP-GFP mutant. Filled arrowheads - MCM4-GFP, open arrowheads - endogenous MCM4. D . Immunofluorescence analysis of promastigotes overexpressing MCM4-GFP and MCM4/PIP-GFP. Cells were examined for direct fluorescence of the protein, 12-14 days after drug-induced selection pressure. Magnification bar represents 5 µm.

    Article Snippet: 30–40 µg His-PCNA (overexpressed and purified from pASK-PCNA by Strep-Tactin chromatography) was incubated with Talon metal affinity resin beads (BD Biosciences) equilibrated with 50 mM Tris.Cl (pH 8.0) containing 300 mM NaCl, using a nutator mixer, at 4°C for 1 h. After removing unbound PCNA by two successive washes, whole cell extract from 1×109 exponentially growing promastigotes [that had been treated with 20 U of DNase I (New England Biolabs, USA) for 15 min with mixing using a nutator mixer during isolation of extract], was added in the presence or absence of 500 µM ATPγS.

    Techniques: Mutagenesis, Expressing, Standard Deviation, Western Blot, Transfection, SDS Page, Immunofluorescence, Fluorescence, Selection