Structured Review

Promega dnase i
MdcY-mediated protection of the  mdc  operator against digestion by DNase I. Lanes 1 to 4, target DNA (0.1 pmol, 2 × 10 5  cpm) was incubated in the absence or in the presence of MdcY (the number above each lane indicates the nanomolar concentration of MdcY protein). In lanes 5 to 7, target DNA and 2.4 nM MdcY were incubated with 50, 100, and 500 μM malonate, respectively. The vertical arrows beside the nucleotide sequence indicate a palindromic structure.
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Images

1) Product Images from "The Malonate Decarboxylase Operon of Acinetobacter calcoaceticus KCCM 40902 Is Regulated by Malonate and the Transcriptional Repressor MdcY"

Article Title: The Malonate Decarboxylase Operon of Acinetobacter calcoaceticus KCCM 40902 Is Regulated by Malonate and the Transcriptional Repressor MdcY

Journal: Journal of Bacteriology

doi:

MdcY-mediated protection of the  mdc  operator against digestion by DNase I. Lanes 1 to 4, target DNA (0.1 pmol, 2 × 10 5  cpm) was incubated in the absence or in the presence of MdcY (the number above each lane indicates the nanomolar concentration of MdcY protein). In lanes 5 to 7, target DNA and 2.4 nM MdcY were incubated with 50, 100, and 500 μM malonate, respectively. The vertical arrows beside the nucleotide sequence indicate a palindromic structure.
Figure Legend Snippet: MdcY-mediated protection of the mdc operator against digestion by DNase I. Lanes 1 to 4, target DNA (0.1 pmol, 2 × 10 5 cpm) was incubated in the absence or in the presence of MdcY (the number above each lane indicates the nanomolar concentration of MdcY protein). In lanes 5 to 7, target DNA and 2.4 nM MdcY were incubated with 50, 100, and 500 μM malonate, respectively. The vertical arrows beside the nucleotide sequence indicate a palindromic structure.

Techniques Used: Incubation, Concentration Assay, Sequencing

2) Product Images from "Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster"

Article Title: Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki281

Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to DNase I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.
Figure Legend Snippet: Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to DNase I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.

Techniques Used: Amplification

3) Product Images from "Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster"

Article Title: Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki281

Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to DNase I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.
Figure Legend Snippet: Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to DNase I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.

Techniques Used: Amplification

4) Product Images from "Identification of the Replication Origins from Cyanothece ATCC 51142 and Their Interactions with the DnaA Protein: From In Silico to In Vitro Studies"

Article Title: Identification of the Replication Origins from Cyanothece ATCC 51142 and Their Interactions with the DnaA Protein: From In Silico to In Vitro Studies

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2015.01370

DNase I footprint of protein His 6 -DnaA (IV) to the oriC s of the circular and linear chromosomes. (A) Along with the increase of His 6 -DnaA (IV), a clearly protected region of 39 nt relative to the second and third DnaA boxes sites in the oriC region of the circular chromosome was found, although the first DnaA box was not bound to the protein [the sequences in (A) are reverse complementary]. The asterisks mark the hypersensitive sites, which are consistent with the locations of two DnaA boxes of the circular chromosome. (B) Within the protected regions of the linear chromosome oriC , the results from the DNase I footprinting assay revealed that His 6 -DnaA (IV) protected two specific regions- an AT-rich region, as well as a region containing two DnaA boxes and an incomplete DnaA box (TATG) [the sequences in (B) are reverse complementary]. The asterisks mark the hypersensitive sites, which are consistent with the locations of two DnaA boxes of the linear chromosome.
Figure Legend Snippet: DNase I footprint of protein His 6 -DnaA (IV) to the oriC s of the circular and linear chromosomes. (A) Along with the increase of His 6 -DnaA (IV), a clearly protected region of 39 nt relative to the second and third DnaA boxes sites in the oriC region of the circular chromosome was found, although the first DnaA box was not bound to the protein [the sequences in (A) are reverse complementary]. The asterisks mark the hypersensitive sites, which are consistent with the locations of two DnaA boxes of the circular chromosome. (B) Within the protected regions of the linear chromosome oriC , the results from the DNase I footprinting assay revealed that His 6 -DnaA (IV) protected two specific regions- an AT-rich region, as well as a region containing two DnaA boxes and an incomplete DnaA box (TATG) [the sequences in (B) are reverse complementary]. The asterisks mark the hypersensitive sites, which are consistent with the locations of two DnaA boxes of the linear chromosome.

Techniques Used: Footprinting

5) Product Images from "Structural analysis of the regulatory mechanism of MarR protein Rv2887 in M. tuberculosis"

Article Title: Structural analysis of the regulatory mechanism of MarR protein Rv2887 in M. tuberculosis

Journal: Scientific Reports

doi: 10.1038/s41598-017-01705-4

MarR family protein Rv2887 binds to a sequence upstream of the Rv0560c gene. ( A ) EMSA experiment using a PCR-amplified DNA probe spanning the upstream region of Rv0560c. Migration of the DNA probe was retarded compared to free-labeled DNA on addition of Rv2887. A labeled random DNA sequence of the same length as the target probe was used as a control (lane 1) ( B ) Dye primer-based DNase I footprinting shows that Rv2887 binds directly to a sequence upstream of Rv0560c. Electropherograms indicating the protection pattern of the region upstream of Rv0560c on digestion with Dnase I after incubation with (I) 0 μg (II) 0.45 μg or (III) 0.9 μg Rv2887 protein. ( C ) The protected DNA sequence. The DNA sequence upstream of Rv0560c showing the Rv2887 binding site (highlighted in light green).
Figure Legend Snippet: MarR family protein Rv2887 binds to a sequence upstream of the Rv0560c gene. ( A ) EMSA experiment using a PCR-amplified DNA probe spanning the upstream region of Rv0560c. Migration of the DNA probe was retarded compared to free-labeled DNA on addition of Rv2887. A labeled random DNA sequence of the same length as the target probe was used as a control (lane 1) ( B ) Dye primer-based DNase I footprinting shows that Rv2887 binds directly to a sequence upstream of Rv0560c. Electropherograms indicating the protection pattern of the region upstream of Rv0560c on digestion with Dnase I after incubation with (I) 0 μg (II) 0.45 μg or (III) 0.9 μg Rv2887 protein. ( C ) The protected DNA sequence. The DNA sequence upstream of Rv0560c showing the Rv2887 binding site (highlighted in light green).

Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, Migration, Labeling, Footprinting, Incubation, Binding Assay

6) Product Images from "NADP, corepressor for the Bacillus catabolite control protein CcpA"

Article Title: NADP, corepressor for the Bacillus catabolite control protein CcpA

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Synergistic stimulation of combinations of HPr-P (Ser-46)/FDP and HPr-P (Ser-46)/NADP on CcpA binding to amyO . ( A ) DNase I footprints of amyO ). After the various concentrations of CcpA (2.2 pM-2.2 nM) were incubated with HPr-P (Ser-46) (0.68 μM) and FDP (3 mM) or HPr-P (Ser-46) (0.68 μM) and NADP (1 mM), amyO fragments were added to the mixture to allow formation of amyO –CcpA complexes. The complexes were digested with DNase I. C1 and C2 represent the DNase I-digested amyO fragments without (C1) and with (C2) CcpA (28 nM), respectively. The DNase I hypersensitivity band is indicated by an arrow. ( B ) Titration of CcpA binding to amyO by CcpA alone (•) and CcpA combined with FDP (♦), NADP (○), HPr-P (Ser-46) (■), HPr-P (Ser-46) + FDP (□), or HPr-P (Ser-46) + NADP (⋄). The DNA-binding isotherms were derived from original footprinting gels such as those shown in A . Fractional occupancy (%) was obtained by quantifying and normalizing the band intensity of diagnostic band (−2G) to that of the reference band (−14A) and plotted as a function of CcpA concentration. Both bands are shown by numbers in the figure.
Figure Legend Snippet: Synergistic stimulation of combinations of HPr-P (Ser-46)/FDP and HPr-P (Ser-46)/NADP on CcpA binding to amyO . ( A ) DNase I footprints of amyO ). After the various concentrations of CcpA (2.2 pM-2.2 nM) were incubated with HPr-P (Ser-46) (0.68 μM) and FDP (3 mM) or HPr-P (Ser-46) (0.68 μM) and NADP (1 mM), amyO fragments were added to the mixture to allow formation of amyO –CcpA complexes. The complexes were digested with DNase I. C1 and C2 represent the DNase I-digested amyO fragments without (C1) and with (C2) CcpA (28 nM), respectively. The DNase I hypersensitivity band is indicated by an arrow. ( B ) Titration of CcpA binding to amyO by CcpA alone (•) and CcpA combined with FDP (♦), NADP (○), HPr-P (Ser-46) (■), HPr-P (Ser-46) + FDP (□), or HPr-P (Ser-46) + NADP (⋄). The DNA-binding isotherms were derived from original footprinting gels such as those shown in A . Fractional occupancy (%) was obtained by quantifying and normalizing the band intensity of diagnostic band (−2G) to that of the reference band (−14A) and plotted as a function of CcpA concentration. Both bands are shown by numbers in the figure.

Techniques Used: Binding Assay, Incubation, Titration, Derivative Assay, Footprinting, Diagnostic Assay, Concentration Assay

Effect of HPr and HPr-P (Ser-46) on CcpA binding to amyO . Various concentrations of the HPr or HPr-P (Ser-46) (0.68–3.4 μM) were combined with the amount of CcpA required for one-half saturation of amyO (28 nM). After the amyO fragment was added to the binding buffer containing CcpA (C), CcpA + HPr, or CcpA + HPr-P (Ser-46) and allowed to form complexes; DNase I was added to digest unprotected DNA. A/G represents A+G ladder of coding strand of the amyO fragment and the arrow indicates a band showing hypersensitivity to DNase I digestion. The location of the amyO site is indicated by the box on the Left .
Figure Legend Snippet: Effect of HPr and HPr-P (Ser-46) on CcpA binding to amyO . Various concentrations of the HPr or HPr-P (Ser-46) (0.68–3.4 μM) were combined with the amount of CcpA required for one-half saturation of amyO (28 nM). After the amyO fragment was added to the binding buffer containing CcpA (C), CcpA + HPr, or CcpA + HPr-P (Ser-46) and allowed to form complexes; DNase I was added to digest unprotected DNA. A/G represents A+G ladder of coding strand of the amyO fragment and the arrow indicates a band showing hypersensitivity to DNase I digestion. The location of the amyO site is indicated by the box on the Left .

Techniques Used: Binding Assay

7) Product Images from "TetR-Type Regulator SLCG_2919 Is a Negative Regulator of Lincomycin Biosynthesis in Streptomyces lincolnensis"

Article Title: TetR-Type Regulator SLCG_2919 Is a Negative Regulator of Lincomycin Biosynthesis in Streptomyces lincolnensis

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.02091-18

Analysis of the precise SLCG_2919 binding site. (A) Determination of SLCG_2919 binding site in the promoter region of SLCG_2920 by DNase I footprinting assay. Top fluorogram shows control reaction without protein. Protection regions were acquired with increasing concentrations (0.5 μM and 0.75 μM) of His 6 -SLCG_2919 protein. (B) EMSAs of SLCG_2919 binding to P1 and P1m (lacking 24-nt sequence). Underlining indicates inverted repeats. (C) Nucleotide sequences of SLCG_2920 promoter region and SLCG_2919 binding site. Bigger black font, SLCG_2920 transcription start site (TSS); black dotted boxes, putative −10 and −35 regions and start codon; underlining, SLCG_2919 binding site. (D) The different base substitution mutagenesis of the binding site (24 nt). (E) EMSAs of SLCG_2919 binding to the fragment P1 (wild type [WT]) and mutated fragments P2, P3, P4, and P5.
Figure Legend Snippet: Analysis of the precise SLCG_2919 binding site. (A) Determination of SLCG_2919 binding site in the promoter region of SLCG_2920 by DNase I footprinting assay. Top fluorogram shows control reaction without protein. Protection regions were acquired with increasing concentrations (0.5 μM and 0.75 μM) of His 6 -SLCG_2919 protein. (B) EMSAs of SLCG_2919 binding to P1 and P1m (lacking 24-nt sequence). Underlining indicates inverted repeats. (C) Nucleotide sequences of SLCG_2920 promoter region and SLCG_2919 binding site. Bigger black font, SLCG_2920 transcription start site (TSS); black dotted boxes, putative −10 and −35 regions and start codon; underlining, SLCG_2919 binding site. (D) The different base substitution mutagenesis of the binding site (24 nt). (E) EMSAs of SLCG_2919 binding to the fragment P1 (wild type [WT]) and mutated fragments P2, P3, P4, and P5.

Techniques Used: Binding Assay, Footprinting, Sequencing, Mutagenesis

8) Product Images from "Effect of Damage Type on Stimulation of Human Excision Nuclease by SWI/SNF Chromatin Remodeling Factor"

Article Title: Effect of Damage Type on Stimulation of Human Excision Nuclease by SWI/SNF Chromatin Remodeling Factor

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.12.4121-4125.2003

DNase I footprinting of naked DNA and nucleosomal (Nuc) substrates. (A) Footprints of the AAF-G substrate and the corresponding undamaged DNA (UM). (B) Footprints of the two UV photoproducts and of unmodified DNA of identical sequence. The nucleosome reconstitution mixtures were treated with DNase I and were directly loaded on a 5% nondenaturing polyacrylamide gel. The free and nucleosomal DNA bands were located by autoradiography, were recovered from the gel, and were then separated on 6% denaturing polyacrylamide gels that were autoradiographed. The ∼10-nt periodicity in nucleosomal DNA is indicated by open arrows, and the positions of G-AAF and the UV photoproducts are marked.
Figure Legend Snippet: DNase I footprinting of naked DNA and nucleosomal (Nuc) substrates. (A) Footprints of the AAF-G substrate and the corresponding undamaged DNA (UM). (B) Footprints of the two UV photoproducts and of unmodified DNA of identical sequence. The nucleosome reconstitution mixtures were treated with DNase I and were directly loaded on a 5% nondenaturing polyacrylamide gel. The free and nucleosomal DNA bands were located by autoradiography, were recovered from the gel, and were then separated on 6% denaturing polyacrylamide gels that were autoradiographed. The ∼10-nt periodicity in nucleosomal DNA is indicated by open arrows, and the positions of G-AAF and the UV photoproducts are marked.

Techniques Used: Footprinting, Sequencing, Autoradiography

9) Product Images from "Purification and In Vitro Characterization of the Serratia marcescens NucC Protein, a Zinc-Binding Transcription Factor Homologous to P2 Ogr"

Article Title: Purification and In Vitro Characterization of the Serratia marcescens NucC Protein, a Zinc-Binding Transcription Factor Homologous to P2 Ogr

Journal: Journal of Bacteriology

doi: 10.1128/JB.185.6.1808-1816.2003

DNase I footprint analysis of NucC and RNA polymerase binding to P2 late promoter P F . The G and G + A lanes indicate Maxam-Gilbert sequencing reactions, and the bracketed regions indicate the approximate boundaries of the observed NucC and RNA polymerase binding sites. (A) DNase I footprint in the absence (−) and presence (+) of 1.3 μM NucC. (B) DNase I footprint in the presence of 85 nM RNA polymerase holoenzyme in the absence of NucC (−) and in the presence of increasing concentrations of NucC (8.7, 43, 87, and 430 nM). This figure was compiled by using Adobe Photoshop.
Figure Legend Snippet: DNase I footprint analysis of NucC and RNA polymerase binding to P2 late promoter P F . The G and G + A lanes indicate Maxam-Gilbert sequencing reactions, and the bracketed regions indicate the approximate boundaries of the observed NucC and RNA polymerase binding sites. (A) DNase I footprint in the absence (−) and presence (+) of 1.3 μM NucC. (B) DNase I footprint in the presence of 85 nM RNA polymerase holoenzyme in the absence of NucC (−) and in the presence of increasing concentrations of NucC (8.7, 43, 87, and 430 nM). This figure was compiled by using Adobe Photoshop.

Techniques Used: Binding Assay, Sequencing

10) Product Images from "Acetyl Coenzyme A Stimulates RNA Polymerase II Transcription and Promoter Binding by Transcription Factor IID in the Absence of Histones"

Article Title: Acetyl Coenzyme A Stimulates RNA Polymerase II Transcription and Promoter Binding by Transcription Factor IID in the Absence of Histones

Journal: Molecular and Cellular Biology

doi:

Acetyl-CoA stimulates TFIID binding to the adenovirus major late promoter, resulting in an extended DNase I footprint. (A) With subsaturating amounts of TFIID, acetyl-CoA stimulates TFIID binding to promoter DNA, as observed by DNase I footprinting. Reaction mixtures (lanes 1 to 3) contained 0.3 μl (∼8 ng) of TFIID and 20 ng of recombinant TFIIA. Acetyl-CoA (Pharmacia) was added to the reaction mixtures at final concentrations of 30 nM (lane 2) or 100 nM (lane 3). The reaction mixtures contained 0.4 nM 287-bp DNA fragment (positions −212 to +75) with five GAL4 sites upstream of the adenovirus major late core promoter (positions −53 to +33) that was 32 P labeled on the 5′ end of the template strand. Footprinting reactions were resolved by denaturing PAGE and analyzed with a PhosphorImager. Positions relative to the transcriptional start site (+1) are indicated on the left. Protected regions and an enhanced band are indicated on the right by brackets and an asterisk, respectively. (B) Acetyl-CoA causes an extension of the TFIID-TFIIA footprint into the region of the template DNA upstream of −70. Reaction mixtures (lanes 2 to 4) contained 0.8 μl (∼22 ng) of TFIID and 20 ng of recombinant TFIIA. Acetyl-CoA (Pharmacia) was added to the reaction mixtures at final concentrations of 30 nM (lane 3) or 100 nM (lanes 4 and 6). Reaction mixtures contained 1 nM 287-bp DNA fragment (positions −212 to +75) with five GAL4 sites upstream of the adenovirus major late core promoter (positions −53 to +33) that was 32 P labeled on the 5′ end of the template strand. Footprinting reactions were resolved by denaturing PAGE and analyzed by autoradiography. Positions relative to the transcriptional start site (+1) are indicated on the left. Protected regions and enhanced bands are indicated on the right by brackets and asterisks, respectively. (C) The sequence of the upstream region is not critical for the extended DNase I footprint in response to acetyl-CoA. Reaction mixtures (lanes 1 and 2) contained 0.6 μl (∼16 ng) of TFIID and 20 ng of recombinant TFIIA. Acetyl-CoA (Pharmacia) was added at a final concentration of 30 nM (lane 2). Reaction mixtures contained 0.4 nM 230-bp DNA fragment (positions −152 to +78) with plasmid DNA sequence upstream of the adenovirus major late core promoter (positions −53 to +33) that was 32 P labeled on the 5′ end of the template strand. Footprinting reactions were resolved by denaturing PAGE and analyzed with a PhosphorImager. Positions relative to the transcriptional start site (+1) are indicated on the left. Protected regions and an enhanced band are indicated on the right by brackets and an asterisk, respectively.
Figure Legend Snippet: Acetyl-CoA stimulates TFIID binding to the adenovirus major late promoter, resulting in an extended DNase I footprint. (A) With subsaturating amounts of TFIID, acetyl-CoA stimulates TFIID binding to promoter DNA, as observed by DNase I footprinting. Reaction mixtures (lanes 1 to 3) contained 0.3 μl (∼8 ng) of TFIID and 20 ng of recombinant TFIIA. Acetyl-CoA (Pharmacia) was added to the reaction mixtures at final concentrations of 30 nM (lane 2) or 100 nM (lane 3). The reaction mixtures contained 0.4 nM 287-bp DNA fragment (positions −212 to +75) with five GAL4 sites upstream of the adenovirus major late core promoter (positions −53 to +33) that was 32 P labeled on the 5′ end of the template strand. Footprinting reactions were resolved by denaturing PAGE and analyzed with a PhosphorImager. Positions relative to the transcriptional start site (+1) are indicated on the left. Protected regions and an enhanced band are indicated on the right by brackets and an asterisk, respectively. (B) Acetyl-CoA causes an extension of the TFIID-TFIIA footprint into the region of the template DNA upstream of −70. Reaction mixtures (lanes 2 to 4) contained 0.8 μl (∼22 ng) of TFIID and 20 ng of recombinant TFIIA. Acetyl-CoA (Pharmacia) was added to the reaction mixtures at final concentrations of 30 nM (lane 3) or 100 nM (lanes 4 and 6). Reaction mixtures contained 1 nM 287-bp DNA fragment (positions −212 to +75) with five GAL4 sites upstream of the adenovirus major late core promoter (positions −53 to +33) that was 32 P labeled on the 5′ end of the template strand. Footprinting reactions were resolved by denaturing PAGE and analyzed by autoradiography. Positions relative to the transcriptional start site (+1) are indicated on the left. Protected regions and enhanced bands are indicated on the right by brackets and asterisks, respectively. (C) The sequence of the upstream region is not critical for the extended DNase I footprint in response to acetyl-CoA. Reaction mixtures (lanes 1 and 2) contained 0.6 μl (∼16 ng) of TFIID and 20 ng of recombinant TFIIA. Acetyl-CoA (Pharmacia) was added at a final concentration of 30 nM (lane 2). Reaction mixtures contained 0.4 nM 230-bp DNA fragment (positions −152 to +78) with plasmid DNA sequence upstream of the adenovirus major late core promoter (positions −53 to +33) that was 32 P labeled on the 5′ end of the template strand. Footprinting reactions were resolved by denaturing PAGE and analyzed with a PhosphorImager. Positions relative to the transcriptional start site (+1) are indicated on the left. Protected regions and an enhanced band are indicated on the right by brackets and an asterisk, respectively.

Techniques Used: Binding Assay, Footprinting, Recombinant, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Sequencing, Concentration Assay, Plasmid Preparation

11) Product Images from "Mode of Action of the Bordetella BvgA Protein: Transcriptional Activation and Repression of the Bordetella bronchiseptica bipA Promoter"

Article Title: Mode of Action of the Bordetella BvgA Protein: Transcriptional Activation and Repression of the Bordetella bronchiseptica bipA Promoter

Journal:

doi: 10.1128/JB.187.18.6290-6299.2005

DNase I protection assays of the bipA promoter by purified BvgA and RNAP. Binding reaction mixtures contained a radiolabeled DNA template strand. Lanes 3 to 6, 7 to 10, and 11 to 14 contained, respectively, 0.04 μM, 0.8 μM, and 1.2 μM
Figure Legend Snippet: DNase I protection assays of the bipA promoter by purified BvgA and RNAP. Binding reaction mixtures contained a radiolabeled DNA template strand. Lanes 3 to 6, 7 to 10, and 11 to 14 contained, respectively, 0.04 μM, 0.8 μM, and 1.2 μM

Techniques Used: Purification, Binding Assay

DNase I footprinting with purified DNA-protein complexes. DNase I footprinting was performed as described for Fig. . The DNA-protein complexes were then purified on a native 4% gel and excised, and the digested DNA fragments were electroeluted.
Figure Legend Snippet: DNase I footprinting with purified DNA-protein complexes. DNase I footprinting was performed as described for Fig. . The DNA-protein complexes were then purified on a native 4% gel and excised, and the digested DNA fragments were electroeluted.

Techniques Used: Footprinting, Purification

12) Product Images from "Transcriptional Activation of Multiple Operons Involved in para-Nitrophenol Degradation by Pseudomonas sp. Strain WBC-3"

Article Title: Transcriptional Activation of Multiple Operons Involved in para-Nitrophenol Degradation by Pseudomonas sp. Strain WBC-3

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.02720-14

The DNase I footprinting analysis of PnpR binding to P pnpAB in which the sense strand (A) and antisense strand (B) are labeled. An amount of 0.025 μM probe P pnpAB covering the entire intergenic region of pnpA and pnpB was incubated with 1.6 μM
Figure Legend Snippet: The DNase I footprinting analysis of PnpR binding to P pnpAB in which the sense strand (A) and antisense strand (B) are labeled. An amount of 0.025 μM probe P pnpAB covering the entire intergenic region of pnpA and pnpB was incubated with 1.6 μM

Techniques Used: Footprinting, Binding Assay, Labeling, Incubation

13) Product Images from "Streptococcus mutans Extracellular DNA Is Upregulated during Growth in Biofilms, Actively Released via Membrane Vesicles, and Influenced by Components of the Protein Secretion Machinery"

Article Title: Streptococcus mutans Extracellular DNA Is Upregulated during Growth in Biofilms, Actively Released via Membrane Vesicles, and Influenced by Components of the Protein Secretion Machinery

Journal: Journal of Bacteriology

doi: 10.1128/JB.01493-14

Effect of DNase I on biofilm formation. S. mutans biofilms were grown in FMC with glucose (18 mM) and sucrose (2 mM) as the carbohydrate sources on hydroxylapatite discs vertically deposited in 24-well plates with inclusion of DNase I (DNase) for 5 and
Figure Legend Snippet: Effect of DNase I on biofilm formation. S. mutans biofilms were grown in FMC with glucose (18 mM) and sucrose (2 mM) as the carbohydrate sources on hydroxylapatite discs vertically deposited in 24-well plates with inclusion of DNase I (DNase) for 5 and

Techniques Used:

14) Product Images from "Streptococcus mutans Extracellular DNA Is Upregulated during Growth in Biofilms, Actively Released via Membrane Vesicles, and Influenced by Components of the Protein Secretion Machinery"

Article Title: Streptococcus mutans Extracellular DNA Is Upregulated during Growth in Biofilms, Actively Released via Membrane Vesicles, and Influenced by Components of the Protein Secretion Machinery

Journal: Journal of Bacteriology

doi: 10.1128/JB.01493-14

Effect of DNase I on biofilm formation. S. mutans biofilms were grown in FMC with glucose (18 mM) and sucrose (2 mM) as the carbohydrate sources on hydroxylapatite discs vertically deposited in 24-well plates with inclusion of DNase I (DNase) for 5 and
Figure Legend Snippet: Effect of DNase I on biofilm formation. S. mutans biofilms were grown in FMC with glucose (18 mM) and sucrose (2 mM) as the carbohydrate sources on hydroxylapatite discs vertically deposited in 24-well plates with inclusion of DNase I (DNase) for 5 and

Techniques Used:

15) Product Images from "VraR Binding to the Promoter Region of agr Inhibits Its Function in Vancomycin-Intermediate Staphylococcus aureus (VISA) and Heterogeneous VISA"

Article Title: VraR Binding to the Promoter Region of agr Inhibits Its Function in Vancomycin-Intermediate Staphylococcus aureus (VISA) and Heterogeneous VISA

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.02740-16

Identification of VraR binding sequences. (A) DNase I footprinting analysis of the agr promoter with VraR. (B) agr promoter sequence with a summary of the DNase I footprinting assay results. The −10 and −35 promoter regions are indicated
Figure Legend Snippet: Identification of VraR binding sequences. (A) DNase I footprinting analysis of the agr promoter with VraR. (B) agr promoter sequence with a summary of the DNase I footprinting assay results. The −10 and −35 promoter regions are indicated

Techniques Used: Binding Assay, Footprinting, Sequencing

16) Product Images from "RovM and CsrA Negatively Regulate Urease Expression in Yersinia pseudotuberculosis"

Article Title: RovM and CsrA Negatively Regulate Urease Expression in Yersinia pseudotuberculosis

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.00348

RovM directly binds to the urease promoter. (A) Gel retardation assay analysis of the interaction between RovM-His 6 and the urease promoter ( P ureABC ), a fragment derived from the coding sequence (CDS) of urease structural gene ureC was used as a negative control. Probe concentrations were 20 ng/μL with increased protein concentration (0.3, 0.6, and 1.2 μM). As a negative control ( NC ), a fragment of ureC (urease structural gene) was subjected to the same protein concentration gradient. (B) DNase I footprinting assay identified RovM binding sites in the urease promoter region. (C) Nucleotide sequences of the urease promoter region [from –800 to –500 relative to the ATG start codon of the first ORF (open reading frame) (Ypk_1131, ureA ) of the urease operon]. The probe used for EMSA was amplified from –754 to –568 relative to the ATG start codon of the first urease ORF (Ypk_1131, ureA ). The red region denotes the RovM binding site identified in the DNase I footprinting assay extending from –754 to –681 (74 bp) upstream of the initiation codon of the first urease ORF ( ureA , Ypk_1131)]. The RovM binding site identified with the DNase I footprinting assay was indicated by shading. Putative –35 and –10 elements of the urease promoter are boxed. +1 denotes the transcription start point. The red number above the nucleotide sequence indicates the position relative to the ATG start codon of the first urease ORF (Ypk_1131, ureA ).
Figure Legend Snippet: RovM directly binds to the urease promoter. (A) Gel retardation assay analysis of the interaction between RovM-His 6 and the urease promoter ( P ureABC ), a fragment derived from the coding sequence (CDS) of urease structural gene ureC was used as a negative control. Probe concentrations were 20 ng/μL with increased protein concentration (0.3, 0.6, and 1.2 μM). As a negative control ( NC ), a fragment of ureC (urease structural gene) was subjected to the same protein concentration gradient. (B) DNase I footprinting assay identified RovM binding sites in the urease promoter region. (C) Nucleotide sequences of the urease promoter region [from –800 to –500 relative to the ATG start codon of the first ORF (open reading frame) (Ypk_1131, ureA ) of the urease operon]. The probe used for EMSA was amplified from –754 to –568 relative to the ATG start codon of the first urease ORF (Ypk_1131, ureA ). The red region denotes the RovM binding site identified in the DNase I footprinting assay extending from –754 to –681 (74 bp) upstream of the initiation codon of the first urease ORF ( ureA , Ypk_1131)]. The RovM binding site identified with the DNase I footprinting assay was indicated by shading. Putative –35 and –10 elements of the urease promoter are boxed. +1 denotes the transcription start point. The red number above the nucleotide sequence indicates the position relative to the ATG start codon of the first urease ORF (Ypk_1131, ureA ).

Techniques Used: Electrophoretic Mobility Shift Assay, Derivative Assay, Sequencing, Negative Control, Protein Concentration, Footprinting, Binding Assay, Amplification

17) Product Images from "A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5? End or within the Genome of Feline Parvovirus and Can Modify Virus Replication"

Article Title: A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5? End or within the Genome of Feline Parvovirus and Can Modify Virus Replication

Journal: Journal of Virology

doi:

Protein binding and protection of sequences within the FPV genome. (A) DNase I protection of the negative and positive strands of the region from nt 290 to 370 of the FPV genome by increasing amounts of DBP40, in the region where the block to DNA polymerization appeared to occur. Numbers represent nucleotides in the complete FPV genome. (B) Sequence of the FPV genome between nt 250 and 340, showing the approximate region protected by the bound DBP40. (C) EMSA showing binding of 20 ng of DBP40 to 32 P-labeled oligonucleotides containing the sequences from the negative (−ve) and positive (+ve) strands of the viral DNA in the region protected and competition with 10- and 100-fold excess of the same unlabeled oligonucleotide.
Figure Legend Snippet: Protein binding and protection of sequences within the FPV genome. (A) DNase I protection of the negative and positive strands of the region from nt 290 to 370 of the FPV genome by increasing amounts of DBP40, in the region where the block to DNA polymerization appeared to occur. Numbers represent nucleotides in the complete FPV genome. (B) Sequence of the FPV genome between nt 250 and 340, showing the approximate region protected by the bound DBP40. (C) EMSA showing binding of 20 ng of DBP40 to 32 P-labeled oligonucleotides containing the sequences from the negative (−ve) and positive (+ve) strands of the viral DNA in the region protected and competition with 10- and 100-fold excess of the same unlabeled oligonucleotide.

Techniques Used: Protein Binding, Blocking Assay, Sequencing, Binding Assay, Labeling

ELISA optical density at 450 nm (OD 450 ) of T7 phage 40 screened from the library by panning on full FPV capsids. The phage supernatant was incubated in wells coated with full FPV capsids, empty FPV capsids, or capsids pretreated with DNase I, nuclease A, or micrococcal nuclease. The bound phage were detected with a horseradish peroxidase-conjugated antibody against the T7 capsid protein.
Figure Legend Snippet: ELISA optical density at 450 nm (OD 450 ) of T7 phage 40 screened from the library by panning on full FPV capsids. The phage supernatant was incubated in wells coated with full FPV capsids, empty FPV capsids, or capsids pretreated with DNase I, nuclease A, or micrococcal nuclease. The bound phage were detected with a horseradish peroxidase-conjugated antibody against the T7 capsid protein.

Techniques Used: Enzyme-linked Immunosorbent Assay, Incubation

). Arrows indicate the DNase I-sensitive sites. Numbering is from the complete FPV genome sequence, starting at the left-hand end.
Figure Legend Snippet: ). Arrows indicate the DNase I-sensitive sites. Numbering is from the complete FPV genome sequence, starting at the left-hand end.

Techniques Used: Sequencing

18) Product Images from "The Nitrogen Regulator GlnR Directly Controls Transcription of the prpDBC Operon Involved in Methylcitrate Cycle in Mycobacterium smegmatis"

Article Title: The Nitrogen Regulator GlnR Directly Controls Transcription of the prpDBC Operon Involved in Methylcitrate Cycle in Mycobacterium smegmatis

Journal: Journal of Bacteriology

doi: 10.1128/JB.00099-19

Binding of GlnR or PrpR with the upstream region of prpDBC . (A) DNase I footprinting assay for GlnR binding motif characterization. (B) EMSA of His-GlnR with biotin labeled EMSA fragment (P1). (C) GGACCGGCACCGTAAC. EMSA of His-GlnR with biotin labeled EMSA fragment (P2). (D and E) AGACTGGCACCATGGT. Binding of the prpDBC biotin-labeled probe with increasing concentrations of His-GlnR (2.4, 1.2, 0.6, 0.3, and 0.015 μM) (D) and His-PrpR (6.4, 3.2, 1.6, 0.8, 0.4, 0 μM) (E).
Figure Legend Snippet: Binding of GlnR or PrpR with the upstream region of prpDBC . (A) DNase I footprinting assay for GlnR binding motif characterization. (B) EMSA of His-GlnR with biotin labeled EMSA fragment (P1). (C) GGACCGGCACCGTAAC. EMSA of His-GlnR with biotin labeled EMSA fragment (P2). (D and E) AGACTGGCACCATGGT. Binding of the prpDBC biotin-labeled probe with increasing concentrations of His-GlnR (2.4, 1.2, 0.6, 0.3, and 0.015 μM) (D) and His-PrpR (6.4, 3.2, 1.6, 0.8, 0.4, 0 μM) (E).

Techniques Used: Binding Assay, Footprinting, Labeling

19) Product Images from "SsaA, a Member of a Novel Class of Transcriptional Regulators, Controls Sansanmycin Production in Streptomyces sp. Strain SS through a Feedback Mechanism"

Article Title: SsaA, a Member of a Novel Class of Transcriptional Regulators, Controls Sansanmycin Production in Streptomyces sp. Strain SS through a Feedback Mechanism

Journal: Journal of Bacteriology

doi: 10.1128/JB.00054-13

Identification of SsaA binding sites. (A) Identification of SsaA binding site in ssaN-P p promoter region by DNase I footprinting. The upper electropherogram shows the control reaction, and the lower one shows DNase I footprints of His 10 -SsaA (2.67 μM) bound to the labeled DNA fragments (50 ng). The protected nucleotide sequences are shaded gray. The nucleotide positions of the protected sequences respective to the ssaP translation start point are shown below the gray shading. (B) Identification of the SsaA consensus binding site by WebLogo. Six sequences were aligned, including 5 sequences detected by the DNase I footprinting experiments and 1 sequence upstream of ssaW that is identical to that of ssaU . The base-pairing nucleotides within the binding site are underlined. (C) Validation of the identified SsaA consensus binding site. The binding of SsaA to the three synthetic duplexes, D1, D2, and D3 (the dashed lines in D3 indicate the deletion of 5 nucleotides), was detected by EMSA analysis. −, probe only; +, probe incubated with 1.75 μM His 10 -SsaA.
Figure Legend Snippet: Identification of SsaA binding sites. (A) Identification of SsaA binding site in ssaN-P p promoter region by DNase I footprinting. The upper electropherogram shows the control reaction, and the lower one shows DNase I footprints of His 10 -SsaA (2.67 μM) bound to the labeled DNA fragments (50 ng). The protected nucleotide sequences are shaded gray. The nucleotide positions of the protected sequences respective to the ssaP translation start point are shown below the gray shading. (B) Identification of the SsaA consensus binding site by WebLogo. Six sequences were aligned, including 5 sequences detected by the DNase I footprinting experiments and 1 sequence upstream of ssaW that is identical to that of ssaU . The base-pairing nucleotides within the binding site are underlined. (C) Validation of the identified SsaA consensus binding site. The binding of SsaA to the three synthetic duplexes, D1, D2, and D3 (the dashed lines in D3 indicate the deletion of 5 nucleotides), was detected by EMSA analysis. −, probe only; +, probe incubated with 1.75 μM His 10 -SsaA.

Techniques Used: Binding Assay, Footprinting, Labeling, Sequencing, Incubation

20) Product Images from "Cleavage of Phosphorothioated DNA and Methylated DNA by the Type IV Restriction Endonuclease ScoMcrA"

Article Title: Cleavage of Phosphorothioated DNA and Methylated DNA by the Type IV Restriction Endonuclease ScoMcrA

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1001253

In vitro DNase I protection and cleavage of a double-stranded 164 bp Dcm-methylated synthetic oligonucleotide by purified His 6 -Sco4631. A. Analysis of 5′ labeled top strand DNA, and B, 5′ labeled bottom strand DNA. Bands a-d on these autoradiographs represent cleavage of the DNA strands by His 6 -tagged Sco4631. Lanes 1–3 are controls lacking either the enzyme (E) or the Dcm methylation (M). The samples in lanes 4 contained both methylated DNA and active His 6 -Sco4631. Lanes 5–8 are sequencing ladders. Lanes 9–12 are controls containing unmethylated DNA and increasing amounts of active His 6 -Sco4631. Lanes 13–16 contained methylated DNA and increasing amounts of the active His 6 -Sco4631. Lanes 9 and 13 contained no enzyme, and lanes 10–12 and lanes 14–16 contained 1.1, 4.5 and 18 µM enzyme, respectively. The vertical sequence to the right of each gel picture indicates the DNA regions that were partially protected from cleavage by DNase I. The horizontal lines point to bases that are not protected and shown in lower case letters. (Please note, panel A shows the correct sequence of the top strand, but in panel B there is a compression of the sequence with the bold bases in the sequence CCAGGTGCGAATAAG not visible.) C. Central sequence of the 164 bp double-stranded oligonucleotide containing the Dcm methylated sequence C5mCWGG (dark blue) and additional sequences protected against DNase I activity (light blue). The fat vertical arrows labeled a and b are the major cut sites, and the thin arrows labeled c and d are minor cut sites indicated in the gels A and B. Arrows with numbers indicate cut sites that were identified by cloning and sequencing (see Figure 3 ). Bases printed light grey are not visible on the gel sections shown.
Figure Legend Snippet: In vitro DNase I protection and cleavage of a double-stranded 164 bp Dcm-methylated synthetic oligonucleotide by purified His 6 -Sco4631. A. Analysis of 5′ labeled top strand DNA, and B, 5′ labeled bottom strand DNA. Bands a-d on these autoradiographs represent cleavage of the DNA strands by His 6 -tagged Sco4631. Lanes 1–3 are controls lacking either the enzyme (E) or the Dcm methylation (M). The samples in lanes 4 contained both methylated DNA and active His 6 -Sco4631. Lanes 5–8 are sequencing ladders. Lanes 9–12 are controls containing unmethylated DNA and increasing amounts of active His 6 -Sco4631. Lanes 13–16 contained methylated DNA and increasing amounts of the active His 6 -Sco4631. Lanes 9 and 13 contained no enzyme, and lanes 10–12 and lanes 14–16 contained 1.1, 4.5 and 18 µM enzyme, respectively. The vertical sequence to the right of each gel picture indicates the DNA regions that were partially protected from cleavage by DNase I. The horizontal lines point to bases that are not protected and shown in lower case letters. (Please note, panel A shows the correct sequence of the top strand, but in panel B there is a compression of the sequence with the bold bases in the sequence CCAGGTGCGAATAAG not visible.) C. Central sequence of the 164 bp double-stranded oligonucleotide containing the Dcm methylated sequence C5mCWGG (dark blue) and additional sequences protected against DNase I activity (light blue). The fat vertical arrows labeled a and b are the major cut sites, and the thin arrows labeled c and d are minor cut sites indicated in the gels A and B. Arrows with numbers indicate cut sites that were identified by cloning and sequencing (see Figure 3 ). Bases printed light grey are not visible on the gel sections shown.

Techniques Used: In Vitro, Methylation, Purification, Labeling, Sequencing, Activity Assay, Clone Assay

21) Product Images from "Changes in the reproductive function and developmental phenotypes in mice following intramuscular injection of an activin betaA-expressing plasmid"

Article Title: Changes in the reproductive function and developmental phenotypes in mice following intramuscular injection of an activin betaA-expressing plasmid

Journal: Reproductive Biology and Endocrinology : RB & E

doi: 10.1186/1477-7827-6-63

pCMV-rAct structure and activin expression . (A) Diagram of the pCMV-rAct construct. Functional elements include the cytomegalovirus (CMV) promoter, the activin cDNA, and the human growth hormone (hGH) poly(A) signal. (B) RT-PCR and (C) Southern blot analysis were performed as described in methods. Marker of pGEM3Z/infI was loaded in lane 1. RNA from the pCMV-rAct-injected mice was loaded in lane 3 after treatment with DNase I, reverse transcriptase, and PCR. RNAs from control mice and from the pCMV-rAct-injected mice without reverse transcription were used as a normal (lane 2) and an internal control (lane 4), respectively. pCMV-rAct plasmid was used as a positive control (lane 5). RT: reverse transcription, pCMV-rAct: pCMV-rAct-injected mice. +: treated (or reacted), -: not treated (or not reacted).
Figure Legend Snippet: pCMV-rAct structure and activin expression . (A) Diagram of the pCMV-rAct construct. Functional elements include the cytomegalovirus (CMV) promoter, the activin cDNA, and the human growth hormone (hGH) poly(A) signal. (B) RT-PCR and (C) Southern blot analysis were performed as described in methods. Marker of pGEM3Z/infI was loaded in lane 1. RNA from the pCMV-rAct-injected mice was loaded in lane 3 after treatment with DNase I, reverse transcriptase, and PCR. RNAs from control mice and from the pCMV-rAct-injected mice without reverse transcription were used as a normal (lane 2) and an internal control (lane 4), respectively. pCMV-rAct plasmid was used as a positive control (lane 5). RT: reverse transcription, pCMV-rAct: pCMV-rAct-injected mice. +: treated (or reacted), -: not treated (or not reacted).

Techniques Used: Expressing, Construct, Functional Assay, Reverse Transcription Polymerase Chain Reaction, Southern Blot, Marker, Injection, Mouse Assay, Polymerase Chain Reaction, Plasmid Preparation, Positive Control

22) Product Images from "The Epstein-Barr virus miR-BHRF1-1 targets RNF4 during productive infection to promote the accumulation of SUMO conjugates and the release of infectious virus"

Article Title: The Epstein-Barr virus miR-BHRF1-1 targets RNF4 during productive infection to promote the accumulation of SUMO conjugates and the release of infectious virus

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006338

Reconstitution of RNF4 hampers the release of virus particles. The productive virus cycle was induced by anti-IgG treatment in Akata-Bx1, Akata-Bx1 expressing the inducible miR-BHRF1-1 sponge and Akata-Bx1 expressing a miR-BHRF1-1 resistant RNF4 cultured in the presence or absence of doxycycline. Intact virus particles released in the supernatants harvested after 3 and 7 days were measured by qPCR after treatment with DNase I. The amount of virus was estimated relative to a standard curve constructed by serial dilutions of a BZLF1 encoding plasmid. Reconstitution of RNF4 expression in cells expressing the miR-BHRF1-1 sponge ( A ) or miR-BHRF1-1 resistant RNF4 ( B ) was accompanied by a significantly reduced virus yield. The mean ± SD of two to three experiments is shown.
Figure Legend Snippet: Reconstitution of RNF4 hampers the release of virus particles. The productive virus cycle was induced by anti-IgG treatment in Akata-Bx1, Akata-Bx1 expressing the inducible miR-BHRF1-1 sponge and Akata-Bx1 expressing a miR-BHRF1-1 resistant RNF4 cultured in the presence or absence of doxycycline. Intact virus particles released in the supernatants harvested after 3 and 7 days were measured by qPCR after treatment with DNase I. The amount of virus was estimated relative to a standard curve constructed by serial dilutions of a BZLF1 encoding plasmid. Reconstitution of RNF4 expression in cells expressing the miR-BHRF1-1 sponge ( A ) or miR-BHRF1-1 resistant RNF4 ( B ) was accompanied by a significantly reduced virus yield. The mean ± SD of two to three experiments is shown.

Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Construct, Plasmid Preparation

23) Product Images from "Ash1l and lnc-Smad3 coordinate Smad3 locus accessibility to modulate iTreg polarization and T cell autoimmunity"

Article Title: Ash1l and lnc-Smad3 coordinate Smad3 locus accessibility to modulate iTreg polarization and T cell autoimmunity

Journal: Nature Communications

doi: 10.1038/ncomms15818

Lnc-Smad3 reduces the accessibility of the Smad3 promoter to Ash1l. ( a ) CHIP analysis of the H3K4me3 and H3K27ac modifications, and Pol II occupancy around lnc-Smad3 and Smad3 loci in CD4 + T cells left unstimulated (day 0) or cultured under iTreg cell-skewing conditions (with TGF-β) for 2 days (day 2). Four regions (capital letters A–D) across lnc-Smad3 gene locus and three regions (capital letters E–G) across Smad3 gene locus were analysed by CHIP assay. Normalized data are shown as percentage of input control. ( b ) HepG2 cells were transfected with a Smad3 promoter reporter construct (1 kb upstream of the transcription start site) and lnc-Smad3 expression vector (lnc-Smad3) or empty control vector (CTR), and stimulated with TGF-β. Luciferase activity was measured 48 h later. Data were normalized to renilla luciferase and presented with respect to CTR, set as 1. ( c ) Chromatin accessibility of the Smad3 promoter region by quantitative PCR with DNase I pretreated nucleus of CD4 + T cells transduced with a control lentivirus (Lenti-CTR) or lnc-Smad3-expressing lentivirus (Lenti-lnc-Smad3) and cultured under iTreg cell-skewing conditions (with TGF-β) for 2 days. Changed fold are concluded using 2 ΔCt with respect to CD4 + T cells transduced with Lenti-CTR, set as 1. ( d ) CHIP analysis of the accumulation of Ash1l and H3K4me3 modification at Smad3 promoter regions in CD4 + T cells transduced and cultured as in c . Normalized data are shown as percentage of input control (% Inp). IgG serves as a CHIP control. Error bars represent s.d. Student's t test. * P
Figure Legend Snippet: Lnc-Smad3 reduces the accessibility of the Smad3 promoter to Ash1l. ( a ) CHIP analysis of the H3K4me3 and H3K27ac modifications, and Pol II occupancy around lnc-Smad3 and Smad3 loci in CD4 + T cells left unstimulated (day 0) or cultured under iTreg cell-skewing conditions (with TGF-β) for 2 days (day 2). Four regions (capital letters A–D) across lnc-Smad3 gene locus and three regions (capital letters E–G) across Smad3 gene locus were analysed by CHIP assay. Normalized data are shown as percentage of input control. ( b ) HepG2 cells were transfected with a Smad3 promoter reporter construct (1 kb upstream of the transcription start site) and lnc-Smad3 expression vector (lnc-Smad3) or empty control vector (CTR), and stimulated with TGF-β. Luciferase activity was measured 48 h later. Data were normalized to renilla luciferase and presented with respect to CTR, set as 1. ( c ) Chromatin accessibility of the Smad3 promoter region by quantitative PCR with DNase I pretreated nucleus of CD4 + T cells transduced with a control lentivirus (Lenti-CTR) or lnc-Smad3-expressing lentivirus (Lenti-lnc-Smad3) and cultured under iTreg cell-skewing conditions (with TGF-β) for 2 days. Changed fold are concluded using 2 ΔCt with respect to CD4 + T cells transduced with Lenti-CTR, set as 1. ( d ) CHIP analysis of the accumulation of Ash1l and H3K4me3 modification at Smad3 promoter regions in CD4 + T cells transduced and cultured as in c . Normalized data are shown as percentage of input control (% Inp). IgG serves as a CHIP control. Error bars represent s.d. Student's t test. * P

Techniques Used: Chromatin Immunoprecipitation, Cell Culture, Transfection, Construct, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Transduction, Modification

24) Product Images from "Fabrication and characterization of DNA-loaded zein nanospheres"

Article Title: Fabrication and characterization of DNA-loaded zein nanospheres

Journal: Journal of Nanobiotechnology

doi: 10.1186/1477-3155-10-44

Agarose gel electrophoresis images of extracted samples for spheres made at various zein to DNA ratios (A): lane 1, ladder; lane 2, stock DNA; lane 3, 20:1 spheres; lane 4, 20:1 supernatant; lane 5, 40:1 spheres; lane 6, 40:1 supernatant; lane 7, 80:1 spheres; lane 8, 80:1 supernatant; lane 9, 160:1 spheres; lane 10, 160:1 supernatant; lane 11, 250:1 spheres; lane 12, 250:1 supernatant; lane 13, stock DNA; lane 14 ladder. Agarose gel image of DNA extracted from spheres ( B ) and supernatants ( C ) at various time points in the PBS release study: lane 1, ladder; lane 2, stock DNA; lane 3, 0 hr; lane 4, 1 hr; lane 5, 3 hr; lane 6, 6 hr; lane 7, 9 hr; lane 8, 12 hr; lane 9, 24 hr; lane 10, ladder; lane 11, 48 hr; lane 12, 72 hr; lane 13, 96 hr; lane 14, 120 hr; lane 15, 144 hr; lane 16, 168 hr; lane 17, stock DNA; lane 18, ladder. Agarose gel electrophoresis images of pDNA in DNase I degradation assay : lane 1, ladder; lane 2, stock DNA; lane 3, Naked DNA + DNase I; lane 4, 80:1 spheres + DNase I; lane 5, blank spheres (zein with no DNA); lane 6, 80:1 spheres ; lane 7, stock DNA; lane 8, ladder.
Figure Legend Snippet: Agarose gel electrophoresis images of extracted samples for spheres made at various zein to DNA ratios (A): lane 1, ladder; lane 2, stock DNA; lane 3, 20:1 spheres; lane 4, 20:1 supernatant; lane 5, 40:1 spheres; lane 6, 40:1 supernatant; lane 7, 80:1 spheres; lane 8, 80:1 supernatant; lane 9, 160:1 spheres; lane 10, 160:1 supernatant; lane 11, 250:1 spheres; lane 12, 250:1 supernatant; lane 13, stock DNA; lane 14 ladder. Agarose gel image of DNA extracted from spheres ( B ) and supernatants ( C ) at various time points in the PBS release study: lane 1, ladder; lane 2, stock DNA; lane 3, 0 hr; lane 4, 1 hr; lane 5, 3 hr; lane 6, 6 hr; lane 7, 9 hr; lane 8, 12 hr; lane 9, 24 hr; lane 10, ladder; lane 11, 48 hr; lane 12, 72 hr; lane 13, 96 hr; lane 14, 120 hr; lane 15, 144 hr; lane 16, 168 hr; lane 17, stock DNA; lane 18, ladder. Agarose gel electrophoresis images of pDNA in DNase I degradation assay : lane 1, ladder; lane 2, stock DNA; lane 3, Naked DNA + DNase I; lane 4, 80:1 spheres + DNase I; lane 5, blank spheres (zein with no DNA); lane 6, 80:1 spheres ; lane 7, stock DNA; lane 8, ladder.

Techniques Used: Agarose Gel Electrophoresis, Degradation Assay

25) Product Images from "MgrA Negatively Regulates Biofilm Formation and Detachment by Repressing the Expression of psm Operons in Staphylococcus aureus"

Article Title: MgrA Negatively Regulates Biofilm Formation and Detachment by Repressing the Expression of psm Operons in Staphylococcus aureus

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.01008-18

(A to C) Mapping of the MgrA recognition site in the psmα promoter by DNase I footprinting. The 336-bp psmα promoter fragment was labeled with 6-FAM, and probes (1 pmol/100 μl) were incubated for 1 h at 25°C with MgrA at 0 μg/100 μl (A), 2 μg/100 μl (B), or 4 μg/100 μl (C) and then digested for 1 min at 37°C with DNase I at 0.1 U/100 μl. The protected region of MgrA is boxed in black. (D) MgrA binding sequence in the psmα promoter region. The MgrA binding region, based on the DNase footprinting analyses, is underlined in black. SD, Shine-Dalgarno sequence.
Figure Legend Snippet: (A to C) Mapping of the MgrA recognition site in the psmα promoter by DNase I footprinting. The 336-bp psmα promoter fragment was labeled with 6-FAM, and probes (1 pmol/100 μl) were incubated for 1 h at 25°C with MgrA at 0 μg/100 μl (A), 2 μg/100 μl (B), or 4 μg/100 μl (C) and then digested for 1 min at 37°C with DNase I at 0.1 U/100 μl. The protected region of MgrA is boxed in black. (D) MgrA binding sequence in the psmα promoter region. The MgrA binding region, based on the DNase footprinting analyses, is underlined in black. SD, Shine-Dalgarno sequence.

Techniques Used: Footprinting, Labeling, Incubation, Binding Assay, Sequencing

26) Product Images from "In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium"

Article Title: In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium

Journal: PLoS ONE

doi: 10.1371/journal.pone.0177751

DdrC protects DNA against degradation by nucleases. Protection of supercoiled pBR322 plasmid (3.5 nM) from DNase I activity (0.1 U) (panel a), linear pBR322 (3.5 nM) from Exonuclease III activity (200 U) (panel b) and phiX174 ssDNA (5.9 nM) from Mung Bean Nuclease activity (1 U) (panel c) by 7 μM, 7 μM, and 2 μM DdrC, respectively. Lanes C: DNA controls without protein. Lanes 1: DNA incubation with nuclease alone. Lanes 2: DNA incubation with DdrC alone. Lanes 3: DNA pre-incubated with DdrC 15 min at 4°C before addition of nuclease. Lanes 4: Reaction products corresponding to lane 3 were further treated with Proteinase K/SDS. Panel a, lane 5: DdrC and DNase I were simultaneously incubated with supercoiled DNA before treatment with Proteinase K/SDS.
Figure Legend Snippet: DdrC protects DNA against degradation by nucleases. Protection of supercoiled pBR322 plasmid (3.5 nM) from DNase I activity (0.1 U) (panel a), linear pBR322 (3.5 nM) from Exonuclease III activity (200 U) (panel b) and phiX174 ssDNA (5.9 nM) from Mung Bean Nuclease activity (1 U) (panel c) by 7 μM, 7 μM, and 2 μM DdrC, respectively. Lanes C: DNA controls without protein. Lanes 1: DNA incubation with nuclease alone. Lanes 2: DNA incubation with DdrC alone. Lanes 3: DNA pre-incubated with DdrC 15 min at 4°C before addition of nuclease. Lanes 4: Reaction products corresponding to lane 3 were further treated with Proteinase K/SDS. Panel a, lane 5: DdrC and DNase I were simultaneously incubated with supercoiled DNA before treatment with Proteinase K/SDS.

Techniques Used: Plasmid Preparation, Activity Assay, Incubation

27) Product Images from "Transcriptome-guided target identification of the TetR-like regulator SACE_5754 and engineered overproduction of erythromycin in Saccharopolyspora erythraea"

Article Title: Transcriptome-guided target identification of the TetR-like regulator SACE_5754 and engineered overproduction of erythromycin in Saccharopolyspora erythraea

Journal: Journal of Biological Engineering

doi: 10.1186/s13036-018-0135-2

Analysis of the SACE_5754-binding sites. a DNase I footprinting assay of SACE_5754-binding site in the P 0388–0389 . Upper fluorogram: control reaction without protein. Protection regions were acquired with increasing concentrations (0.5 μM and 0.2 μM) of His 6 -SACE_5754 protein. b SACE_5754 binds to the probe P1and mutated probe P2 and P3 by EMSAs. c DNase I footprinting assay of SACE_5754-binding site in the P 3599 . Upper fluorogram: control reaction without protein. Protection regions were acquired with increasing concentrations (0.2 μM and 0.5 μM) of His 6 -SACE_5754 protein. d SACE_5754 binds to the probe P4 and mutated probe P5 and P6 by EMSAs. e DNase I footprinting assay of SACE_5754-binding site in the P 6148–6149 . Upper fluorogram: control reaction without protein. Protection regions were acquired with increasing concentrations (0.05 μM and 0.2 μM) of His 6 -SACE_5754 protein. f SACE_5754 binds to the probe P5 and mutated probe P6 and P7 by EMSAs
Figure Legend Snippet: Analysis of the SACE_5754-binding sites. a DNase I footprinting assay of SACE_5754-binding site in the P 0388–0389 . Upper fluorogram: control reaction without protein. Protection regions were acquired with increasing concentrations (0.5 μM and 0.2 μM) of His 6 -SACE_5754 protein. b SACE_5754 binds to the probe P1and mutated probe P2 and P3 by EMSAs. c DNase I footprinting assay of SACE_5754-binding site in the P 3599 . Upper fluorogram: control reaction without protein. Protection regions were acquired with increasing concentrations (0.2 μM and 0.5 μM) of His 6 -SACE_5754 protein. d SACE_5754 binds to the probe P4 and mutated probe P5 and P6 by EMSAs. e DNase I footprinting assay of SACE_5754-binding site in the P 6148–6149 . Upper fluorogram: control reaction without protein. Protection regions were acquired with increasing concentrations (0.05 μM and 0.2 μM) of His 6 -SACE_5754 protein. f SACE_5754 binds to the probe P5 and mutated probe P6 and P7 by EMSAs

Techniques Used: Binding Assay, Footprinting

28) Product Images from "Thyroglobulin Repression of Thyroid Transcription Factor 1 (TTF-1) Gene Expression Is Mediated by Decreased DNA Binding of Nuclear Factor I Proteins Which Control Constitutive TTF-1 Expression"

Article Title: Thyroglobulin Repression of Thyroid Transcription Factor 1 (TTF-1) Gene Expression Is Mediated by Decreased DNA Binding of Nuclear Factor I Proteins Which Control Constitutive TTF-1 Expression

Journal: Molecular and Cellular Biology

doi:

TG treatment decreases the protection of two areas identified by DNase I protection assay in the proximal TTF-1 promoter. The coding (top) and noncoding (bottom) strands of the DNA fragment from bp −264 to −153 of the TTF-1 promoter were end labeled with [γ- 32 P]ATP. Footprinting analysis was performed as described in Materials and Methods in the absence (lanes 1, 5, 6, and 9) or presence (lanes 4 and 8) of 30 μg of nuclear extract (N.E.) from FRTL-5 cells treated with 10 mg of TG per ml for 48 h (lanes 4 and 8) or left untreated (lanes 2, 3, and 7). Three protected areas were identified and defined as A, B, and C sites (denoted by black bars). TG treatment results in decreased protection of the B and C sites (compare lanes 2 and 3 with lane 4 and compare lane 7 with lane 8). The extract in lane 2 was from cells with no TSH in the medium; that in lane 3 was from TSH-treated cells. TSH did not significantly alter the protected areas.
Figure Legend Snippet: TG treatment decreases the protection of two areas identified by DNase I protection assay in the proximal TTF-1 promoter. The coding (top) and noncoding (bottom) strands of the DNA fragment from bp −264 to −153 of the TTF-1 promoter were end labeled with [γ- 32 P]ATP. Footprinting analysis was performed as described in Materials and Methods in the absence (lanes 1, 5, 6, and 9) or presence (lanes 4 and 8) of 30 μg of nuclear extract (N.E.) from FRTL-5 cells treated with 10 mg of TG per ml for 48 h (lanes 4 and 8) or left untreated (lanes 2, 3, and 7). Three protected areas were identified and defined as A, B, and C sites (denoted by black bars). TG treatment results in decreased protection of the B and C sites (compare lanes 2 and 3 with lane 4 and compare lane 7 with lane 8). The extract in lane 2 was from cells with no TSH in the medium; that in lane 3 was from TSH-treated cells. TSH did not significantly alter the protected areas.

Techniques Used: Labeling, Footprinting

29) Product Images from "Importance of Tetramer Formation by the Nitrogen Assimilation Control Protein for Strong Repression of Glutamate Dehydrogenase Formation in Klebsiella pneumoniae"

Article Title: Importance of Tetramer Formation by the Nitrogen Assimilation Control Protein for Strong Repression of Glutamate Dehydrogenase Formation in Klebsiella pneumoniae

Journal:

doi: 10.1128/JB.187.24.8291-8299.2005

DNase I footprint of NAC WT and NAC L111K bound to the nac promoter region. Radioactively labeled pCB1426 DNA was digested with DNase I in the presence of NAC WT (lanes 2 to 6 [0, 0.9, 1.4, 2.2, and 4.4 pmol, respectively]) or NAC L111K (lanes 7 to 11 [4.7,
Figure Legend Snippet: DNase I footprint of NAC WT and NAC L111K bound to the nac promoter region. Radioactively labeled pCB1426 DNA was digested with DNase I in the presence of NAC WT (lanes 2 to 6 [0, 0.9, 1.4, 2.2, and 4.4 pmol, respectively]) or NAC L111K (lanes 7 to 11 [4.7,

Techniques Used: Labeling

30) Product Images from "RcsAB and Fur Coregulate the Iron-Acquisition System via entC in Klebsiella pneumoniae NTUH-K2044 in Response to Iron Availability"

Article Title: RcsAB and Fur Coregulate the Iron-Acquisition System via entC in Klebsiella pneumoniae NTUH-K2044 in Response to Iron Availability

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2020.00282

DNase I footprints of RcsAB and Fur at the entC promoter. The promoter DNA regions of entC were labeled with FAM and incubated with increasing amounts of purified His 6 -RcsB (0, 1, 3 μg) (A) and His 6 -Fur (B) (0, 0.5, 1 μg). The footprint regions are boxed within lines and marked.
Figure Legend Snippet: DNase I footprints of RcsAB and Fur at the entC promoter. The promoter DNA regions of entC were labeled with FAM and incubated with increasing amounts of purified His 6 -RcsB (0, 1, 3 μg) (A) and His 6 -Fur (B) (0, 0.5, 1 μg). The footprint regions are boxed within lines and marked.

Techniques Used: Labeling, Incubation, Purification

31) Product Images from "Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster"

Article Title: Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki281

Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to DNase I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.
Figure Legend Snippet: Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to DNase I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.

Techniques Used: Amplification

32) Product Images from "Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster"

Article Title: Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki281

Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to DNase I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.
Figure Legend Snippet: Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to DNase I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.

Techniques Used: Amplification

33) Product Images from "Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster"

Article Title: Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki281

Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to DNase I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.
Figure Legend Snippet: Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to DNase I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.

Techniques Used: Amplification

34) Product Images from "The Transcriptional Repressor, MtrR, of the mtrCDE Efflux Pump Operon of Neisseria gonorrhoeae Can Also Serve as an Activator of “off Target” Gene (glnE) Expression"

Article Title: The Transcriptional Repressor, MtrR, of the mtrCDE Efflux Pump Operon of Neisseria gonorrhoeae Can Also Serve as an Activator of “off Target” Gene (glnE) Expression

Journal: Antibiotics (Basel, Switzerland)

doi: 10.3390/antibiotics4020188

The nucleotide sequence upstream of glnE and MtrR-binding sites. The 301 bp sequence of the DNA upstream of glnE and the first two codons (encoding M and S, respectively) is shown with the annotated −10 and −35 hexamer sequences of the glnE promoter identified by a line under the sequences. The putative extended −10 element is shown in blue. The alternative −35 hexamer is shown by the dashed line above the sequence. The boxed regions represent predicted MtrR binding sites that were identified based on sequence similarity to that of a site upstream of mtrCDE [ 8 , 10 ] or rpoH [ 7 ]. The grey box represents a sequence with 53% identity to the region upstream of rpoH [ 7 ] while the yellow, black, and red boxes represent sequences with 55%, 67%, and 52%, respectively, identity to regions upstream of mtrCDE [ 8 , 10 ]. The MtrR-binding sites identified by DNase I protection ( Figure 3 ) are noted by the solid line above the sense strand or below the anti-sense strand; the two sites on the anti-sense strand are denoted as A′ and B′ with the DNase I hypersensitive site in A′ shown by an *. The adjacent seven nucleotide imperfect inverted element is shown in green and red.
Figure Legend Snippet: The nucleotide sequence upstream of glnE and MtrR-binding sites. The 301 bp sequence of the DNA upstream of glnE and the first two codons (encoding M and S, respectively) is shown with the annotated −10 and −35 hexamer sequences of the glnE promoter identified by a line under the sequences. The putative extended −10 element is shown in blue. The alternative −35 hexamer is shown by the dashed line above the sequence. The boxed regions represent predicted MtrR binding sites that were identified based on sequence similarity to that of a site upstream of mtrCDE [ 8 , 10 ] or rpoH [ 7 ]. The grey box represents a sequence with 53% identity to the region upstream of rpoH [ 7 ] while the yellow, black, and red boxes represent sequences with 55%, 67%, and 52%, respectively, identity to regions upstream of mtrCDE [ 8 , 10 ]. The MtrR-binding sites identified by DNase I protection ( Figure 3 ) are noted by the solid line above the sense strand or below the anti-sense strand; the two sites on the anti-sense strand are denoted as A′ and B′ with the DNase I hypersensitive site in A′ shown by an *. The adjacent seven nucleotide imperfect inverted element is shown in green and red.

Techniques Used: Sequencing, Binding Assay

Identification of the MtrR-binding site in the glnE upstream DNA. ( A ) The binding specificity of MtrR for the DNA shown in Figure 1 was determined by competitive EMSA; ( B ) The MtrR-binding sites within this sequence were identified by DNase I protection assays that employed increasing amounts of purified MtrR-MBP (0, 5, 10, and 15 μg) with both sense and anti-sense probes. The protected regions on each probe are identified by the black bars and the two sites on the anti-sense strand are labeled as A′ and B′. Regions containing DNase I hypersensitive sites, which could contain more than one nucleotide, on the sense and antisense strands are denoted by *. The sequencing reactions for each probe are adjacent to the DNase I protection reactions and oriented G, A, T, C.
Figure Legend Snippet: Identification of the MtrR-binding site in the glnE upstream DNA. ( A ) The binding specificity of MtrR for the DNA shown in Figure 1 was determined by competitive EMSA; ( B ) The MtrR-binding sites within this sequence were identified by DNase I protection assays that employed increasing amounts of purified MtrR-MBP (0, 5, 10, and 15 μg) with both sense and anti-sense probes. The protected regions on each probe are identified by the black bars and the two sites on the anti-sense strand are labeled as A′ and B′. Regions containing DNase I hypersensitive sites, which could contain more than one nucleotide, on the sense and antisense strands are denoted by *. The sequencing reactions for each probe are adjacent to the DNase I protection reactions and oriented G, A, T, C.

Techniques Used: Binding Assay, Sequencing, Purification, Labeling

35) Product Images from "A DNase from a Fungal Phytopathogen Is a Virulence Factor Likely Deployed as Counter Defense against Host-Secreted Extracellular DNA"

Article Title: A DNase from a Fungal Phytopathogen Is a Virulence Factor Likely Deployed as Counter Defense against Host-Secreted Extracellular DNA

Journal: mBio

doi: 10.1128/mBio.02805-18

Corn root cap cells secrete DNA that is degraded by DNase I. (A) Corn roots were touched to glass slides, causing border cells (arrows) to slough off. Slides were stained with Sytox green, which stains extracellular DNA (arrowheads) or DNA in dead cells. Left, fluorescence; middle, differential interference contrast (DIC); right, merged. Scale bar is 20 μm. (B) DNase treatment degrades extracellular DNA. Left, corn roots and root cap border cells (arrowheads), Sytox green staining outside border cells (arrows); right, no exDNA staining after DNase treatment. Scale bar is 50 μm.
Figure Legend Snippet: Corn root cap cells secrete DNA that is degraded by DNase I. (A) Corn roots were touched to glass slides, causing border cells (arrows) to slough off. Slides were stained with Sytox green, which stains extracellular DNA (arrowheads) or DNA in dead cells. Left, fluorescence; middle, differential interference contrast (DIC); right, merged. Scale bar is 20 μm. (B) DNase treatment degrades extracellular DNA. Left, corn roots and root cap border cells (arrowheads), Sytox green staining outside border cells (arrows); right, no exDNA staining after DNase treatment. Scale bar is 50 μm.

Techniques Used: Staining, Fluorescence

The nuc1 mutant is rescued by the addition of DNase I. Roots treated with the mutant plus DNase I show necrotic symptoms (red arrows), while those without DNase I do not. The mock control was water instead of spores; the addition of DNase I to the control had no deleterious effect on the roots.
Figure Legend Snippet: The nuc1 mutant is rescued by the addition of DNase I. Roots treated with the mutant plus DNase I show necrotic symptoms (red arrows), while those without DNase I do not. The mock control was water instead of spores; the addition of DNase I to the control had no deleterious effect on the roots.

Techniques Used: Mutagenesis

36) Product Images from "A broad-range PCR technique for the diagnosis of infective endocarditis"

Article Title: A broad-range PCR technique for the diagnosis of infective endocarditis

Journal: Brazilian Journal of Microbiology

doi: 10.1016/j.bjm.2017.03.019

Nested-PCR using the primer pairs MW9/NW17 and NW11/NW12. (A) First-round PCR amplification with primer pairs MW9/NW17. Lane 1 DNA ladder; lane 2–7 contained different suspensions of  Streptococcus pneumoniae : lane 2 – 10 6  CFU/mL; lane 3 – 10 5  CFU/mL; lane 4 – 10 4  CFU/mL; lane 5 – 10 3  CFU/mL; lane 6 – 10 2  CFU/mL; lane 7 – 10 CFU/mL; lane 8 – negative control. (B) Second-round of PCR amplification with primer pairs NW11/NW12. Lane 1 DNA ladder; lane 2, 3 contained 1 μL from the first-round PCR; lane 2 – suspension 10 2  CFU/mL of  Streptococcus pneumoniae ; lane 3 – suspension 10 CFU/mL of  Streptococcus pneumoniae ; lane 4 contained 1 μL of the negative reaction mixture from the first round. (C) Second-round of PCR amplification of the negative reaction mixture with primer pairs NW11/NW12. Lane 1 DNA ladder; lane 2–6 contained 1 μL of the negative reaction mixture from the first round; lane 2 negative reaction mixture without pretreatment; lane 3–6 negative reaction mixture treated with 1 U, 1.25 U, 1.5 U and 2 U of DNase I, respectively.
Figure Legend Snippet: Nested-PCR using the primer pairs MW9/NW17 and NW11/NW12. (A) First-round PCR amplification with primer pairs MW9/NW17. Lane 1 DNA ladder; lane 2–7 contained different suspensions of Streptococcus pneumoniae : lane 2 – 10 6  CFU/mL; lane 3 – 10 5  CFU/mL; lane 4 – 10 4  CFU/mL; lane 5 – 10 3  CFU/mL; lane 6 – 10 2  CFU/mL; lane 7 – 10 CFU/mL; lane 8 – negative control. (B) Second-round of PCR amplification with primer pairs NW11/NW12. Lane 1 DNA ladder; lane 2, 3 contained 1 μL from the first-round PCR; lane 2 – suspension 10 2  CFU/mL of Streptococcus pneumoniae ; lane 3 – suspension 10 CFU/mL of Streptococcus pneumoniae ; lane 4 contained 1 μL of the negative reaction mixture from the first round. (C) Second-round of PCR amplification of the negative reaction mixture with primer pairs NW11/NW12. Lane 1 DNA ladder; lane 2–6 contained 1 μL of the negative reaction mixture from the first round; lane 2 negative reaction mixture without pretreatment; lane 3–6 negative reaction mixture treated with 1 U, 1.25 U, 1.5 U and 2 U of DNase I, respectively.

Techniques Used: Nested PCR, Polymerase Chain Reaction, Amplification, Negative Control

37) Product Images from "Physical and Functional Interaction between DNA Ligase III? and Poly(ADP-Ribose) Polymerase 1 in DNA Single-Strand Break Repair"

Article Title: Physical and Functional Interaction between DNA Ligase III? and Poly(ADP-Ribose) Polymerase 1 in DNA Single-Strand Break Repair

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.16.5919-5927.2003

Binding of DNA ligase III and PARP-1 to DNA single-strand interruptions. Effects of NAD on DNA binding by PARP-1 are shown. (A) Analysis of the binding of DNA ligase III to a DNA single-strand break by surface plasmon resonance. (Top panel) Schematic representation of the nicked hairpin oligonucleotide immobilized on the chip surface. (Bottom panel) Representative sensorgram showing the binding and release of untagged full-length DNA ligase III (LigIII), injected at the concentrations indicated, from the nicked hairpin oligonucleotide immobilized on the chip surface. (B) Visualization of DNA ligase III bound to a DNA single-strand break by DNase I footprinting. Intact DNA ligase III (LigIII; 20 nM) was preincubated with the labeled, nicked DNA substrate (16 nM) indicated and then incubated with DNase I as described in Materials and Methods. After separation by denaturing gel electrophoresis, labeled oligonucleotides were detected by autoradiography. −, no enzyme. The vertical line indicates the region protected from DNase I activity. (C) Effect of DNA strand break binding by DNA ligase III and PARP-1 on T4 polynucleotide kinase activity. Intact DNA ligase III (150 nM) or PARP-1 (150 nM) was preincubated with the indicated DNA substrate containing a single-nucleotide (1 nt) gap in the presence (+) or absence (−) of NAD as indicated prior to incubation with polynucleotide kinase and [γ- 32 P]ATP as described in Materials and Methods. After separation by denaturing gel electrophoresis, labeled 29-mer oligonucleotide was detected and quantitated by phosphorimager analysis. Lane C, no PARP-1 or DNA ligase III.
Figure Legend Snippet: Binding of DNA ligase III and PARP-1 to DNA single-strand interruptions. Effects of NAD on DNA binding by PARP-1 are shown. (A) Analysis of the binding of DNA ligase III to a DNA single-strand break by surface plasmon resonance. (Top panel) Schematic representation of the nicked hairpin oligonucleotide immobilized on the chip surface. (Bottom panel) Representative sensorgram showing the binding and release of untagged full-length DNA ligase III (LigIII), injected at the concentrations indicated, from the nicked hairpin oligonucleotide immobilized on the chip surface. (B) Visualization of DNA ligase III bound to a DNA single-strand break by DNase I footprinting. Intact DNA ligase III (LigIII; 20 nM) was preincubated with the labeled, nicked DNA substrate (16 nM) indicated and then incubated with DNase I as described in Materials and Methods. After separation by denaturing gel electrophoresis, labeled oligonucleotides were detected by autoradiography. −, no enzyme. The vertical line indicates the region protected from DNase I activity. (C) Effect of DNA strand break binding by DNA ligase III and PARP-1 on T4 polynucleotide kinase activity. Intact DNA ligase III (150 nM) or PARP-1 (150 nM) was preincubated with the indicated DNA substrate containing a single-nucleotide (1 nt) gap in the presence (+) or absence (−) of NAD as indicated prior to incubation with polynucleotide kinase and [γ- 32 P]ATP as described in Materials and Methods. After separation by denaturing gel electrophoresis, labeled 29-mer oligonucleotide was detected and quantitated by phosphorimager analysis. Lane C, no PARP-1 or DNA ligase III.

Techniques Used: Binding Assay, SPR Assay, Chromatin Immunoprecipitation, Injection, Footprinting, Labeling, Incubation, Nucleic Acid Electrophoresis, Autoradiography, Activity Assay

DNA ligase III preferentially binds to poly(ADP-ribosyl)ated PARP-1 in vitro. Effects of DNA damage on the association between PARP-1 and DNA ligase III-XRCC1 in vivo are shown. (A) Purified PARP-1 (500 nM) was preincubated with a labeled DNA duplex containing a single ligatable nick (300 nM) in the presence (+) or absence (−) of NAD. After treatment with DNase I, intact DNA ligase III (10 nM) or a truncated version lacking the zinc finger (10 nM) was added to the reaction mixture as indicated. Proteins immunoprecipitated (IP) by PARP-1 antibody were separated by SDS-PAGE and then detected by immunblotting (IB) with the indicated antibody. Upper panel, intact DNA ligase III (Lig III); middle panel, truncated version of DNA ligase III lacking the zinc finger (ΔZf-Lig III); lower panel, unmodified PARP-1 y(ADP-ribosyl)ated PARP-1 [P-(ADPR) n ]. (B) Effect of H 2 O 2  treatment on the association of PARP-1, DNA ligase IIIα, and XRCC1. Whole cell extracts (WCE) were prepared from undamaged (−) or damaged (+) HeLa cells as described in Materials and Methods. Equivalent aliquots of the cells to be damaged were pretreated with 1,5-isoquinolinediol (DiQ; 100 μM) as indicated for 1 h prior to and during H 2 O 2  treatment. Proteins immunoprecipitated by PARP-1 antibody were separated by SDS-PAGE and then detected by immunoblotting with the indicated antibody. DNA ligase IIIα and XRCC1 in the extracts from undamaged cells were detected by direct immunoblotting.
Figure Legend Snippet: DNA ligase III preferentially binds to poly(ADP-ribosyl)ated PARP-1 in vitro. Effects of DNA damage on the association between PARP-1 and DNA ligase III-XRCC1 in vivo are shown. (A) Purified PARP-1 (500 nM) was preincubated with a labeled DNA duplex containing a single ligatable nick (300 nM) in the presence (+) or absence (−) of NAD. After treatment with DNase I, intact DNA ligase III (10 nM) or a truncated version lacking the zinc finger (10 nM) was added to the reaction mixture as indicated. Proteins immunoprecipitated (IP) by PARP-1 antibody were separated by SDS-PAGE and then detected by immunblotting (IB) with the indicated antibody. Upper panel, intact DNA ligase III (Lig III); middle panel, truncated version of DNA ligase III lacking the zinc finger (ΔZf-Lig III); lower panel, unmodified PARP-1 y(ADP-ribosyl)ated PARP-1 [P-(ADPR) n ]. (B) Effect of H 2 O 2 treatment on the association of PARP-1, DNA ligase IIIα, and XRCC1. Whole cell extracts (WCE) were prepared from undamaged (−) or damaged (+) HeLa cells as described in Materials and Methods. Equivalent aliquots of the cells to be damaged were pretreated with 1,5-isoquinolinediol (DiQ; 100 μM) as indicated for 1 h prior to and during H 2 O 2 treatment. Proteins immunoprecipitated by PARP-1 antibody were separated by SDS-PAGE and then detected by immunoblotting with the indicated antibody. DNA ligase IIIα and XRCC1 in the extracts from undamaged cells were detected by direct immunoblotting.

Techniques Used: In Vitro, In Vivo, Purification, Labeling, Immunoprecipitation, SDS Page

38) Product Images from "Metabolomics Reveals a Role for the Chromatin-Binding Protein HMGN5 in Glutathione Metabolism"

Article Title: Metabolomics Reveals a Role for the Chromatin-Binding Protein HMGN5 in Glutathione Metabolism

Journal: PLoS ONE

doi: 10.1371/journal.pone.0084583

Gene expression and DNase I hypersensitivity analysis of Gpx6 and Hk1 . A) Relative expression of Gpx6 and Hk1 measured by real-time PCR. B) Maps depicting the genomic regions selected for analysis by DNase I digestion. Black rectangles indicate exon regions within the gene. Bars below the maps indicate the regions chosen for DNase I hypersensitivity analysis. (C) Recovery of Gpx6 amplicons following digestion with varying concentrations of DNase I (D). Recovery of Hk1 amplicons following digestion with varying concentrations of DNase I. P-values represent the significance testing for the effect of genotype as determined by two-way ANOVA, values in bold are significant at p
Figure Legend Snippet: Gene expression and DNase I hypersensitivity analysis of Gpx6 and Hk1 . A) Relative expression of Gpx6 and Hk1 measured by real-time PCR. B) Maps depicting the genomic regions selected for analysis by DNase I digestion. Black rectangles indicate exon regions within the gene. Bars below the maps indicate the regions chosen for DNase I hypersensitivity analysis. (C) Recovery of Gpx6 amplicons following digestion with varying concentrations of DNase I (D). Recovery of Hk1 amplicons following digestion with varying concentrations of DNase I. P-values represent the significance testing for the effect of genotype as determined by two-way ANOVA, values in bold are significant at p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

39) Product Images from "Regulation of Sulfur Assimilation Pathways in Burkholderia cenocepacia through Control of Genes by the SsuR Transcription Factor ▿"

Article Title: Regulation of Sulfur Assimilation Pathways in Burkholderia cenocepacia through Control of Genes by the SsuR Transcription Factor ▿

Journal: Journal of Bacteriology

doi: 10.1128/JB.00483-10

DNase I footprinting analysis of the SsuR-regulated promoter region ssuD P . The DNA fragment subjected to footprinting was amplified with primers SSUD1fw and SSUD2rev of which either the former or the latter was 5′ labeled with 32 P to label the
Figure Legend Snippet: DNase I footprinting analysis of the SsuR-regulated promoter region ssuD P . The DNA fragment subjected to footprinting was amplified with primers SSUD1fw and SSUD2rev of which either the former or the latter was 5′ labeled with 32 P to label the

Techniques Used: Footprinting, Amplification, Labeling

40) Product Images from "Identification of an Adeno-Associated Virus Rep Protein Binding Site in the Adenovirus E2a Promoter"

Article Title: Identification of an Adeno-Associated Virus Rep Protein Binding Site in the Adenovirus E2a Promoter

Journal:

doi: 10.1128/JVI.79.1.28-38.2005

Ad5  E2a  and AAV p5 promoter regions protected by Rep68. (A) Schematic diagram of the Ad5  E2a  promoter showing principal transcription factor binding sites and the transcription start site (arrow). The DNase I-protected region is bracketed. (B) Sequences
Figure Legend Snippet: Ad5 E2a and AAV p5 promoter regions protected by Rep68. (A) Schematic diagram of the Ad5 E2a promoter showing principal transcription factor binding sites and the transcription start site (arrow). The DNase I-protected region is bracketed. (B) Sequences

Techniques Used: Binding Assay

Rep68 protects a region of the Ad5 E2a promoter from DNase I digestion. (A) DNase I protection assays were performed on the 303-bp fragment (∼600 pmol) containing labeled lower strand (lanes 1 to 4) or labeled upper strand (lanes 5 to 8) with
Figure Legend Snippet: Rep68 protects a region of the Ad5 E2a promoter from DNase I digestion. (A) DNase I protection assays were performed on the 303-bp fragment (∼600 pmol) containing labeled lower strand (lanes 1 to 4) or labeled upper strand (lanes 5 to 8) with

Techniques Used: Labeling

Related Articles

Incubation:

Article Title: Structural analysis of the regulatory mechanism of MarR protein Rv2887 in M. tuberculosis
Article Snippet: .. After incubation for 30 min at 25 °C, 10 µl solution containing about 0.015 units DNase I (Promega) and 100 nM freshly prepared CaCl2 was added and further incubated for 1 min at 25 °C. .. The reaction was stopped by adding 140 µl DNase I stop solution (200 mM unbuffered sodium acetate, 30 mM EDTA and 0.15% SDS).

Article Title: TetR-Type Regulator SLCG_2919 Is a Negative Regulator of Lincomycin Biosynthesis in Streptomyces lincolnensis
Article Snippet: .. The mixture was incubated at 25°C for 60 s and terminated by addition of DNase I stop solution and heating for 10 min at 65°C. .. DNA samples were analyzed with a 3730XL DNA genetic analyzer (Applied Biosystems) after purification, and data analyses were performed using the GeneMarker software program v2.2.

other:

Article Title: Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster
Article Snippet: Sensitivity to DNase I is a conventional gauge for the compactness of chromatin.

Article Title: Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster
Article Snippet: The distribution of chromatin resistance to DNase I in brains is also not supported by the linear model (P = 0.77), the best fit was observed for the quadratic (P = 0.06), cubic (P = 0.04) and S-shaped curve (P = 0.05) models.

Article Title: The Malonate Decarboxylase Operon of Acinetobacter calcoaceticus KCCM 40902 Is Regulated by Malonate and the Transcriptional Repressor MdcY
Article Snippet: The reaction was stopped by the addition 36 μl of DNase I stop solution (200 mM NaCl, 30 mM EDTA, 1% sodium dodecyl sulfate [SDS], 100 μl of yeast RNA per ml).

Article Title: Identification of the Replication Origins from Cyanothece ATCC 51142 and Their Interactions with the DnaA Protein: From In Silico to In Vitro Studies
Article Snippet: The reaction was stopped by adding 140 μL of DNase I stop solution (200 mM unbuffered sodium acetate, 30 mM of EDTA, and 0.15% SDS).

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  • 92
    Promega dnase i
    MdcY-mediated protection of the  mdc  operator against digestion by DNase I. Lanes 1 to 4, target DNA (0.1 pmol, 2 × 10 5  cpm) was incubated in the absence or in the presence of MdcY (the number above each lane indicates the nanomolar concentration of MdcY protein). In lanes 5 to 7, target DNA and 2.4 nM MdcY were incubated with 50, 100, and 500 μM malonate, respectively. The vertical arrows beside the nucleotide sequence indicate a palindromic structure.
    Dnase I, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 560 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i/product/Promega
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    Promega dnase i footprinting
    RcsAB-dependent expression of hmsT . Primer extension (a), LacZ fusion (b), EMSA (c), <t>DNase</t> I <t>footprinting</t> (d) experiments were performed as described in Fig 2 .
    Dnase I Footprinting, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MdcY-mediated protection of the  mdc  operator against digestion by DNase I. Lanes 1 to 4, target DNA (0.1 pmol, 2 × 10 5  cpm) was incubated in the absence or in the presence of MdcY (the number above each lane indicates the nanomolar concentration of MdcY protein). In lanes 5 to 7, target DNA and 2.4 nM MdcY were incubated with 50, 100, and 500 μM malonate, respectively. The vertical arrows beside the nucleotide sequence indicate a palindromic structure.

    Journal: Journal of Bacteriology

    Article Title: The Malonate Decarboxylase Operon of Acinetobacter calcoaceticus KCCM 40902 Is Regulated by Malonate and the Transcriptional Repressor MdcY

    doi:

    Figure Lengend Snippet: MdcY-mediated protection of the mdc operator against digestion by DNase I. Lanes 1 to 4, target DNA (0.1 pmol, 2 × 10 5 cpm) was incubated in the absence or in the presence of MdcY (the number above each lane indicates the nanomolar concentration of MdcY protein). In lanes 5 to 7, target DNA and 2.4 nM MdcY were incubated with 50, 100, and 500 μM malonate, respectively. The vertical arrows beside the nucleotide sequence indicate a palindromic structure.

    Article Snippet: The reaction was stopped by the addition 36 μl of DNase I stop solution (200 mM NaCl, 30 mM EDTA, 1% sodium dodecyl sulfate [SDS], 100 μl of yeast RNA per ml).

    Techniques: Incubation, Concentration Assay, Sequencing

    Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to DNase I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.

    Journal: Nucleic Acids Research

    Article Title: Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster

    doi: 10.1093/nar/gki281

    Figure Lengend Snippet: Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to DNase I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.

    Article Snippet: The distribution of chromatin resistance to DNase I in brains is also not supported by the linear model (P = 0.77), the best fit was observed for the quadratic (P = 0.06), cubic (P = 0.04) and S-shaped curve (P = 0.05) models.

    Techniques: Amplification

    RcsAB-dependent expression of hmsT . Primer extension (a), LacZ fusion (b), EMSA (c), DNase I footprinting (d) experiments were performed as described in Fig 2 .

    Journal: Scientific Reports

    Article Title: RcsAB is a major repressor of Yersinia biofilm development through directly acting on hmsCDE, hmsT, and hmsHFRS

    doi: 10.1038/srep09566

    Figure Lengend Snippet: RcsAB-dependent expression of hmsT . Primer extension (a), LacZ fusion (b), EMSA (c), DNase I footprinting (d) experiments were performed as described in Fig 2 .

    Article Snippet: DNase I footprinting For DNase I footprinting , the target DNA fragment with a single 32 P-labeled end was incubated with increasing amounts of purified His-RcsB-p with addition of 24 pmol of purified MBP-RcsA, which was followed by partial digestion of RQ1 RNase-Free DNase I (Promega).

    Techniques: Expressing, Footprinting

    RcsAB-dependent expression of hmsCDE . (a) Primer extension. The relative mRNA levels of hmsC in indicated strains were determined by primer extension. The Sanger sequence ladders (lanes G, C, A, and T) and the primer extension products of hmsC were analyzed with an 8 M urea-6% acrylamide sequencing gel. The transcription start site of hmsC was indicated by arrow with nucleotide A, and the minus number under arrow indicated the nucleotide position upstream of hmsC start codon. (b) LacZ fusion. The hmsC : lacZ transcriptional fusion vector was transformed into in indicated strains, and then hmsC promoter activities (miller units of β-galactosidase activity) were determined in bacterial cellular extracts. (c) EMSA. The radioactively labeled DNA fragments were incubated with indicated purified proteins and then subjected to a native 4% polyacrylamide gel electrophoresis. (d) DNase I footprinting. Labeled coding or non-coding DNA probes were incubated with indicated purified proteins and then subjected to DNase I digestion. The digested DNA samples were analyzed in an 8 M urea-6% polyacrylamide gel. The footprint regions were indicated with vertical bars. Lanes C, T, A, and G represented Sanger sequencing reactions. The DNA-binding of His-RcsB-p in presence of MBP-RcsA (involved in EMSA and DNase I footprinting) and that of His-RcsB-p alone (in EMSA) were tested. (d) Promoter structure. Shown were with translation/transcription starts, core promoter −10 and −35 elements, SD sequences, RcsAB sites, and RcsAB box-like sequences.

    Journal: Scientific Reports

    Article Title: RcsAB is a major repressor of Yersinia biofilm development through directly acting on hmsCDE, hmsT, and hmsHFRS

    doi: 10.1038/srep09566

    Figure Lengend Snippet: RcsAB-dependent expression of hmsCDE . (a) Primer extension. The relative mRNA levels of hmsC in indicated strains were determined by primer extension. The Sanger sequence ladders (lanes G, C, A, and T) and the primer extension products of hmsC were analyzed with an 8 M urea-6% acrylamide sequencing gel. The transcription start site of hmsC was indicated by arrow with nucleotide A, and the minus number under arrow indicated the nucleotide position upstream of hmsC start codon. (b) LacZ fusion. The hmsC : lacZ transcriptional fusion vector was transformed into in indicated strains, and then hmsC promoter activities (miller units of β-galactosidase activity) were determined in bacterial cellular extracts. (c) EMSA. The radioactively labeled DNA fragments were incubated with indicated purified proteins and then subjected to a native 4% polyacrylamide gel electrophoresis. (d) DNase I footprinting. Labeled coding or non-coding DNA probes were incubated with indicated purified proteins and then subjected to DNase I digestion. The digested DNA samples were analyzed in an 8 M urea-6% polyacrylamide gel. The footprint regions were indicated with vertical bars. Lanes C, T, A, and G represented Sanger sequencing reactions. The DNA-binding of His-RcsB-p in presence of MBP-RcsA (involved in EMSA and DNase I footprinting) and that of His-RcsB-p alone (in EMSA) were tested. (d) Promoter structure. Shown were with translation/transcription starts, core promoter −10 and −35 elements, SD sequences, RcsAB sites, and RcsAB box-like sequences.

    Article Snippet: DNase I footprinting For DNase I footprinting , the target DNA fragment with a single 32 P-labeled end was incubated with increasing amounts of purified His-RcsB-p with addition of 24 pmol of purified MBP-RcsA, which was followed by partial digestion of RQ1 RNase-Free DNase I (Promega).

    Techniques: Expressing, Sequencing, Plasmid Preparation, Transformation Assay, Activity Assay, Labeling, Incubation, Purification, Polyacrylamide Gel Electrophoresis, Footprinting, Binding Assay

    RcsAB-dependent expression of hmsHFRS . Primer extension (a), LacZ fusion (b), EMSA (c), and DNase I footprinting (d) experiments were performed as described in Fig 2 .

    Journal: Scientific Reports

    Article Title: RcsAB is a major repressor of Yersinia biofilm development through directly acting on hmsCDE, hmsT, and hmsHFRS

    doi: 10.1038/srep09566

    Figure Lengend Snippet: RcsAB-dependent expression of hmsHFRS . Primer extension (a), LacZ fusion (b), EMSA (c), and DNase I footprinting (d) experiments were performed as described in Fig 2 .

    Article Snippet: DNase I footprinting For DNase I footprinting , the target DNA fragment with a single 32 P-labeled end was incubated with increasing amounts of purified His-RcsB-p with addition of 24 pmol of purified MBP-RcsA, which was followed by partial digestion of RQ1 RNase-Free DNase I (Promega).

    Techniques: Expressing, Footprinting