dnase i Thermo Fisher Search Results


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  • 99
    Thermo Fisher dnase i enzyme
    <t>DNase</t> I treatment is effective against C. jejuni biofilms on stainless steel surfaces and in the presence of organic materials in aerobic conditions . The ability of DNase I to inhibit biofilm formation of C. jejuni NCTC 11168 on sterile, stainless steel coupons (A) or in the presence of chicken juice, mimicking a conditioned surface (B) . TTC staining was used to measure biofilm formation in the presence of chicken juice (B) . DNase I is able to significantly decrease biofilm formation in both conditions. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P
    Dnase I Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dnase i
    <t>DNase</t> I treatment is effective against C. jejuni biofilms on stainless steel surfaces and in the presence of organic materials in aerobic conditions . The ability of DNase I to inhibit biofilm formation of C. jejuni NCTC 11168 on sterile, stainless steel coupons (A) or in the presence of chicken juice, mimicking a conditioned surface (B) . TTC staining was used to measure biofilm formation in the presence of chicken juice (B) . DNase I is able to significantly decrease biofilm formation in both conditions. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P
    Dnase I, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 26477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dnase i
    Effects of noncomplementary dNTPs on <t>DNase</t> I footprints and stable complex formation by HIV-1 RT
    Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 61107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rnase free dnase i
    Consensus GGUG-containing RNA oligonucleotide promotes the inhibitory effect of TLS on CBP/p300 HAT activities a , Co-immunoprecipitation (IP) of p300 and TLS from <t>RNase</t> A-treated HeLa cells. b, P300 HAT activity was measured using micrococcal nuclease (MNase) or <t>DNase</t> I pre-treated GST and GST-TLS in the presence of GGUG- or CCUC-oligonucleotide. * p
    Rnase Free Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8524 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher a594 dnaase i
    Consensus GGUG-containing RNA oligonucleotide promotes the inhibitory effect of TLS on CBP/p300 HAT activities a , Co-immunoprecipitation (IP) of p300 and TLS from <t>RNase</t> A-treated HeLa cells. b, P300 HAT activity was measured using micrococcal nuclease (MNase) or <t>DNase</t> I pre-treated GST and GST-TLS in the presence of GGUG- or CCUC-oligonucleotide. * p
    A594 Dnaase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher turbo dnase i
    Effect of the K297R mutation on progeny virus release. iSLK-BAC16 and iSLK-BAC-K297R cells were induced with Dox and butyrate for indicated time. The extracellular virions were collected from culture media and treated with Turbo DNase I. Viral DNAs were extracted and KSHV genomic DNA copy numbers were estimated by qPCR along with external standards of known concentrations of the viral DNA with primers against the ORF73 gene (A). Intracellular KSHV genomic DNAs were extracted from harvested cells and quantitated by qPCR as above (B). (*,  p
    Turbo Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher ambion dnase i
    Effect of the K297R mutation on progeny virus release. iSLK-BAC16 and iSLK-BAC-K297R cells were induced with Dox and butyrate for indicated time. The extracellular virions were collected from culture media and treated with Turbo DNase I. Viral DNAs were extracted and KSHV genomic DNA copy numbers were estimated by qPCR along with external standards of known concentrations of the viral DNA with primers against the ORF73 gene (A). Intracellular KSHV genomic DNAs were extracted from harvested cells and quantitated by qPCR as above (B). (*,  p
    Ambion Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dnase i buffer
    Analysis of the Mur34 binding site by <t>DNase</t> I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.
    Dnase I Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 454 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher m v dnase i
    Analysis of the Mur34 binding site by <t>DNase</t> I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.
    M V Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dnase i grade ii
    MCC22 reduced pro-inflammatory cytokine and increased the anti-inflammatory cytokine IL-10 expression in the spinal cord of HbSS mice. HbSS mice were treated with i.p. injections of 8 μmol/kg (10 mg/kg) MCC22 (n=4) or vehicle (n=4) for 9 consecutive days. The spinal cord was removed from HbAA mice and HbSS mice administered vehicle or MCC22. The <t>RNA</t> was isolated, <t>DNase</t> treated, converted to cDNA, and used in real time PCR with primers for IL-1β (A) , IL-6 (B) , TNFα (C) , and IL-10 (D) . Concentrations for each cytokine were determined based on a standard set for each primer pair, and samples were normalized to β-actin expression. Significant differences in expression were determined between HbAA and HbSS vehicle-treated mice or HbSS MCC22-treated mice (Bonferroni t-tests; * p
    Dnase I Grade Ii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1337 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher gt dnase i
    MCC22 reduced pro-inflammatory cytokine and increased the anti-inflammatory cytokine IL-10 expression in the spinal cord of HbSS mice. HbSS mice were treated with i.p. injections of 8 μmol/kg (10 mg/kg) MCC22 (n=4) or vehicle (n=4) for 9 consecutive days. The spinal cord was removed from HbAA mice and HbSS mice administered vehicle or MCC22. The <t>RNA</t> was isolated, <t>DNase</t> treated, converted to cDNA, and used in real time PCR with primers for IL-1β (A) , IL-6 (B) , TNFα (C) , and IL-10 (D) . Concentrations for each cytokine were determined based on a standard set for each primer pair, and samples were normalized to β-actin expression. Significant differences in expression were determined between HbAA and HbSS vehicle-treated mice or HbSS MCC22-treated mice (Bonferroni t-tests; * p
    Gt Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dnase i rnasefree
    MCC22 reduced pro-inflammatory cytokine and increased the anti-inflammatory cytokine IL-10 expression in the spinal cord of HbSS mice. HbSS mice were treated with i.p. injections of 8 μmol/kg (10 mg/kg) MCC22 (n=4) or vehicle (n=4) for 9 consecutive days. The spinal cord was removed from HbAA mice and HbSS mice administered vehicle or MCC22. The <t>RNA</t> was isolated, <t>DNase</t> treated, converted to cDNA, and used in real time PCR with primers for IL-1β (A) , IL-6 (B) , TNFα (C) , and IL-10 (D) . Concentrations for each cytokine were determined based on a standard set for each primer pair, and samples were normalized to β-actin expression. Significant differences in expression were determined between HbAA and HbSS vehicle-treated mice or HbSS MCC22-treated mice (Bonferroni t-tests; * p
    Dnase I Rnasefree, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher dnase i endonuclease
    Effect of calpain-like silencing during PCD. (A) DNA fragmentation in calpain-like silenced trophozoites after PCD induction by G418. Confocal microscopy analysis of trophozoites showing the TUNEL staining, samples were counter-stained with DAPI. (A) Negative control, untreated trophozoites; (B) Positive control, trophozoites treated with 20 μg of <t>DNase</t> I endonuclease for 30 min; (C) Trophozoites after a 9-h incubation with 10 μg/ml of G418; (D,E) Trophozoites pre-incubated for 24 h with NRS (D) or calpain-like (E) siRNAs sequences and then 9 h with 10 μg/ml G418; (B) Densitometric analysis of (A). (C) Effect of calpain-like silencing on the cell viability of E. histolytica incubated with G418; (A) Negative control, untreated trophozoites; (B) Trophozoites after a 9-h incubation with 10 μg/ml of G418; (C,D) Trophozoites pre-incubated for 24 h with NRS (C) or calpain-like (D) siRNAs sequences and then 9 h with 10 μg/ml G418. *indicate statistically significant difference: P
    Dnase I Endonuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    MACHEREY NAGEL dnase i digestion
    Effect of calpain-like silencing during PCD. (A) DNA fragmentation in calpain-like silenced trophozoites after PCD induction by G418. Confocal microscopy analysis of trophozoites showing the TUNEL staining, samples were counter-stained with DAPI. (A) Negative control, untreated trophozoites; (B) Positive control, trophozoites treated with 20 μg of <t>DNase</t> I endonuclease for 30 min; (C) Trophozoites after a 9-h incubation with 10 μg/ml of G418; (D,E) Trophozoites pre-incubated for 24 h with NRS (D) or calpain-like (E) siRNAs sequences and then 9 h with 10 μg/ml G418; (B) Densitometric analysis of (A). (C) Effect of calpain-like silencing on the cell viability of E. histolytica incubated with G418; (A) Negative control, untreated trophozoites; (B) Trophozoites after a 9-h incubation with 10 μg/ml of G418; (C,D) Trophozoites pre-incubated for 24 h with NRS (C) or calpain-like (D) siRNAs sequences and then 9 h with 10 μg/ml G418. *indicate statistically significant difference: P
    Dnase I Digestion, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    DNase I treatment is effective against C. jejuni biofilms on stainless steel surfaces and in the presence of organic materials in aerobic conditions . The ability of DNase I to inhibit biofilm formation of C. jejuni NCTC 11168 on sterile, stainless steel coupons (A) or in the presence of chicken juice, mimicking a conditioned surface (B) . TTC staining was used to measure biofilm formation in the presence of chicken juice (B) . DNase I is able to significantly decrease biofilm formation in both conditions. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Journal: Frontiers in Microbiology

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

    doi: 10.3389/fmicb.2015.00699

    Figure Lengend Snippet: DNase I treatment is effective against C. jejuni biofilms on stainless steel surfaces and in the presence of organic materials in aerobic conditions . The ability of DNase I to inhibit biofilm formation of C. jejuni NCTC 11168 on sterile, stainless steel coupons (A) or in the presence of chicken juice, mimicking a conditioned surface (B) . TTC staining was used to measure biofilm formation in the presence of chicken juice (B) . DNase I is able to significantly decrease biofilm formation in both conditions. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Article Snippet: Enzyme treatment of C. jejuni biofilms For DNase I treatments, unless otherwise stated, a volume of 4 μl DNase I enzyme (Fermentas), giving a final concentration within the biofilm of 4 U/ml v/v and 4 μl of DNase I buffer (Fermentas) were added to each test tube, along with 1 ml of diluted cell suspension at either the start of the static incubation or after 12, 24, 36, or 48 h of static incubation.

    Techniques: Staining, Standard Deviation, MANN-WHITNEY

    DNase I is able to rapidly degrade C. jejuni NCTC 11168 biofilms . (A) DNase I (4 units/ml) was added at defined intervals to aerobically incubated NCTC 11168 cultures over a 48 h static incubation and biofilm degradation assessed by crystal violet staining. (B) Following a 48 h static incubation to allow biofilm formation, DNase I was added to biofilms for between 5 and 120 min before biofilm degradation was assessed. (C) The concentration of DNase I required for biofilm control was also assessed using DNase I concentrations of between 0.01 and 5 U/ml. In each graph, “11168” represents an untreated biofilm culture of C. jejuni NCTC 11168 and “control” represents a tube containing sterile Brucella medium only. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Journal: Frontiers in Microbiology

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

    doi: 10.3389/fmicb.2015.00699

    Figure Lengend Snippet: DNase I is able to rapidly degrade C. jejuni NCTC 11168 biofilms . (A) DNase I (4 units/ml) was added at defined intervals to aerobically incubated NCTC 11168 cultures over a 48 h static incubation and biofilm degradation assessed by crystal violet staining. (B) Following a 48 h static incubation to allow biofilm formation, DNase I was added to biofilms for between 5 and 120 min before biofilm degradation was assessed. (C) The concentration of DNase I required for biofilm control was also assessed using DNase I concentrations of between 0.01 and 5 U/ml. In each graph, “11168” represents an untreated biofilm culture of C. jejuni NCTC 11168 and “control” represents a tube containing sterile Brucella medium only. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Article Snippet: Enzyme treatment of C. jejuni biofilms For DNase I treatments, unless otherwise stated, a volume of 4 μl DNase I enzyme (Fermentas), giving a final concentration within the biofilm of 4 U/ml v/v and 4 μl of DNase I buffer (Fermentas) were added to each test tube, along with 1 ml of diluted cell suspension at either the start of the static incubation or after 12, 24, 36, or 48 h of static incubation.

    Techniques: Incubation, Staining, Concentration Assay, Standard Deviation, MANN-WHITNEY

    Treatment of pre-existing biofilms with DNase I leads to inhibition of biofilm regrowth . C. jejuni NCTC 11168 biofilms were allowed to form for 48 h in sterile borosilicate glass test tubes. To assess biofilm re-growth following DNase I treatment, two sets of tubes were treated with 4 U/ml DNase I for 15 min then washed with sterile PBS. Tubes were then supplemented with either fresh Brucella media (fifth bar) or fresh C. jejuni NCTC 11168 culture (sixth bar) and incubated for a further 48 h. The following controls were also prepared: C. jejuni NCTC 11168 biofilm formation following primary culture (first bar, white), tubes supplemented with sterile Brucella media (second bar, black), C. jejuni NCTC 11168 biofilm formation following only secondary culture (third bar, light gray), and 48 h-old C. jejuni NCTC 11168 biofilm, washed with PBS, then supplemented with fresh C. jejuni NCTC 11168 culture (fourth bar, dark gray). Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Journal: Frontiers in Microbiology

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

    doi: 10.3389/fmicb.2015.00699

    Figure Lengend Snippet: Treatment of pre-existing biofilms with DNase I leads to inhibition of biofilm regrowth . C. jejuni NCTC 11168 biofilms were allowed to form for 48 h in sterile borosilicate glass test tubes. To assess biofilm re-growth following DNase I treatment, two sets of tubes were treated with 4 U/ml DNase I for 15 min then washed with sterile PBS. Tubes were then supplemented with either fresh Brucella media (fifth bar) or fresh C. jejuni NCTC 11168 culture (sixth bar) and incubated for a further 48 h. The following controls were also prepared: C. jejuni NCTC 11168 biofilm formation following primary culture (first bar, white), tubes supplemented with sterile Brucella media (second bar, black), C. jejuni NCTC 11168 biofilm formation following only secondary culture (third bar, light gray), and 48 h-old C. jejuni NCTC 11168 biofilm, washed with PBS, then supplemented with fresh C. jejuni NCTC 11168 culture (fourth bar, dark gray). Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Article Snippet: Enzyme treatment of C. jejuni biofilms For DNase I treatments, unless otherwise stated, a volume of 4 μl DNase I enzyme (Fermentas), giving a final concentration within the biofilm of 4 U/ml v/v and 4 μl of DNase I buffer (Fermentas) were added to each test tube, along with 1 ml of diluted cell suspension at either the start of the static incubation or after 12, 24, 36, or 48 h of static incubation.

    Techniques: Inhibition, Incubation, Standard Deviation, MANN-WHITNEY

    Restriction endonuclease treatment of C. jejuni biofilms reduces biofilm formation . Static cultures of C. jejuni NCTC 11168 (A,B) and 81116 (C,D) were prepared then supplemented with either DNase I, RNase, or a single restriction endonuclease. Cultures were incubated for 48 h at 37°C in aerobic conditions. A range of restriction enzymes was selected, based on varying levels of DNA fragmentation following digestion of C. jejuni NCTC 11168 (B) and 81116 (D) genomic DNA. Restriction enzyme and DNase I treatment of NCTC 11168 biofilms lead to a reduction in biofilm formation. The same trend was observed for C. jejuni 81116, although only DNase I and Hae III digestion were significantly different from the control. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Journal: Frontiers in Microbiology

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

    doi: 10.3389/fmicb.2015.00699

    Figure Lengend Snippet: Restriction endonuclease treatment of C. jejuni biofilms reduces biofilm formation . Static cultures of C. jejuni NCTC 11168 (A,B) and 81116 (C,D) were prepared then supplemented with either DNase I, RNase, or a single restriction endonuclease. Cultures were incubated for 48 h at 37°C in aerobic conditions. A range of restriction enzymes was selected, based on varying levels of DNA fragmentation following digestion of C. jejuni NCTC 11168 (B) and 81116 (D) genomic DNA. Restriction enzyme and DNase I treatment of NCTC 11168 biofilms lead to a reduction in biofilm formation. The same trend was observed for C. jejuni 81116, although only DNase I and Hae III digestion were significantly different from the control. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Article Snippet: Enzyme treatment of C. jejuni biofilms For DNase I treatments, unless otherwise stated, a volume of 4 μl DNase I enzyme (Fermentas), giving a final concentration within the biofilm of 4 U/ml v/v and 4 μl of DNase I buffer (Fermentas) were added to each test tube, along with 1 ml of diluted cell suspension at either the start of the static incubation or after 12, 24, 36, or 48 h of static incubation.

    Techniques: Incubation, Standard Deviation, MANN-WHITNEY

    Effects of noncomplementary dNTPs on DNase I footprints and stable complex formation by HIV-1 RT

    Journal:

    Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

    doi: 10.1016/j.jmb.2007.03.006

    Figure Lengend Snippet: Effects of noncomplementary dNTPs on DNase I footprints and stable complex formation by HIV-1 RT

    Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

    Techniques:

    Effects of foscarnet on DNase I protection and stable complex formation by HIV-1 RT

    Journal:

    Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

    doi: 10.1016/j.jmb.2007.03.006

    Figure Lengend Snippet: Effects of foscarnet on DNase I protection and stable complex formation by HIV-1 RT

    Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

    Techniques:

    DNase I protection on P/Ts terminated with dT analogues

    Journal:

    Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

    doi: 10.1016/j.jmb.2007.03.006

    Figure Lengend Snippet: DNase I protection on P/Ts terminated with dT analogues

    Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

    Techniques:

    Effects of the next complementary dNTP on DNase I protection and stable complex formation by HIV-1 RT

    Journal:

    Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

    doi: 10.1016/j.jmb.2007.03.006

    Figure Lengend Snippet: Effects of the next complementary dNTP on DNase I protection and stable complex formation by HIV-1 RT

    Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

    Techniques:

    DNase I cleavage patterns and footprint distribution for overrepresented footprints surrounding TSSs. ( A ) Mean per-nucleotide DNase I cleavage profile from aligning the annotated TSSs of 5050 genes (+/− 1 kb regions). ( B ) Top heat map plotted for DNase I cleavage patterns of 5050 genes at +/− 1 kb TSS flanking regions by K-means clustering, which were subsequently divided into four distinct clusters, marked with red, blue, green and purple bars. The bottom mean DNase I cleavage patterns derived from four distinct clusters, where the line colors correspond to the marked colors of the heatmap. ( C ) Distribution of digital footprints (FDR

    Journal: Nucleic Acids Research

    Article Title: Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

    doi: 10.1093/nar/gkv334

    Figure Lengend Snippet: DNase I cleavage patterns and footprint distribution for overrepresented footprints surrounding TSSs. ( A ) Mean per-nucleotide DNase I cleavage profile from aligning the annotated TSSs of 5050 genes (+/− 1 kb regions). ( B ) Top heat map plotted for DNase I cleavage patterns of 5050 genes at +/− 1 kb TSS flanking regions by K-means clustering, which were subsequently divided into four distinct clusters, marked with red, blue, green and purple bars. The bottom mean DNase I cleavage patterns derived from four distinct clusters, where the line colors correspond to the marked colors of the heatmap. ( C ) Distribution of digital footprints (FDR

    Article Snippet: RT-qPCR reactions, including 10 μl of reaction mixture with 50 ng DNase I digestion product, 2 × 0.4 μl primers (forward and reverse, 10 μM), 0.2 μl ROX reference dye II (50×), 5 μl SYBR Premix Ex Taq II (2×) and dH2 O, were amplified using an Applied Biosystems 7500 Real-time PCR System for 1 min at 95°C, followed by 40 cycles of 95°C for 5 s, 55°C for 20 s and 72°C for 34 s. The degree of DNase I digestion was determined based on changes in Ct values.

    Techniques: Derivative Assay

    Diversity of DNase I cleavage patterns and function annotation of target genes for the overrepresented motifs in genomic footprints. ( A ) DNase I cleavage density per nucleotide calculated for footprint instances from two culture conditions. Shaded regions delineate the overrepresented motifs derived from the footprint region. The MEME logo of overrepresented motifs derived from footprints is shown below the graph. ( B ) GO function enrichment for the target genes under the DPY_motif 3 and DPY_motif 7. The genes containing at least one motif instance inside the 1-kb region of the annotated TSSs were selected. The genes under the same motif were analyzed using ClueGo. Functional group networks are represented by nodes linked with each other based on their kappa score level ( > 0.3). The node size represents the percentage of associated genes with the enrichment significance of the term (Term P -value

    Journal: Nucleic Acids Research

    Article Title: Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

    doi: 10.1093/nar/gkv334

    Figure Lengend Snippet: Diversity of DNase I cleavage patterns and function annotation of target genes for the overrepresented motifs in genomic footprints. ( A ) DNase I cleavage density per nucleotide calculated for footprint instances from two culture conditions. Shaded regions delineate the overrepresented motifs derived from the footprint region. The MEME logo of overrepresented motifs derived from footprints is shown below the graph. ( B ) GO function enrichment for the target genes under the DPY_motif 3 and DPY_motif 7. The genes containing at least one motif instance inside the 1-kb region of the annotated TSSs were selected. The genes under the same motif were analyzed using ClueGo. Functional group networks are represented by nodes linked with each other based on their kappa score level ( > 0.3). The node size represents the percentage of associated genes with the enrichment significance of the term (Term P -value

    Article Snippet: RT-qPCR reactions, including 10 μl of reaction mixture with 50 ng DNase I digestion product, 2 × 0.4 μl primers (forward and reverse, 10 μM), 0.2 μl ROX reference dye II (50×), 5 μl SYBR Premix Ex Taq II (2×) and dH2 O, were amplified using an Applied Biosystems 7500 Real-time PCR System for 1 min at 95°C, followed by 40 cycles of 95°C for 5 s, 55°C for 20 s and 72°C for 34 s. The degree of DNase I digestion was determined based on changes in Ct values.

    Techniques: Derivative Assay, Functional Assay

    The DNase I cleavage patterns of five family types of TFs parallel the co-crystal structures of protein and DNA interaction. ( A ) Strand-specific DNase-seq signal for DNase I cleavage imbalance between the plus and minus motif sequences of five family types of the TFs independent of strand orientation. The upper panels show the heat maps of per-nucleotide DNase I cleavage derived from all instances of plus (red) and minus (blue) TFBS motifs within DHSs under DPY conditions ranked according to the probability of MILLIPEDE (FIMO P

    Journal: Nucleic Acids Research

    Article Title: Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

    doi: 10.1093/nar/gkv334

    Figure Lengend Snippet: The DNase I cleavage patterns of five family types of TFs parallel the co-crystal structures of protein and DNA interaction. ( A ) Strand-specific DNase-seq signal for DNase I cleavage imbalance between the plus and minus motif sequences of five family types of the TFs independent of strand orientation. The upper panels show the heat maps of per-nucleotide DNase I cleavage derived from all instances of plus (red) and minus (blue) TFBS motifs within DHSs under DPY conditions ranked according to the probability of MILLIPEDE (FIMO P

    Article Snippet: RT-qPCR reactions, including 10 μl of reaction mixture with 50 ng DNase I digestion product, 2 × 0.4 μl primers (forward and reverse, 10 μM), 0.2 μl ROX reference dye II (50×), 5 μl SYBR Premix Ex Taq II (2×) and dH2 O, were amplified using an Applied Biosystems 7500 Real-time PCR System for 1 min at 95°C, followed by 40 cycles of 95°C for 5 s, 55°C for 20 s and 72°C for 34 s. The degree of DNase I digestion was determined based on changes in Ct values.

    Techniques: Derivative Assay

    Analysis of RNA isolated from purified virions and dense bodies (DBs). Virions and DBs were purified from RNase-ONE-treated virus stocks, dialysed and treated with micrococcal nuclease; RNA was extracted, and during the RNA extraction the column was treated with DNase-I. (a) RNA concentrations of virions and DBs. Presence of (b) viral IE RNA transcripts and (c) lncRNA2.7 transcripts determined by TaqMan PCR assays. Values are mean± sem of three experiments performed in triplicate. RT, reverse transcriptase.

    Journal: The Journal of General Virology

    Article Title: Human cytomegalovirus microRNAs are carried by virions and dense bodies and are delivered to target cells

    doi: 10.1099/jgv.0.000736

    Figure Lengend Snippet: Analysis of RNA isolated from purified virions and dense bodies (DBs). Virions and DBs were purified from RNase-ONE-treated virus stocks, dialysed and treated with micrococcal nuclease; RNA was extracted, and during the RNA extraction the column was treated with DNase-I. (a) RNA concentrations of virions and DBs. Presence of (b) viral IE RNA transcripts and (c) lncRNA2.7 transcripts determined by TaqMan PCR assays. Values are mean± sem of three experiments performed in triplicate. RT, reverse transcriptase.

    Article Snippet: RNA was isolated with the miRNeasy mini kit (Qiagen), including the DNase-I treatment or isolated with Trizol-LS reagent (Life Technologies).

    Techniques: Isolation, Purification, RNA Extraction, Polymerase Chain Reaction

    Procedure to study protein-nucleic acid interactions. The oligonucleotide is designed to contain a 5'-biotin tag, a 3'-fluorescent tag and a UV sensitive group in the middle. (A) Crosslink initiated by exposing to UV (305+16 nm). (B) After three hours of UV activation, denatured samples were subjected to SDS-PAGE analysis, where crosslinked species were confirmed by fluorescent imaging and all bands were visualized by Commassie staining technique. (C) Interesting spots were picked for protease enzymatic in-gel digestion to yield peptide mixtures. (D) Crosslinked peptides were extracted by magnetic streptavidin beads and uncrosslinked peptides were washed away. (E) The crosslinked peptides were subjected to DNase I degradation to minimize the attaching oligonucleotide moieties. (F) Crosslinked peptides with the remaining nucleic acid attached were extracted by reverse phase ZipTip C18 cartridge and analyzed by Q-tof ESI. (G) Raw data were collected and processed by Protein-Lynx to generate a PKL file, which was used as input to CLPM to identify matches with theoretical peptides.

    Journal: BMC Bioinformatics

    Article Title: CLPM: A Cross-Linked Peptide Mapping Algorithm for Mass Spectrometric Analysis

    doi: 10.1186/1471-2105-6-S2-S9

    Figure Lengend Snippet: Procedure to study protein-nucleic acid interactions. The oligonucleotide is designed to contain a 5'-biotin tag, a 3'-fluorescent tag and a UV sensitive group in the middle. (A) Crosslink initiated by exposing to UV (305+16 nm). (B) After three hours of UV activation, denatured samples were subjected to SDS-PAGE analysis, where crosslinked species were confirmed by fluorescent imaging and all bands were visualized by Commassie staining technique. (C) Interesting spots were picked for protease enzymatic in-gel digestion to yield peptide mixtures. (D) Crosslinked peptides were extracted by magnetic streptavidin beads and uncrosslinked peptides were washed away. (E) The crosslinked peptides were subjected to DNase I degradation to minimize the attaching oligonucleotide moieties. (F) Crosslinked peptides with the remaining nucleic acid attached were extracted by reverse phase ZipTip C18 cartridge and analyzed by Q-tof ESI. (G) Raw data were collected and processed by Protein-Lynx to generate a PKL file, which was used as input to CLPM to identify matches with theoretical peptides.

    Article Snippet: • DNase I (Ambion).

    Techniques: Activation Assay, SDS Page, Imaging, Staining

    Possible ion structures for the fragmentation of tryptic peptide crosslinked to the dinucleotide (dGdU) after DNase I digestion as proposed by Golden et al [31] . The blue line on the right of each diagram represents the peptide moiety in the heteroconjugate.

    Journal: BMC Bioinformatics

    Article Title: CLPM: A Cross-Linked Peptide Mapping Algorithm for Mass Spectrometric Analysis

    doi: 10.1186/1471-2105-6-S2-S9

    Figure Lengend Snippet: Possible ion structures for the fragmentation of tryptic peptide crosslinked to the dinucleotide (dGdU) after DNase I digestion as proposed by Golden et al [31] . The blue line on the right of each diagram represents the peptide moiety in the heteroconjugate.

    Article Snippet: • DNase I (Ambion).

    Techniques:

    RhA3A induces low frequencies of G-to-A mutations in SHIVΔ vif nascent reverse transcripts. TZM-Bl cells were infected with DNAse-I treated SHIVΔ vif or SHIV virions and DNA was extracted after 24 h. A 300-bp segment of nef was amplified,

    Journal: Virology

    Article Title: Differential virus restriction patterns of rhesus macaque and human APOBEC3A: implications for lentivirus evolution

    doi: 10.1016/j.virol.2011.07.017

    Figure Lengend Snippet: RhA3A induces low frequencies of G-to-A mutations in SHIVΔ vif nascent reverse transcripts. TZM-Bl cells were infected with DNAse-I treated SHIVΔ vif or SHIV virions and DNA was extracted after 24 h. A 300-bp segment of nef was amplified,

    Article Snippet: The resulting supernatant was DNase-I-treated (Fermentas) at 37°C for 30 min to minimize plasmid carry-over.

    Techniques: Infection, Amplification

    DNase I expression and activity. (A) The protein expression pattern of DNase I in kidney sections from (NZBxNZW)F1 mice as revealed by IHC, IF, IEM, and confocal microscopy (DNase I in red and Trap1 in green as a cytoplasmic marker). (B) DNase I endonuclease activity as revealed by zymography on samples from whole kidney lysates of representative Group 1, 2, and 3 (NZBxNZW)F1 mice, as well as a control (R, recombinant DNase I) and a serum (S) sample. (C) Urinary DNase I endonuclease activity in different groups of (NZBxNZW)F1 mice as revealed by zymography.

    Journal: The Journal of Pathology: Clinical Research

    Article Title: Lupus nephritis: low urinary DNase I levels reflect loss of renal DNase I and may be utilized as a biomarker of disease progression

    doi: 10.1002/cjp2.99

    Figure Lengend Snippet: DNase I expression and activity. (A) The protein expression pattern of DNase I in kidney sections from (NZBxNZW)F1 mice as revealed by IHC, IF, IEM, and confocal microscopy (DNase I in red and Trap1 in green as a cytoplasmic marker). (B) DNase I endonuclease activity as revealed by zymography on samples from whole kidney lysates of representative Group 1, 2, and 3 (NZBxNZW)F1 mice, as well as a control (R, recombinant DNase I) and a serum (S) sample. (C) Urinary DNase I endonuclease activity in different groups of (NZBxNZW)F1 mice as revealed by zymography.

    Article Snippet: In addition, a radial diffusion assay was used to measure DNase I activity in 1% agarose gel with 30 µg/ml heat‐denatured salmon sperm DNA (Invitrogen) in the DNase I reaction buffer.

    Techniques: Expressing, Activity Assay, Mouse Assay, Immunohistochemistry, Confocal Microscopy, Marker, Zymography, Recombinant

    Kinetics of loop DNA digestion with DNase I in nucleoids from P0, P7, P80 and P540 rat neurons. Nucleoids were treated with DNase I at 0.92 U/ml. Each time-point value corresponds to the average of independent experiments using separate animals as the source of nucleoids (P0 n = 4; P7 n = 5; P80 n = 5; P540 n = 4). The data sets for P7 and P540 were taken from our previous work [21] . The topological zones relative to the NM for the corresponding kinetics were established considering the local slopes between pairs of time points ( Table 3 ) and the corresponding S.D.

    Journal: PLoS ONE

    Article Title: Continued Stabilization of the Nuclear Higher-Order Structure of Post-Mitotic Neurons In Vivo

    doi: 10.1371/journal.pone.0021360

    Figure Lengend Snippet: Kinetics of loop DNA digestion with DNase I in nucleoids from P0, P7, P80 and P540 rat neurons. Nucleoids were treated with DNase I at 0.92 U/ml. Each time-point value corresponds to the average of independent experiments using separate animals as the source of nucleoids (P0 n = 4; P7 n = 5; P80 n = 5; P540 n = 4). The data sets for P7 and P540 were taken from our previous work [21] . The topological zones relative to the NM for the corresponding kinetics were established considering the local slopes between pairs of time points ( Table 3 ) and the corresponding S.D.

    Article Snippet: Standard PCR was carried out using 1.25 U GoTaq Flexi DNA polymerase (Promega) and 60 ng of nuclear matrix-bound DNA obtained from each DNase I digestion point, using an Applied Biosystems 2720 thermal cycler, and the same amplification program was used for all pairs of primers ( ): initial denaturising step at 94°C for 5 min, denaturising step at 94°C for 45 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min for 35 cycles, with a final extension at 72°C for 10 min.

    Techniques:

    Developing surface-tethered nuclease sensor (SNS) for the in situ mapping of membrane-bound nuclease (MN) activity on the cell membrane. (A) SNS is a fluorophore (here Cy3) conjugated with a biotin and a quencher-labeled dsDNA. The fluorophore is freed from quenching when the dsDNA is degraded by MN, thus reporting the MN activity by fluorescence on site. (B) SNS-coated surface reported DNase I in solution at a series of concentrations. (C) The detection limit for soluble DNase I by SNS is calibrated to be 0.01 unit/ml (rounding 0.05/8.3 to 0.01). (D) An SNS-coated surface reported highly organized MN activity on the ventral membrane of adherent MDA-MB-231 cells. (E) The structure feature of the SNS pattern is finer than 1 μm.

    Journal: Journal of biophotonics

    Article Title: Optical sensor revealed abnormal nuclease spatial activity on cancer cell membrane

    doi: 10.1002/jbio.201800351

    Figure Lengend Snippet: Developing surface-tethered nuclease sensor (SNS) for the in situ mapping of membrane-bound nuclease (MN) activity on the cell membrane. (A) SNS is a fluorophore (here Cy3) conjugated with a biotin and a quencher-labeled dsDNA. The fluorophore is freed from quenching when the dsDNA is degraded by MN, thus reporting the MN activity by fluorescence on site. (B) SNS-coated surface reported DNase I in solution at a series of concentrations. (C) The detection limit for soluble DNase I by SNS is calibrated to be 0.01 unit/ml (rounding 0.05/8.3 to 0.01). (D) An SNS-coated surface reported highly organized MN activity on the ventral membrane of adherent MDA-MB-231 cells. (E) The structure feature of the SNS pattern is finer than 1 μm.

    Article Snippet: Soluble nuclease DNase I (89836, ThermoFisher Scientific) solutions at a series of concentrations were added to SNS coated petridishes.

    Techniques: In Situ, Activity Assay, Labeling, Fluorescence, Multiple Displacement Amplification

    Luciferase gene expression and DNA accessibility as a function of r charge using pre-casted RNA gels. (A) G4 dendrimers and (B) CTAB. The synthesized amounts of RNA are displayed and samples were not pretreated with Dnase I. References are displayed in B where lane 1 shows the sample consisting only of DNA and without any compacting agent or transcriptional activity. Lane 2 shows the control sample containing DNA and the in vitro transcription mixture in the absence of compacting agents. Gels were post-stained using GelStar.

    Journal: PLoS ONE

    Article Title: DNA Compaction Induced by a Cationic Polymer or Surfactant Impact Gene Expression and DNA Degradation

    doi: 10.1371/journal.pone.0092692

    Figure Lengend Snippet: Luciferase gene expression and DNA accessibility as a function of r charge using pre-casted RNA gels. (A) G4 dendrimers and (B) CTAB. The synthesized amounts of RNA are displayed and samples were not pretreated with Dnase I. References are displayed in B where lane 1 shows the sample consisting only of DNA and without any compacting agent or transcriptional activity. Lane 2 shows the control sample containing DNA and the in vitro transcription mixture in the absence of compacting agents. Gels were post-stained using GelStar.

    Article Snippet: Degradation of DNA Dnase I (Turbo Dnase I, Ambion) was used to elucidate how the degree of compaction affects the protection against enzymatic digestion of DNA.

    Techniques: Luciferase, Expressing, Synthesized, Activity Assay, In Vitro, Staining

    Protection of DNA against Dnase I digestion by CTAB using a gel stained with EtBr. A gel electrophoresis gel where samples in lanes 1 and 6 contain linearized plasmid DNA only (control). The remaining lanes contain CTAB/DNA of the r charge values indicated. The samples loaded onto lanes 6–10 were incubated with Dnase I for 20 min following DNA condensation. At r charge ≤1.0, the DNA is completely degraded. At higher concentrations of CTAB, DNA degradation is inhibited.

    Journal: PLoS ONE

    Article Title: DNA Compaction Induced by a Cationic Polymer or Surfactant Impact Gene Expression and DNA Degradation

    doi: 10.1371/journal.pone.0092692

    Figure Lengend Snippet: Protection of DNA against Dnase I digestion by CTAB using a gel stained with EtBr. A gel electrophoresis gel where samples in lanes 1 and 6 contain linearized plasmid DNA only (control). The remaining lanes contain CTAB/DNA of the r charge values indicated. The samples loaded onto lanes 6–10 were incubated with Dnase I for 20 min following DNA condensation. At r charge ≤1.0, the DNA is completely degraded. At higher concentrations of CTAB, DNA degradation is inhibited.

    Article Snippet: Degradation of DNA Dnase I (Turbo Dnase I, Ambion) was used to elucidate how the degree of compaction affects the protection against enzymatic digestion of DNA.

    Techniques: Staining, Nucleic Acid Electrophoresis, Plasmid Preparation, Incubation

    Protection of DNA against Dnase I digestion using gels stained with GelStar. (A) G4/DNA complexes with r charge = 0.4 are used and the untreated complex is loaded on lane 1. The 2 nd lane displays the dissociated complex after treatment with 10 μg mL −1 heparin for 30 min. All other samples (lanes 3–7) are treated with 1 unit of Dnase I for the indicated time periods. To the samples in lanes 4–7, heparin was added after the Dnase I enzyme was heat inactivated. (B) Linearized plasmid DNA only is loaded onto lane 1 and the sample loaded onto lane 2 contains DNA, treated with Dnase I for 30 min. Samples loaded onto lanes 3–7 contain DNA and CTAB ( r charge = 7.5). Samples loaded onto lanes 4–7 are treated with Dnase I for the time periods indicated.

    Journal: PLoS ONE

    Article Title: DNA Compaction Induced by a Cationic Polymer or Surfactant Impact Gene Expression and DNA Degradation

    doi: 10.1371/journal.pone.0092692

    Figure Lengend Snippet: Protection of DNA against Dnase I digestion using gels stained with GelStar. (A) G4/DNA complexes with r charge = 0.4 are used and the untreated complex is loaded on lane 1. The 2 nd lane displays the dissociated complex after treatment with 10 μg mL −1 heparin for 30 min. All other samples (lanes 3–7) are treated with 1 unit of Dnase I for the indicated time periods. To the samples in lanes 4–7, heparin was added after the Dnase I enzyme was heat inactivated. (B) Linearized plasmid DNA only is loaded onto lane 1 and the sample loaded onto lane 2 contains DNA, treated with Dnase I for 30 min. Samples loaded onto lanes 3–7 contain DNA and CTAB ( r charge = 7.5). Samples loaded onto lanes 4–7 are treated with Dnase I for the time periods indicated.

    Article Snippet: Degradation of DNA Dnase I (Turbo Dnase I, Ambion) was used to elucidate how the degree of compaction affects the protection against enzymatic digestion of DNA.

    Techniques: Staining, Plasmid Preparation

    Expression of CES genes in opossum. Liver and intestinal cDNAs were reverse transcribed from DNase I-treated RNA, and they were used as templates in RT-PCR to analyze CES gene expression. Lanes 2 and 3 are RT-PCR products amplified from liver (L) and intestine (I) cDNAs for the CES1 gene; lanes 4 and 5, RT-PCR products from liver (L) and intestine (I) cDNAs for the CES2.1 gene; lanes 6 and 7, RT-PCR products from liver (L) and intestine (I) for cDNAs for the CES2.2 gene; and lanes 8 and 9, RT-PCR products from liver (L) and intestine (I) for cDNAs for the CES2.3 gene. M shows the DNA size ladder.

    Journal: BMC Evolutionary Biology

    Article Title: Opossum carboxylesterases: sequences, phylogeny and evidence for CES gene duplication events predating the marsupial-eutherian common ancestor

    doi: 10.1186/1471-2148-8-54

    Figure Lengend Snippet: Expression of CES genes in opossum. Liver and intestinal cDNAs were reverse transcribed from DNase I-treated RNA, and they were used as templates in RT-PCR to analyze CES gene expression. Lanes 2 and 3 are RT-PCR products amplified from liver (L) and intestine (I) cDNAs for the CES1 gene; lanes 4 and 5, RT-PCR products from liver (L) and intestine (I) cDNAs for the CES2.1 gene; lanes 6 and 7, RT-PCR products from liver (L) and intestine (I) for cDNAs for the CES2.2 gene; and lanes 8 and 9, RT-PCR products from liver (L) and intestine (I) for cDNAs for the CES2.3 gene. M shows the DNA size ladder.

    Article Snippet: DNase I-treated RNA was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification

    Minor satellite RNA associate with CENP-A-containing chromatin. ( A ) RNA NChIP from nuclear extracts of asynchronously growing (AS) or nocodazole-arrested (NOC) MEL cells using antibodies against CENP-A, H3K9Me3 or H3Ac. Following DNaseI digestion, minor satellite RNA (minsat) and rDNA transcripts (rDNA) were analysed by RT-PCR, from the immunoprecipitated (IP) or supernatant fractions (SUP), in reactions containing (+) or not (−) RT. Control reactions in the absence of immunoprecipitating antibody (beads) were used as a negative control. ( B ) RNA pull-down experiments using in vitro transcribed minor satellite RNA (minsatF), followed by western blot (WB) analysis with the indicated antibodies. As a control, a similar experiment was performed with in vitro transcribed minor satellite RNA from the reverse strand (minsatR), therefore not complementary to the biotinylated DNA/LNA oligonucleotide used, or in the absence of RNA.

    Journal: Nucleic Acids Research

    Article Title: Non-coding murine centromeric transcripts associate with and potentiate Aurora B kinase

    doi: 10.1093/nar/gkp529

    Figure Lengend Snippet: Minor satellite RNA associate with CENP-A-containing chromatin. ( A ) RNA NChIP from nuclear extracts of asynchronously growing (AS) or nocodazole-arrested (NOC) MEL cells using antibodies against CENP-A, H3K9Me3 or H3Ac. Following DNaseI digestion, minor satellite RNA (minsat) and rDNA transcripts (rDNA) were analysed by RT-PCR, from the immunoprecipitated (IP) or supernatant fractions (SUP), in reactions containing (+) or not (−) RT. Control reactions in the absence of immunoprecipitating antibody (beads) were used as a negative control. ( B ) RNA pull-down experiments using in vitro transcribed minor satellite RNA (minsatF), followed by western blot (WB) analysis with the indicated antibodies. As a control, a similar experiment was performed with in vitro transcribed minor satellite RNA from the reverse strand (minsatR), therefore not complementary to the biotinylated DNA/LNA oligonucleotide used, or in the absence of RNA.

    Article Snippet: Genomic DNA was removed by digestion with 2 U of DNaseI (Ambion). cDNA were synthesized from an equivalent of 500 000 cells ( A) or 100 000 cells ( B and C), using random hexamers and Superscript II reverse transcriptase (Invitrogen), and amplified by PCR.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Negative Control, In Vitro, Western Blot

    NETs contribute to the pathogenesis of myocarditis. (a) Confocal immunofluorescence images of an EMB of patient 6 suffering from PVB19-positive myocarditis (see Table 1 ). Top: Images show staining of H2A-H2B-DNA, MPO, and H3Cit as well as the merged image. Bar, 20 µm. Bottom: Magnification of a selected area of the merged confocal image (box) from the top panel. Arrows indicate NETs. Bar, 10 µm. (b and c) EAM score (b) as well as evaluation of the heart/body weight ratio (c) at day 21 after induction of EAM or sham immunization (sham; mean EAM score, 0.5 ± 0.19; mean heart/body weight ratio, 0.57 ± 0.01). Mice were treated with DNase (EAM + DNase: mean EAM score, 0.8 ± 0.19; mean heart/body weight ratio, 0.62 ± 0.005), Cl-amid (EAM + Cl-amid: mean EAM score, 0.9 ± 0.20; mean heart/body weight ratio, 0.62 ± 0.01), or vehicle as control (EAM + vehicle: mean EAM score, 1.7 ± 0.25; mean heart/body weight ratio, 0.71 ± 0.04) as indicated. n = 8 for sham group; n = 26 for EAM groups. (d) Left: Representative confocal images of cardiac sections of immunized WT mice at day 21 after induction of EAM (EAM + vehicle). Images show staining of DNA, NE, and H3Cit as well as the merged image. Bars, 20 µm. Right: Representative confocal magnified image of NETs. Images show staining of DNA, NE, and H3Cit as well as the merged image, colocalization (coloc) of DNA/NE, and DNA/H3Cit as indicated. Bars, 10 µm. (e) Representative confocal images of cardiac sections of EAM mice at day 21 after induction of EAM and blockade of MK for 21 d. Images show staining of DNA, NE, and H3Cit as well as the merged image. Bars, 20 µm. (f–h) Histological analysis of extravasated PMN into the cardiac tissue at day 21 after induction of EAM without blocking MK (EAM + vehicle) or after application of an anti-N-MK blocking Ab (EAM + anti-N-MK). Diagrams show the number of extravasated PMNs per mm 2 (f: vehicle, 768 ± 55 PMN/mm 2 ; anti-N-MK, 158 ± 86 PMN/mm 2 ), the number of NET + PMNs per mm 2 (g: vehicle, 40 ± 6 NET + PMN; anti-N-MK = 5 ± 2 NET + PMN), and the number of NET + PMNs as a percentage of all extravasated PMNs (h: vehicle, 5.1 ± 0.5 NET + PMN [%]; anti-N-MK, 0.9 ± 0.6 NET + PMN [%]). n = 6 mice. To determine P values, ANOVA on ranks with Dunn’s multiple comparisons test was performed for c and d, and an unpaired Student's t test was used for f–h. For all panels, *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Midkine drives cardiac inflammation by promoting neutrophil trafficking and NETosis in myocarditis

    doi: 10.1084/jem.20181102

    Figure Lengend Snippet: NETs contribute to the pathogenesis of myocarditis. (a) Confocal immunofluorescence images of an EMB of patient 6 suffering from PVB19-positive myocarditis (see Table 1 ). Top: Images show staining of H2A-H2B-DNA, MPO, and H3Cit as well as the merged image. Bar, 20 µm. Bottom: Magnification of a selected area of the merged confocal image (box) from the top panel. Arrows indicate NETs. Bar, 10 µm. (b and c) EAM score (b) as well as evaluation of the heart/body weight ratio (c) at day 21 after induction of EAM or sham immunization (sham; mean EAM score, 0.5 ± 0.19; mean heart/body weight ratio, 0.57 ± 0.01). Mice were treated with DNase (EAM + DNase: mean EAM score, 0.8 ± 0.19; mean heart/body weight ratio, 0.62 ± 0.005), Cl-amid (EAM + Cl-amid: mean EAM score, 0.9 ± 0.20; mean heart/body weight ratio, 0.62 ± 0.01), or vehicle as control (EAM + vehicle: mean EAM score, 1.7 ± 0.25; mean heart/body weight ratio, 0.71 ± 0.04) as indicated. n = 8 for sham group; n = 26 for EAM groups. (d) Left: Representative confocal images of cardiac sections of immunized WT mice at day 21 after induction of EAM (EAM + vehicle). Images show staining of DNA, NE, and H3Cit as well as the merged image. Bars, 20 µm. Right: Representative confocal magnified image of NETs. Images show staining of DNA, NE, and H3Cit as well as the merged image, colocalization (coloc) of DNA/NE, and DNA/H3Cit as indicated. Bars, 10 µm. (e) Representative confocal images of cardiac sections of EAM mice at day 21 after induction of EAM and blockade of MK for 21 d. Images show staining of DNA, NE, and H3Cit as well as the merged image. Bars, 20 µm. (f–h) Histological analysis of extravasated PMN into the cardiac tissue at day 21 after induction of EAM without blocking MK (EAM + vehicle) or after application of an anti-N-MK blocking Ab (EAM + anti-N-MK). Diagrams show the number of extravasated PMNs per mm 2 (f: vehicle, 768 ± 55 PMN/mm 2 ; anti-N-MK, 158 ± 86 PMN/mm 2 ), the number of NET + PMNs per mm 2 (g: vehicle, 40 ± 6 NET + PMN; anti-N-MK = 5 ± 2 NET + PMN), and the number of NET + PMNs as a percentage of all extravasated PMNs (h: vehicle, 5.1 ± 0.5 NET + PMN [%]; anti-N-MK, 0.9 ± 0.6 NET + PMN [%]). n = 6 mice. To determine P values, ANOVA on ranks with Dunn’s multiple comparisons test was performed for c and d, and an unpaired Student's t test was used for f–h. For all panels, *, P

    Article Snippet: RNA was isolated using TRIzol (15596026; Thermo Fisher) according to the manufacturer’s protocol, and genomic DNA was digested using the DNase I Amplification Grade kit (18068015; Thermo Fisher).

    Techniques: Immunofluorescence, Staining, Mouse Assay, Blocking Assay

    Curcumin induces Bex genes in N2a neuroblastoma cells in a dose-dependent manner. ( a ) N2a cells (3 × 10 5 cells) were cultured for two days in 25 cm 2 flask, serum starved for 2 hours and treated either with 10, 25 or 50 μM of curcumin or with equal amount of DMSO (controls) in serum free media for 2 hours. Total RNA was isolated by Trizol reagent and treated with DNase I to remove any DNA contamination. Reverse transcription on 5 μg of DNA-free RNA was performed and Bex cDNAs were amplified either for 32, 34 or 36 PCR cycles. Agarose gel electrophoresis shows a dose-dependent induction of Bex genes by curcumin. The original gel images are shown in Supplementary Fig. S9 . Densitometric analysis of Bex1 ( b ), Bex2 ( c ), Bex4 ( d ) and Bex6 ( e ) mRNA amplicons was performed using Image lab software and values obtained were normalized with respective GAPDH band intensity, and were plotted as histograms of mean ± standard error of mean from three independent experiments. P-values displayed were calculated by two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Journal: Scientific Reports

    Article Title: Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells

    doi: 10.1038/srep41420

    Figure Lengend Snippet: Curcumin induces Bex genes in N2a neuroblastoma cells in a dose-dependent manner. ( a ) N2a cells (3 × 10 5 cells) were cultured for two days in 25 cm 2 flask, serum starved for 2 hours and treated either with 10, 25 or 50 μM of curcumin or with equal amount of DMSO (controls) in serum free media for 2 hours. Total RNA was isolated by Trizol reagent and treated with DNase I to remove any DNA contamination. Reverse transcription on 5 μg of DNA-free RNA was performed and Bex cDNAs were amplified either for 32, 34 or 36 PCR cycles. Agarose gel electrophoresis shows a dose-dependent induction of Bex genes by curcumin. The original gel images are shown in Supplementary Fig. S9 . Densitometric analysis of Bex1 ( b ), Bex2 ( c ), Bex4 ( d ) and Bex6 ( e ) mRNA amplicons was performed using Image lab software and values obtained were normalized with respective GAPDH band intensity, and were plotted as histograms of mean ± standard error of mean from three independent experiments. P-values displayed were calculated by two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Article Snippet: Total RNA was treated with 2 units of amplification grade DNase I (Life technologies, USA) for 15 minutes at room temperature.

    Techniques: Cell Culture, Isolation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Software, Two Tailed Test

    Curcumin is a specific inducer of all the Bex genes. ( a ) Approximately 80% confluent N2a cells were serum starved for 2 hours and treated with either 2.5, 5, 10 μM of MPTQ or 25 μM of curcumin in serum free DMEM for 4 hours. Equivalent amount of DMSO treated N2a cells were considered as controls. Total RNA was isolated, DNase I treated and expression of Bex mRNAs was studied by RT-PCR analysis. GAPDH was used as loading control. The original gel images are shown in Supplementary Fig. S11 . Densitometric analysis of Bex1 ( b ), Bex2 ( c ), Bex3 ( d ), Bex4 ( e ) and Bex6 ( f ) PCR products was performed and normalized with corresponding GAPDH band intensity. Values are displayed as histograms of mean ± standard deviation from three independent experiments, which indicates induction of all endogenous Bex genes are observed in curcumin but not in MPTQ treated N2a cells. P-values displayed were calculated by using two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Journal: Scientific Reports

    Article Title: Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells

    doi: 10.1038/srep41420

    Figure Lengend Snippet: Curcumin is a specific inducer of all the Bex genes. ( a ) Approximately 80% confluent N2a cells were serum starved for 2 hours and treated with either 2.5, 5, 10 μM of MPTQ or 25 μM of curcumin in serum free DMEM for 4 hours. Equivalent amount of DMSO treated N2a cells were considered as controls. Total RNA was isolated, DNase I treated and expression of Bex mRNAs was studied by RT-PCR analysis. GAPDH was used as loading control. The original gel images are shown in Supplementary Fig. S11 . Densitometric analysis of Bex1 ( b ), Bex2 ( c ), Bex3 ( d ), Bex4 ( e ) and Bex6 ( f ) PCR products was performed and normalized with corresponding GAPDH band intensity. Values are displayed as histograms of mean ± standard deviation from three independent experiments, which indicates induction of all endogenous Bex genes are observed in curcumin but not in MPTQ treated N2a cells. P-values displayed were calculated by using two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Article Snippet: Total RNA was treated with 2 units of amplification grade DNase I (Life technologies, USA) for 15 minutes at room temperature.

    Techniques: Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation, Two Tailed Test

    Optimization of curcumin treatment duration for the induction of Bex genes in N2a cells. ( a ) N2a cells (3 × 10 5 cells) were cultured for two days in 25 cm 2 flask, serum starved for 2 hours and treated with 25 μM of curcumin for 2, 4, 8 or 24 hours. Control cells were treated with equivalent amount of DMSO for 2 hours. Total RNA was isolated at indicated time points using Trizol reagent and treated with DNase I for 15 minutes at room temperature. RT-PCR analysis of DNA-free Bex mRNA was performed for 32, 34 or 36 PCR cycles. Agarose gel electrophoresis clearly demonstrates a time-dependent induction of Bex genes by curcumin. The original gel images are shown in Supplementary Fig. S10 . Intensity of Bex1 ( b ), Bex2 ( c ), Bex3 ( d ), Bex4 ( e ) and Bex6 ( f ) PCR products were obtained and normalized with corresponding GAPDH band intensity, and displayed as histograms of mean ± standard error of mean from three independent experiments. P-values displayed were calculated by using two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Journal: Scientific Reports

    Article Title: Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells

    doi: 10.1038/srep41420

    Figure Lengend Snippet: Optimization of curcumin treatment duration for the induction of Bex genes in N2a cells. ( a ) N2a cells (3 × 10 5 cells) were cultured for two days in 25 cm 2 flask, serum starved for 2 hours and treated with 25 μM of curcumin for 2, 4, 8 or 24 hours. Control cells were treated with equivalent amount of DMSO for 2 hours. Total RNA was isolated at indicated time points using Trizol reagent and treated with DNase I for 15 minutes at room temperature. RT-PCR analysis of DNA-free Bex mRNA was performed for 32, 34 or 36 PCR cycles. Agarose gel electrophoresis clearly demonstrates a time-dependent induction of Bex genes by curcumin. The original gel images are shown in Supplementary Fig. S10 . Intensity of Bex1 ( b ), Bex2 ( c ), Bex3 ( d ), Bex4 ( e ) and Bex6 ( f ) PCR products were obtained and normalized with corresponding GAPDH band intensity, and displayed as histograms of mean ± standard error of mean from three independent experiments. P-values displayed were calculated by using two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Article Snippet: Total RNA was treated with 2 units of amplification grade DNase I (Life technologies, USA) for 15 minutes at room temperature.

    Techniques: Cell Culture, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Two Tailed Test

    The unmethylated human IGF2R intron 2 CpG island contains a DNase1 hypersensitive site. (A) Map of human IGF2R intron 2 (17.686 kb) showing the direction of IGF2R transcription. A map of the fragment used to generate the mouse transgenes is shown underneath.

    Journal: Genomics

    Article Title: Identification of the human homolog of the imprinted mouse Air non-coding RNA

    doi: 10.1016/j.ygeno.2008.08.004

    Figure Lengend Snippet: The unmethylated human IGF2R intron 2 CpG island contains a DNase1 hypersensitive site. (A) Map of human IGF2R intron 2 (17.686 kb) showing the direction of IGF2R transcription. A map of the fragment used to generate the mouse transgenes is shown underneath.

    Article Snippet: 20 μg of total DNase1 (DNA-freeTM, Ambion) treated RNA was electrophoresed in 1% agarose/formaldehyde gels.

    Techniques:

    Primer-template dependent inhibitory effect on mRNA amplification . RNA samples extracted from cells harvested at 2,1 mg (dry weight)/mL were treated with DNase I according to protocol II. Different set of primers (as described in table I) was used to amplify CNA2 (lane 2), ACT1 (lane 3), TPS2 (lane 4), PDA1 (lane 5), CNA1 (lane 6), TPS1 (lane 7) mRNAs. 100 bp DNA ladder (lane P) and RT-minus control using ACT1 primers (lane 1).

    Journal: BMC Molecular Biology

    Article Title: Comparing protocols for preparation of DNA-free total yeast RNA suitable for RT-PCR

    doi: 10.1186/1471-2199-6-9

    Figure Lengend Snippet: Primer-template dependent inhibitory effect on mRNA amplification . RNA samples extracted from cells harvested at 2,1 mg (dry weight)/mL were treated with DNase I according to protocol II. Different set of primers (as described in table I) was used to amplify CNA2 (lane 2), ACT1 (lane 3), TPS2 (lane 4), PDA1 (lane 5), CNA1 (lane 6), TPS1 (lane 7) mRNAs. 100 bp DNA ladder (lane P) and RT-minus control using ACT1 primers (lane 1).

    Article Snippet: Protocol II was performed using the DNase I amplification grade kit (Life Technologies, Inc.) following the recommended procedure.

    Techniques: Amplification

    Effect of EDTA concentration on CNA1 mRNA amplification by RT-PCR . RNA samples extracted from cells harvested at 1.2 mg (dry weight/mL) were treated with DNase I according to protocol II. The reactions were stopped by the addition of 1.25 mM (lane 1), 1.5 mM (lane 2), 1.75 mM (lane 3); 2.0 mM (lane 4) and 2.25 mM (lane 5) of EDTA and treated samples were amplified by RT-PCR using CNA1 primers. P, 100 bp DNA Ladder (BioLabs-Inc).

    Journal: BMC Molecular Biology

    Article Title: Comparing protocols for preparation of DNA-free total yeast RNA suitable for RT-PCR

    doi: 10.1186/1471-2199-6-9

    Figure Lengend Snippet: Effect of EDTA concentration on CNA1 mRNA amplification by RT-PCR . RNA samples extracted from cells harvested at 1.2 mg (dry weight/mL) were treated with DNase I according to protocol II. The reactions were stopped by the addition of 1.25 mM (lane 1), 1.5 mM (lane 2), 1.75 mM (lane 3); 2.0 mM (lane 4) and 2.25 mM (lane 5) of EDTA and treated samples were amplified by RT-PCR using CNA1 primers. P, 100 bp DNA Ladder (BioLabs-Inc).

    Article Snippet: Protocol II was performed using the DNase I amplification grade kit (Life Technologies, Inc.) following the recommended procedure.

    Techniques: Concentration Assay, Amplification, Reverse Transcription Polymerase Chain Reaction

    Consensus GGUG-containing RNA oligonucleotide promotes the inhibitory effect of TLS on CBP/p300 HAT activities a , Co-immunoprecipitation (IP) of p300 and TLS from RNase A-treated HeLa cells. b, P300 HAT activity was measured using micrococcal nuclease (MNase) or DNase I pre-treated GST and GST-TLS in the presence of GGUG- or CCUC-oligonucleotide. * p

    Journal: Nature

    Article Title: Induced ncRNAs Allosterically Modify RNA Binding Proteins in cis to Inhibit Transcription

    doi: 10.1038/nature06992

    Figure Lengend Snippet: Consensus GGUG-containing RNA oligonucleotide promotes the inhibitory effect of TLS on CBP/p300 HAT activities a , Co-immunoprecipitation (IP) of p300 and TLS from RNase A-treated HeLa cells. b, P300 HAT activity was measured using micrococcal nuclease (MNase) or DNase I pre-treated GST and GST-TLS in the presence of GGUG- or CCUC-oligonucleotide. * p

    Article Snippet: The soluble DNA-bound RNA fraction was collected after centrifugation at 4,000 g for 15 min. RNA was extracted using Trizol (Invitrogen) and treated with RNase-free DNase I (DNA-free; Ambion).

    Techniques: HAT Assay, Immunoprecipitation, Activity Assay

    Effect of the K297R mutation on progeny virus release. iSLK-BAC16 and iSLK-BAC-K297R cells were induced with Dox and butyrate for indicated time. The extracellular virions were collected from culture media and treated with Turbo DNase I. Viral DNAs were extracted and KSHV genomic DNA copy numbers were estimated by qPCR along with external standards of known concentrations of the viral DNA with primers against the ORF73 gene (A). Intracellular KSHV genomic DNAs were extracted from harvested cells and quantitated by qPCR as above (B). (*,  p

    Journal: PLoS Pathogens

    Article Title: Mono-ubiquitylated ORF45 Mediates Association of KSHV Particles with Internal Lipid Rafts for Viral Assembly and Egress

    doi: 10.1371/journal.ppat.1005332

    Figure Lengend Snippet: Effect of the K297R mutation on progeny virus release. iSLK-BAC16 and iSLK-BAC-K297R cells were induced with Dox and butyrate for indicated time. The extracellular virions were collected from culture media and treated with Turbo DNase I. Viral DNAs were extracted and KSHV genomic DNA copy numbers were estimated by qPCR along with external standards of known concentrations of the viral DNA with primers against the ORF73 gene (A). Intracellular KSHV genomic DNAs were extracted from harvested cells and quantitated by qPCR as above (B). (*, p

    Article Snippet: For the preparation of DNA from intact virions, 200 μl of virus stocks was pretreated with 2 μl of Turbo DNase I (Ambion) for 1 h at 37°C.

    Techniques: Mutagenesis, BAC Assay, Real-time Polymerase Chain Reaction

    Analysis of the Mur34 binding site by DNase I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.

    Journal: PLoS ONE

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471

    doi: 10.1371/journal.pone.0076068

    Figure Lengend Snippet: Analysis of the Mur34 binding site by DNase I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.

    Article Snippet: For binding site analysis, the reaction mixture contained 500 cps 32 P-lablelled DNA fragments (50 nM), after the binding of protein with DNA, the reaction mixture was incubated in ice bath for 5 min prior to addition of 2.5 µl DNase I buffer and 0.3 U of DNase I (Fermentas), then was carried out for further incubation at 30°C for 1 min.

    Techniques: Binding Assay, Footprinting, Labeling, Sequencing, Purification

    Gene expression analysis of the  mur  genes. (A) Transcription analysis of intergenic region of the selected  mur  genes. Top, ethidium bromide-stained agarose gels showing RT-PCR fragments from intergenic regions.  mur10 ← mur11  means that the detected region between  mur10  and  mur11 , and the arrows showed the possible orientation of transcription. In each gel, the left band was positive control using genomic DNA as template, the middle band showed the PCR sample using cDNA as template, the right band is negative control using template from total RNA sample digested with DNase I. (B) Time course of the transcription difference of  mur11  and  mur27  for DM-5 and the wild type strain. (C). The transcription difference of DM-5 and the wild type strain for 96 h incubation was used for the comparative analysis.

    Journal: PLoS ONE

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471

    doi: 10.1371/journal.pone.0076068

    Figure Lengend Snippet: Gene expression analysis of the mur genes. (A) Transcription analysis of intergenic region of the selected mur genes. Top, ethidium bromide-stained agarose gels showing RT-PCR fragments from intergenic regions. mur10 ← mur11 means that the detected region between mur10 and mur11 , and the arrows showed the possible orientation of transcription. In each gel, the left band was positive control using genomic DNA as template, the middle band showed the PCR sample using cDNA as template, the right band is negative control using template from total RNA sample digested with DNase I. (B) Time course of the transcription difference of mur11 and mur27 for DM-5 and the wild type strain. (C). The transcription difference of DM-5 and the wild type strain for 96 h incubation was used for the comparative analysis.

    Article Snippet: For binding site analysis, the reaction mixture contained 500 cps 32 P-lablelled DNA fragments (50 nM), after the binding of protein with DNA, the reaction mixture was incubated in ice bath for 5 min prior to addition of 2.5 µl DNase I buffer and 0.3 U of DNase I (Fermentas), then was carried out for further incubation at 30°C for 1 min.

    Techniques: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Positive Control, Polymerase Chain Reaction, Negative Control, Incubation

    The H2A/H4 nucleosomal HAT activity in Tetrahymena is not p55 dependent. (A) HAT activity of RP-HPLC fractions following reconstitution by stepwise dialysis. A peak of free, nonnucleosomal HAT activity (I*) was contained in fraction 2. In contrast, a well-separated peak of nucleosomal HAT activity was contained in fractions 5 to 7 (II*). (B) Substrate specificity of these fractions. Note that nucleosomal HAT activity (II*) was H2A/H4 specific; in contrast, free-histone HAT activity was H3 specific. (C) Western blot analysis of the fractions. As expected, free-histone HAT activity (I*) (fractions 2 and 3) contained essentially all of the p55 signal. In contrast, the peak of nucleosomal H2A/H4 HAT activity (fractions 5 to 7) was devoid of p55, suggesting that this polypeptide cannot be responsible for the nucleosomal H2A/H4 HAT activity (see text). Note that fractions 2 and 3 also contained the majority of Ada5/Spt20 signal. Lane C contains DNase I extract as input.

    Journal: Molecular and Cellular Biology

    Article Title: A Novel H2A/H4 Nucleosomal Histone Acetyltransferase in Tetrahymena thermophila

    doi:

    Figure Lengend Snippet: The H2A/H4 nucleosomal HAT activity in Tetrahymena is not p55 dependent. (A) HAT activity of RP-HPLC fractions following reconstitution by stepwise dialysis. A peak of free, nonnucleosomal HAT activity (I*) was contained in fraction 2. In contrast, a well-separated peak of nucleosomal HAT activity was contained in fractions 5 to 7 (II*). (B) Substrate specificity of these fractions. Note that nucleosomal HAT activity (II*) was H2A/H4 specific; in contrast, free-histone HAT activity was H3 specific. (C) Western blot analysis of the fractions. As expected, free-histone HAT activity (I*) (fractions 2 and 3) contained essentially all of the p55 signal. In contrast, the peak of nucleosomal H2A/H4 HAT activity (fractions 5 to 7) was devoid of p55, suggesting that this polypeptide cannot be responsible for the nucleosomal H2A/H4 HAT activity (see text). Note that fractions 2 and 3 also contained the majority of Ada5/Spt20 signal. Lane C contains DNase I extract as input.

    Article Snippet: For DNase I extraction, freshly prepared macronuclei (108 macronuclei/ml) were resuspended in DNase I extraction buffer, which consisted of 25 mM Tris-HCl (pH 8.0), 15 mM NaCl, 10 mM MgCl2 , 0.1 mM CaCl2 , 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.05 mM dithiothreitol (DTT), and 500 U of DNase I (Gibco BRL) per ml, and incubated on ice for 90 min. After extraction, macronuclear remnants and insoluble debris were removed by centrifugation at 50,500 × g for 30 min at 2°C.

    Techniques: HAT Assay, Activity Assay, High Performance Liquid Chromatography, Western Blot

    Two distinct type A HAT activities exist in Tetrahymena macronuclei. (A) Sucrose-gradient sedimentation of HAT activities characterized with either a mixture of free (nonnucleosomal) histones (peak I) or nucleosomal histones (peak II); histone substrates were from chickens. (B) Western blot analysis using yAda1p and yAda5p/ySpt20p antisera as well as Tetrahymena p55 (K36) peptide antibodies. (C) Acetylated histone products were examined following gel electrophoresis and fluorography. Lanes C contain DNase I extract as input.

    Journal: Molecular and Cellular Biology

    Article Title: A Novel H2A/H4 Nucleosomal Histone Acetyltransferase in Tetrahymena thermophila

    doi:

    Figure Lengend Snippet: Two distinct type A HAT activities exist in Tetrahymena macronuclei. (A) Sucrose-gradient sedimentation of HAT activities characterized with either a mixture of free (nonnucleosomal) histones (peak I) or nucleosomal histones (peak II); histone substrates were from chickens. (B) Western blot analysis using yAda1p and yAda5p/ySpt20p antisera as well as Tetrahymena p55 (K36) peptide antibodies. (C) Acetylated histone products were examined following gel electrophoresis and fluorography. Lanes C contain DNase I extract as input.

    Article Snippet: For DNase I extraction, freshly prepared macronuclei (108 macronuclei/ml) were resuspended in DNase I extraction buffer, which consisted of 25 mM Tris-HCl (pH 8.0), 15 mM NaCl, 10 mM MgCl2 , 0.1 mM CaCl2 , 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.05 mM dithiothreitol (DTT), and 500 U of DNase I (Gibco BRL) per ml, and incubated on ice for 90 min. After extraction, macronuclear remnants and insoluble debris were removed by centrifugation at 50,500 × g for 30 min at 2°C.

    Techniques: HAT Assay, Sedimentation, Western Blot, Nucleic Acid Electrophoresis

    DNase I expression and activity. (A) The protein expression pattern of DNase I in kidney sections from (NZBxNZW)F1 mice as revealed by IHC, IF, IEM, and confocal microscopy (DNase I in red and Trap1 in green as a cytoplasmic marker). (B) DNase I endonuclease activity as revealed by zymography on samples from whole kidney lysates of representative Group 1, 2, and 3 (NZBxNZW)F1 mice, as well as a control (R, recombinant DNase I) and a serum (S) sample. (C) Urinary DNase I endonuclease activity in different groups of (NZBxNZW)F1 mice as revealed by zymography.

    Journal: The Journal of Pathology: Clinical Research

    Article Title: Lupus nephritis: low urinary DNase I levels reflect loss of renal DNase I and may be utilized as a biomarker of disease progression

    doi: 10.1002/cjp2.99

    Figure Lengend Snippet: DNase I expression and activity. (A) The protein expression pattern of DNase I in kidney sections from (NZBxNZW)F1 mice as revealed by IHC, IF, IEM, and confocal microscopy (DNase I in red and Trap1 in green as a cytoplasmic marker). (B) DNase I endonuclease activity as revealed by zymography on samples from whole kidney lysates of representative Group 1, 2, and 3 (NZBxNZW)F1 mice, as well as a control (R, recombinant DNase I) and a serum (S) sample. (C) Urinary DNase I endonuclease activity in different groups of (NZBxNZW)F1 mice as revealed by zymography.

    Article Snippet: DNase I enzymatic activity was analyzed in 10% polyacrylamide gels in DNase I reactivation buffer (40 m m Tris pH 7.6, 2 n m CaCl2 , 2 m m Mg Cl2 ) containing 88 µg/ml heat‐denatured salmon sperm DNA (Invitrogen) for 24 h , at 37 °C.

    Techniques: Expressing, Activity Assay, Mouse Assay, Immunohistochemistry, Confocal Microscopy, Marker, Zymography, Recombinant

    MCC22 reduced pro-inflammatory cytokine and increased the anti-inflammatory cytokine IL-10 expression in the spinal cord of HbSS mice. HbSS mice were treated with i.p. injections of 8 μmol/kg (10 mg/kg) MCC22 (n=4) or vehicle (n=4) for 9 consecutive days. The spinal cord was removed from HbAA mice and HbSS mice administered vehicle or MCC22. The RNA was isolated, DNase treated, converted to cDNA, and used in real time PCR with primers for IL-1β (A) , IL-6 (B) , TNFα (C) , and IL-10 (D) . Concentrations for each cytokine were determined based on a standard set for each primer pair, and samples were normalized to β-actin expression. Significant differences in expression were determined between HbAA and HbSS vehicle-treated mice or HbSS MCC22-treated mice (Bonferroni t-tests; * p

    Journal: Pain

    Article Title: The bivalent ligand MCC22 potently attenuates nociception in a murine model of sickle cell disease

    doi: 10.1097/j.pain.0000000000001225

    Figure Lengend Snippet: MCC22 reduced pro-inflammatory cytokine and increased the anti-inflammatory cytokine IL-10 expression in the spinal cord of HbSS mice. HbSS mice were treated with i.p. injections of 8 μmol/kg (10 mg/kg) MCC22 (n=4) or vehicle (n=4) for 9 consecutive days. The spinal cord was removed from HbAA mice and HbSS mice administered vehicle or MCC22. The RNA was isolated, DNase treated, converted to cDNA, and used in real time PCR with primers for IL-1β (A) , IL-6 (B) , TNFα (C) , and IL-10 (D) . Concentrations for each cytokine were determined based on a standard set for each primer pair, and samples were normalized to β-actin expression. Significant differences in expression were determined between HbAA and HbSS vehicle-treated mice or HbSS MCC22-treated mice (Bonferroni t-tests; * p

    Article Snippet: RNA was isolated from whole spinal cords using Trizol and was DNAse treated (Invitrogen, Amsterdam) and was converted into cDNA using oligo(dT)12–18 primers with the Advantage RT for PCR Kit (Takara).

    Techniques: Expressing, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction

    Effect of calpain-like silencing during PCD. (A) DNA fragmentation in calpain-like silenced trophozoites after PCD induction by G418. Confocal microscopy analysis of trophozoites showing the TUNEL staining, samples were counter-stained with DAPI. (A) Negative control, untreated trophozoites; (B) Positive control, trophozoites treated with 20 μg of DNase I endonuclease for 30 min; (C) Trophozoites after a 9-h incubation with 10 μg/ml of G418; (D,E) Trophozoites pre-incubated for 24 h with NRS (D) or calpain-like (E) siRNAs sequences and then 9 h with 10 μg/ml G418; (B) Densitometric analysis of (A). (C) Effect of calpain-like silencing on the cell viability of E. histolytica incubated with G418; (A) Negative control, untreated trophozoites; (B) Trophozoites after a 9-h incubation with 10 μg/ml of G418; (C,D) Trophozoites pre-incubated for 24 h with NRS (C) or calpain-like (D) siRNAs sequences and then 9 h with 10 μg/ml G418. *indicate statistically significant difference: P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: A Calpain-Like Protein Is Involved in the Execution Phase of Programmed Cell Death of Entamoeba histolytica

    doi: 10.3389/fcimb.2018.00339

    Figure Lengend Snippet: Effect of calpain-like silencing during PCD. (A) DNA fragmentation in calpain-like silenced trophozoites after PCD induction by G418. Confocal microscopy analysis of trophozoites showing the TUNEL staining, samples were counter-stained with DAPI. (A) Negative control, untreated trophozoites; (B) Positive control, trophozoites treated with 20 μg of DNase I endonuclease for 30 min; (C) Trophozoites after a 9-h incubation with 10 μg/ml of G418; (D,E) Trophozoites pre-incubated for 24 h with NRS (D) or calpain-like (E) siRNAs sequences and then 9 h with 10 μg/ml G418; (B) Densitometric analysis of (A). (C) Effect of calpain-like silencing on the cell viability of E. histolytica incubated with G418; (A) Negative control, untreated trophozoites; (B) Trophozoites after a 9-h incubation with 10 μg/ml of G418; (C,D) Trophozoites pre-incubated for 24 h with NRS (C) or calpain-like (D) siRNAs sequences and then 9 h with 10 μg/ml G418. *indicate statistically significant difference: P

    Article Snippet: As a positive control, trophozoites were treated with 20 mg/ml DNase I endonuclease (Invitrogen) for 30 min, and non-treated trophozoites were used as a negative control.

    Techniques: Confocal Microscopy, TUNEL Assay, Staining, Negative Control, Positive Control, Incubation