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  • 94
    Agilent technologies brilliant iii ultra fast sybr green qpcr master mix
    Design of ORA oligonucleotide construct for NER. (A) Construction of the ORA oligonucleotide that contains a CPD lesion. The 30-nt synthetic ssDNA fragment containing a CPD lesion (marked as a T-T dimer) was hybridized with a 5′-biotinylated (49 nt) and a 3′-ddC protected (98 nt) fragment to form a hairpin dsDNA construct after ligation. The resulting construct contains a central 77-bp dsDNA region with a 19-nt ssDNA in the 5′-terminal region and a 4-nt hairpin at the end of the 3′ arm. The CPD residue locates approximately at the center of the dsDNA stretch, 33 bp from the 3′ end and 42 bp from the 5′ end of the dsDNA region. Including the 19-nt ssDNA sequence, the 5′ arm of the CPD-containing construct is 61 nt/bp in length. Primers for <t>qPCR</t> quantification are shown in arrow lines, where amplification by primers a/b represents the total amount of the oligonucleotide and by a/c the amount of the repaired oligonucleotides. The percentages of oligonucleotide repaired were calculated by the following equation: % Repaired = (2 −ΔCt ) × 100, ΔCt = Ct Control (a/b) − Ct Test (a/c) . (B–E) Calibration of CPD-containing oligonucleotide constructs. qPCR was carried out using 1 μl of 5 nM ligated normal TT- (B) or CPD-containing construct (C) as template. PCR amplifications with primers a and b serve as the internal control for the total number of substrate molecules, whereas amplifications with primers a and c quantify the amount of the repaired products. Based on the ΔCt value (ac-ab) of the TT construct, the ligation efficiency (% ligation = 1/2 ΔCt × 100) was 1.5% (B). Since CPD adducts block the activity of Taq polymerase (C), the ΔCt value (ac-ab) of the CPD construct is greater than that of the normal TT control (D), yielding a ΔΔCt value (CPD-TT) of 10.71 that converts to a 1,670-fold blockage by CPD (Fold Difference = 2 ΔΔCt ) (E). Using this 2 ΔΔCt method, we were able to obtain differential repair efficiencies of the CPD construct in cells with altered genetic backgrounds. Data are presented as mean ± S.D. from <t>three</t> independent experiments.
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    Agilent technologies brilliant iii ultra fast sybr green qrt pcr master mix
    peTrpL and Tc are involved in the differential posttranscriptional regulation of smeABR. (A) <t>qRT-PCR</t> analysis of changes in the smeR levels 10 min after IPTG addition to two parallel 2011 Δ trpL (pSRKGm-peTrpL) cultures. Tc (1.5 μg/ml) was added together with IPTG to one of the cultures (indicated). (B) RT-PCR analysis with a forward primer located in smeB and reverse primer located in smeR . The PCR template input is indicated at the top. (C) Changes in the smeA levels 10 and 20 min after IPTG addition. See also descriptions for panel A. (D and E) mRNA stability determination by qRT-PCR using smeR and smeA specific primer pairs. To 2011 Δ trpL (pSRKGm-peTrpL) cultures, IPTG and/or Tc (1.5 μg/ml) was added and 10 min thereafter, rifampin was added. The relative mRNA level values after stop of transcription by rifampin were determined and plotted against the time. The calculated half-lives are indicated. (F and G) Northern blot analysis of RNA from the experiments described for panels D and E. RNAs detected by the used probes are indicated on the left side. 16S rRNA was used as a loading control. The conditions used and time after rifampin addition are given at the top, the calculated half-lives at the bottom (see also Fig. S1 in the supplemental material). Ten minutes after IPTG and Tc addition (0 min in respect to rifampin addition), the tricistronic smeABR mRNA was detected with the smeR -specific probe (internally radiolabeled 128-nt in vitro transcript) but not with the smeA - and smeB -directed probes (radiolabeled DNA probes generated by random priming). This could be explained by the higher sensitivity and stronger binding of the RNA probe. In all graphs, data from <t>three</t> independent cultures are presented as means ± standard deviations.
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    Agilent technologies brilliant iii ultra fast sybr green qrt pcr master mix kit
    The frmRAB operon is induced after exposure of E . coli MG 1655 to concentrations of TMAO ≥ 5 m M . Anaerobic batch cultures were exposed to different concentrations (0–40 m M ) of TMAO . After 30 min, total RNA was isolated for <t>qRT‐PCR</t> of the frmR mRNA . The data shown and the mean and standard deviation for the fold increase relative to the 0 m M TMAO culture and are typical of <t>three</t> independent experiments.
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    The frmRAB operon is induced after exposure of E . coli MG 1655 to concentrations of TMAO ≥ 5 m M . Anaerobic batch cultures were exposed to different concentrations (0–40 m M ) of TMAO . After 30 min, total RNA was isolated for <t>qRT‐PCR</t> of the frmR mRNA . The data shown and the mean and standard deviation for the fold increase relative to the 0 m M TMAO culture and are typical of <t>three</t> independent experiments.
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    Agilent technologies brilliant iii sybr green master mix
    The frmRAB operon is induced after exposure of E . coli MG 1655 to concentrations of TMAO ≥ 5 m M . Anaerobic batch cultures were exposed to different concentrations (0–40 m M ) of TMAO . After 30 min, total RNA was isolated for <t>qRT‐PCR</t> of the frmR mRNA . The data shown and the mean and standard deviation for the fold increase relative to the 0 m M TMAO culture and are typical of <t>three</t> independent experiments.
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    The frmRAB operon is induced after exposure of E . coli MG 1655 to concentrations of TMAO ≥ 5 m M . Anaerobic batch cultures were exposed to different concentrations (0–40 m M ) of TMAO . After 30 min, total RNA was isolated for <t>qRT‐PCR</t> of the frmR mRNA . The data shown and the mean and standard deviation for the fold increase relative to the 0 m M TMAO culture and are typical of <t>three</t> independent experiments.
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    The frmRAB operon is induced after exposure of E . coli MG 1655 to concentrations of TMAO ≥ 5 m M . Anaerobic batch cultures were exposed to different concentrations (0–40 m M ) of TMAO . After 30 min, total RNA was isolated for <t>qRT‐PCR</t> of the frmR mRNA . The data shown and the mean and standard deviation for the fold increase relative to the 0 m M TMAO culture and are typical of <t>three</t> independent experiments.
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    Agilent technologies brilliant iii sybr green quantitative rt pcr qrt pcr master mix
    The frmRAB operon is induced after exposure of E . coli MG 1655 to concentrations of TMAO ≥ 5 m M . Anaerobic batch cultures were exposed to different concentrations (0–40 m M ) of TMAO . After 30 min, total RNA was isolated for <t>qRT‐PCR</t> of the frmR mRNA . The data shown and the mean and standard deviation for the fold increase relative to the 0 m M TMAO culture and are typical of <t>three</t> independent experiments.
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    MiR-specific <t>qPCR</t> on synthetic templates with DNA primers . A The effect of primer concentration on Cq value of ssc-let-7d and ssc-miR-26a miR-specific qPCR assays. Real-time PCR assays were performed in parallel at <t>three</t> different concentrations (125, 250 and 500 nM) of the forward and of the reverse primers. B Amplification curves of an eight log 10 dilution series of a synthetic ssc-let-7d template in the ssc-let-7d miR-specific qPCR assays. All samples contained a final concentration of 0.2 ng/μl salmon sperm DNA. C Extrapolation of Cq as function of the log 10 of the number of templates for the same experiment as in B was a straight line (R 2 = 0.9993) with slope of -3.341 (PCR efficiency = 99%) over eight log 10 dilution of the template. D Melting curve analysis of the same experiment. No template control is labeled ntc. Melting curve analysis was performed from 60°C to 99°C.
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    Agilent technologies brilliant iii ultra fast sybr green qpcr master mix reagents
    TGA2 controls the UV-B-induced expression of GSTU7, GSTU8 and GSTU25 genes, via recruitment to their promoters. Wild type (WT), tga256 triple mutant ( tga256 ) and complemented plants ( tga256 /TGA2 line #1) were irradiated with UV-B for 5 hours (+). Untreated plants were used as control (-). (A) The expression of GSTU7 , GSTU8 , and GSTU25 genes was evaluated by <t>RT-qPCR.</t> Data is presented as mean values of GSTU gene expression relative to the expression of the housekeeping YLS8 gene. Error bars represent standard deviation from <t>three</t> biological replicates (4-5 seedlings each). Different letters above bars indicate significant differences (ANOVA/Fisher’s LSD test, p
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    TGA2 controls the UV-B-induced expression of GSTU7, GSTU8 and GSTU25 genes, via recruitment to their promoters. Wild type (WT), tga256 triple mutant ( tga256 ) and complemented plants ( tga256 /TGA2 line #1) were irradiated with UV-B for 5 hours (+). Untreated plants were used as control (-). (A) The expression of GSTU7 , GSTU8 , and GSTU25 genes was evaluated by <t>RT-qPCR.</t> Data is presented as mean values of GSTU gene expression relative to the expression of the housekeeping YLS8 gene. Error bars represent standard deviation from <t>three</t> biological replicates (4-5 seedlings each). Different letters above bars indicate significant differences (ANOVA/Fisher’s LSD test, p
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    TGA2 controls the UV-B-induced expression of GSTU7, GSTU8 and GSTU25 genes, via recruitment to their promoters. Wild type (WT), tga256 triple mutant ( tga256 ) and complemented plants ( tga256 /TGA2 line #1) were irradiated with UV-B for 5 hours (+). Untreated plants were used as control (-). (A) The expression of GSTU7 , GSTU8 , and GSTU25 genes was evaluated by <t>RT-qPCR.</t> Data is presented as mean values of GSTU gene expression relative to the expression of the housekeeping YLS8 gene. Error bars represent standard deviation from <t>three</t> biological replicates (4-5 seedlings each). Different letters above bars indicate significant differences (ANOVA/Fisher’s LSD test, p
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    TGA2 controls the UV-B-induced expression of GSTU7, GSTU8 and GSTU25 genes, via recruitment to their promoters. Wild type (WT), tga256 triple mutant ( tga256 ) and complemented plants ( tga256 /TGA2 line #1) were irradiated with UV-B for 5 hours (+). Untreated plants were used as control (-). (A) The expression of GSTU7 , GSTU8 , and GSTU25 genes was evaluated by <t>RT-qPCR.</t> Data is presented as mean values of GSTU gene expression relative to the expression of the housekeeping YLS8 gene. Error bars represent standard deviation from <t>three</t> biological replicates (4-5 seedlings each). Different letters above bars indicate significant differences (ANOVA/Fisher’s LSD test, p
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    Design of ORA oligonucleotide construct for NER. (A) Construction of the ORA oligonucleotide that contains a CPD lesion. The 30-nt synthetic ssDNA fragment containing a CPD lesion (marked as a T-T dimer) was hybridized with a 5′-biotinylated (49 nt) and a 3′-ddC protected (98 nt) fragment to form a hairpin dsDNA construct after ligation. The resulting construct contains a central 77-bp dsDNA region with a 19-nt ssDNA in the 5′-terminal region and a 4-nt hairpin at the end of the 3′ arm. The CPD residue locates approximately at the center of the dsDNA stretch, 33 bp from the 3′ end and 42 bp from the 5′ end of the dsDNA region. Including the 19-nt ssDNA sequence, the 5′ arm of the CPD-containing construct is 61 nt/bp in length. Primers for qPCR quantification are shown in arrow lines, where amplification by primers a/b represents the total amount of the oligonucleotide and by a/c the amount of the repaired oligonucleotides. The percentages of oligonucleotide repaired were calculated by the following equation: % Repaired = (2 −ΔCt ) × 100, ΔCt = Ct Control (a/b) − Ct Test (a/c) . (B–E) Calibration of CPD-containing oligonucleotide constructs. qPCR was carried out using 1 μl of 5 nM ligated normal TT- (B) or CPD-containing construct (C) as template. PCR amplifications with primers a and b serve as the internal control for the total number of substrate molecules, whereas amplifications with primers a and c quantify the amount of the repaired products. Based on the ΔCt value (ac-ab) of the TT construct, the ligation efficiency (% ligation = 1/2 ΔCt × 100) was 1.5% (B). Since CPD adducts block the activity of Taq polymerase (C), the ΔCt value (ac-ab) of the CPD construct is greater than that of the normal TT control (D), yielding a ΔΔCt value (CPD-TT) of 10.71 that converts to a 1,670-fold blockage by CPD (Fold Difference = 2 ΔΔCt ) (E). Using this 2 ΔΔCt method, we were able to obtain differential repair efficiencies of the CPD construct in cells with altered genetic backgrounds. Data are presented as mean ± S.D. from three independent experiments.

    Journal: Scientific Reports

    Article Title: A Rapid Assay for Measuring Nucleotide Excision Repair by Oligonucleotide Retrieval

    doi: 10.1038/srep04894

    Figure Lengend Snippet: Design of ORA oligonucleotide construct for NER. (A) Construction of the ORA oligonucleotide that contains a CPD lesion. The 30-nt synthetic ssDNA fragment containing a CPD lesion (marked as a T-T dimer) was hybridized with a 5′-biotinylated (49 nt) and a 3′-ddC protected (98 nt) fragment to form a hairpin dsDNA construct after ligation. The resulting construct contains a central 77-bp dsDNA region with a 19-nt ssDNA in the 5′-terminal region and a 4-nt hairpin at the end of the 3′ arm. The CPD residue locates approximately at the center of the dsDNA stretch, 33 bp from the 3′ end and 42 bp from the 5′ end of the dsDNA region. Including the 19-nt ssDNA sequence, the 5′ arm of the CPD-containing construct is 61 nt/bp in length. Primers for qPCR quantification are shown in arrow lines, where amplification by primers a/b represents the total amount of the oligonucleotide and by a/c the amount of the repaired oligonucleotides. The percentages of oligonucleotide repaired were calculated by the following equation: % Repaired = (2 −ΔCt ) × 100, ΔCt = Ct Control (a/b) − Ct Test (a/c) . (B–E) Calibration of CPD-containing oligonucleotide constructs. qPCR was carried out using 1 μl of 5 nM ligated normal TT- (B) or CPD-containing construct (C) as template. PCR amplifications with primers a and b serve as the internal control for the total number of substrate molecules, whereas amplifications with primers a and c quantify the amount of the repaired products. Based on the ΔCt value (ac-ab) of the TT construct, the ligation efficiency (% ligation = 1/2 ΔCt × 100) was 1.5% (B). Since CPD adducts block the activity of Taq polymerase (C), the ΔCt value (ac-ab) of the CPD construct is greater than that of the normal TT control (D), yielding a ΔΔCt value (CPD-TT) of 10.71 that converts to a 1,670-fold blockage by CPD (Fold Difference = 2 ΔΔCt ) (E). Using this 2 ΔΔCt method, we were able to obtain differential repair efficiencies of the CPD construct in cells with altered genetic backgrounds. Data are presented as mean ± S.D. from three independent experiments.

    Article Snippet: DNA and primers were mixed with Brilliant III Ultra-Fast SYBR Green qPCR Master Mix (Agilent) following manufacturer's instruction and PCR cycling was typically run with the following protocol: hot-start, 95°C, 3 min; cycling, 95°C, 5 sec and 60°C, 15 sec, 50 cycles; completion, 72°C, 1 min; melting curve, 60°C to 95°C, recording 0.2°C/sec.

    Techniques: Construct, Ligation, Sequencing, Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Blocking Assay, Activity Assay

    NER deficiency in human XPA fibroblasts validated by ORA. (A) NER efficiencies of human SV40-transformed, normal (GM00637) versus XPA (GM04429) fibroblast cells. Cells were transfected with CPD-containing oligonucleotides. After 48 h incubation at 37°C, nuclei were isolated and oligonucleotides were retrieved from the purified nuclei for quantification. Data are presented as mean ± S.D. (n = 3) and the P value was calculated by Student's t -test. (B) NER efficiencies of human primary fibroblasts. Human primary fibroblast cells, normal (AG01440) versus XPA (AG06971), were transfected with CPD-containing oligonucleotide and incubated at 37°C for 48 h and 96 h prior to retrieval. Data derived from qPCR analyses of the retrieved oligonucleotides from three independent experiments are presented as % repaired of the CPD-containing constructs per cell (mean ± S.D., n = 3). The P value was obtained by Student's t -test. The absolute values likely reflect differences in kinetics of repair between primary and immortalized cells. (C) Validation of NER deficiencies by CPD-specific ELISA. After irradiating the cells with 25 J/m 2 of UV-C, CPD lesions were quantified by ELISA at 0 h, 24 h, 48 h and 96 h for the normal fibroblasts and only at 0 h, 24 h and 48 h for the XPA cells since XPA fibroblasts were dying at 96 h. The results are shown as % of remaining CPD relative to 0 h after irradiation and data are presented as mean ± S.D. (n = 3).

    Journal: Scientific Reports

    Article Title: A Rapid Assay for Measuring Nucleotide Excision Repair by Oligonucleotide Retrieval

    doi: 10.1038/srep04894

    Figure Lengend Snippet: NER deficiency in human XPA fibroblasts validated by ORA. (A) NER efficiencies of human SV40-transformed, normal (GM00637) versus XPA (GM04429) fibroblast cells. Cells were transfected with CPD-containing oligonucleotides. After 48 h incubation at 37°C, nuclei were isolated and oligonucleotides were retrieved from the purified nuclei for quantification. Data are presented as mean ± S.D. (n = 3) and the P value was calculated by Student's t -test. (B) NER efficiencies of human primary fibroblasts. Human primary fibroblast cells, normal (AG01440) versus XPA (AG06971), were transfected with CPD-containing oligonucleotide and incubated at 37°C for 48 h and 96 h prior to retrieval. Data derived from qPCR analyses of the retrieved oligonucleotides from three independent experiments are presented as % repaired of the CPD-containing constructs per cell (mean ± S.D., n = 3). The P value was obtained by Student's t -test. The absolute values likely reflect differences in kinetics of repair between primary and immortalized cells. (C) Validation of NER deficiencies by CPD-specific ELISA. After irradiating the cells with 25 J/m 2 of UV-C, CPD lesions were quantified by ELISA at 0 h, 24 h, 48 h and 96 h for the normal fibroblasts and only at 0 h, 24 h and 48 h for the XPA cells since XPA fibroblasts were dying at 96 h. The results are shown as % of remaining CPD relative to 0 h after irradiation and data are presented as mean ± S.D. (n = 3).

    Article Snippet: DNA and primers were mixed with Brilliant III Ultra-Fast SYBR Green qPCR Master Mix (Agilent) following manufacturer's instruction and PCR cycling was typically run with the following protocol: hot-start, 95°C, 3 min; cycling, 95°C, 5 sec and 60°C, 15 sec, 50 cycles; completion, 72°C, 1 min; melting curve, 60°C to 95°C, recording 0.2°C/sec.

    Techniques: Transformation Assay, Transfection, Incubation, Isolation, Purification, Derivative Assay, Real-time Polymerase Chain Reaction, Construct, Enzyme-linked Immunosorbent Assay, Irradiation

    NER activities of human embryonic kidney cell 293T and human colon cancer cell SW480 calibrated by ORA. (A) NER activities in a single HEK293T cell are presented as % CPD-containing oligonucleotide repaired at incremental time points. Data are presented as mean ± S.D. from three independent experiments. (B) Repair of CPD-containing oligonucleotides in HEK 293T cells. HEK293T cells (5 × 10 5 ) were transfected with 0.02 nM of CPD oligonucleotide and incubated at 37°C. Cells were harvested at increasing incubation times, followed by oligonucleotide retrieval and qPCR analysis. Data are shown as the number of oligonucleotides retrieved per cell and the number of oligonucleotides repaired per cell at different time points from three independent experiments (mean ± S.D.). (C) Comparison of NER activities measured by ORA and ELISA between 293T and SW480 cells. The CPD repair efficiencies measured by ORA or the CPD removal efficiencies measured by ELISA are obtained 24 h after oligonucleotide transfection or 24 h after UV-C irradiation, respectively, and are presented as % repaired determined from three independent experiments (mean ± S.D.). (D) The survival of 293T and SW480 cells after UV-C irradiation. Each data point is presented as mean ± S.D. from three independent experiments.

    Journal: Scientific Reports

    Article Title: A Rapid Assay for Measuring Nucleotide Excision Repair by Oligonucleotide Retrieval

    doi: 10.1038/srep04894

    Figure Lengend Snippet: NER activities of human embryonic kidney cell 293T and human colon cancer cell SW480 calibrated by ORA. (A) NER activities in a single HEK293T cell are presented as % CPD-containing oligonucleotide repaired at incremental time points. Data are presented as mean ± S.D. from three independent experiments. (B) Repair of CPD-containing oligonucleotides in HEK 293T cells. HEK293T cells (5 × 10 5 ) were transfected with 0.02 nM of CPD oligonucleotide and incubated at 37°C. Cells were harvested at increasing incubation times, followed by oligonucleotide retrieval and qPCR analysis. Data are shown as the number of oligonucleotides retrieved per cell and the number of oligonucleotides repaired per cell at different time points from three independent experiments (mean ± S.D.). (C) Comparison of NER activities measured by ORA and ELISA between 293T and SW480 cells. The CPD repair efficiencies measured by ORA or the CPD removal efficiencies measured by ELISA are obtained 24 h after oligonucleotide transfection or 24 h after UV-C irradiation, respectively, and are presented as % repaired determined from three independent experiments (mean ± S.D.). (D) The survival of 293T and SW480 cells after UV-C irradiation. Each data point is presented as mean ± S.D. from three independent experiments.

    Article Snippet: DNA and primers were mixed with Brilliant III Ultra-Fast SYBR Green qPCR Master Mix (Agilent) following manufacturer's instruction and PCR cycling was typically run with the following protocol: hot-start, 95°C, 3 min; cycling, 95°C, 5 sec and 60°C, 15 sec, 50 cycles; completion, 72°C, 1 min; melting curve, 60°C to 95°C, recording 0.2°C/sec.

    Techniques: Transfection, Incubation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Irradiation

    Sequence recognition by MAX/MGA is critical for recruiting PCGF6-PRC1 to its target genes. ( A ) MAX-dependent proliferation of ESCs. Schematic representation of FLAG-tagged wild type (WT) and mutant [L31V and E32D substitution (VD) and basic region deletion (Δb)] MAX proteins (left). Failure to rescue growth defects of Max -KO ESCs by either mutant MAX. Mock-transfected Max conditional KO ESCs (WT) stopped growing 4 days after doxycycline treatment (KO) (right). Stable expression of FLAG-tagged WT [KO+MAX(WT)] rescued the growth defect but VD or Δb mutants [KO+MAX(VD) and KO+MAX(Δb)] did not. ( B ) Association of mutant MAX proteins with other components of the PCGF6-PRC1 complex. Immunoprecipitation-immunoblot (IP-IB) analysis revealed the association of FLAG-tagged MAX WT, VD or Δb with HA-tagged PCGF6, RING1B and L3MBTL2. Max -KO ESCs that expressed HA-tagged PCGF6 and FLAG-tagged wild type or mutant MAX were subjected to IP with anti-FLAG antibody. Resulting precipitates (IP) and lysates (Input) were immunoblotted with antibodies against FLAG, HA, RING1B or L3MBTL2. ( C ) Binding of FLAG-tagged WT or mutant MAX to target of ChIPed DNA is depicted as a percentage of input DNA. Error bars represent standard deviation determined from at least three independent experiments. ( D ) Binding of HA-tagged genes in Max -KO ESCs. Local levels of FLAG-tagged WT or mutant MAX at Ddx4 or Tdrd1 promoter regions were determined by ChIP-qPCR. The relative amount PCGF6 to target genes in Max -KO ESCs that express WT or mutant MAX. Local levels of HA-tagged PCGF6 at Ddx4 or Tdrd1 promoter regions were determined by ChIP-qPCR. ( E ) Forced tethering of MAX to a TetO array induced activation of PCGF6-PRC1 recruitment. ChIP analysis for TetR, HA-tag (PCGF6), H2AK119ub1, H3K27me3, H3K27ac and H3 across the TetO-containing locus in ESCs revealed TetR-MAX-mediated local activation of the PCGF6-PRC1 pathway. ChIP experiments were performed at least in biological duplicate with error bars showing SEM. DOI: http://dx.doi.org/10.7554/eLife.21064.011

    Journal: eLife

    Article Title: PCGF6-PRC1 suppresses premature differentiation of mouse embryonic stem cells by regulating germ cell-related genes

    doi: 10.7554/eLife.21064

    Figure Lengend Snippet: Sequence recognition by MAX/MGA is critical for recruiting PCGF6-PRC1 to its target genes. ( A ) MAX-dependent proliferation of ESCs. Schematic representation of FLAG-tagged wild type (WT) and mutant [L31V and E32D substitution (VD) and basic region deletion (Δb)] MAX proteins (left). Failure to rescue growth defects of Max -KO ESCs by either mutant MAX. Mock-transfected Max conditional KO ESCs (WT) stopped growing 4 days after doxycycline treatment (KO) (right). Stable expression of FLAG-tagged WT [KO+MAX(WT)] rescued the growth defect but VD or Δb mutants [KO+MAX(VD) and KO+MAX(Δb)] did not. ( B ) Association of mutant MAX proteins with other components of the PCGF6-PRC1 complex. Immunoprecipitation-immunoblot (IP-IB) analysis revealed the association of FLAG-tagged MAX WT, VD or Δb with HA-tagged PCGF6, RING1B and L3MBTL2. Max -KO ESCs that expressed HA-tagged PCGF6 and FLAG-tagged wild type or mutant MAX were subjected to IP with anti-FLAG antibody. Resulting precipitates (IP) and lysates (Input) were immunoblotted with antibodies against FLAG, HA, RING1B or L3MBTL2. ( C ) Binding of FLAG-tagged WT or mutant MAX to target of ChIPed DNA is depicted as a percentage of input DNA. Error bars represent standard deviation determined from at least three independent experiments. ( D ) Binding of HA-tagged genes in Max -KO ESCs. Local levels of FLAG-tagged WT or mutant MAX at Ddx4 or Tdrd1 promoter regions were determined by ChIP-qPCR. The relative amount PCGF6 to target genes in Max -KO ESCs that express WT or mutant MAX. Local levels of HA-tagged PCGF6 at Ddx4 or Tdrd1 promoter regions were determined by ChIP-qPCR. ( E ) Forced tethering of MAX to a TetO array induced activation of PCGF6-PRC1 recruitment. ChIP analysis for TetR, HA-tag (PCGF6), H2AK119ub1, H3K27me3, H3K27ac and H3 across the TetO-containing locus in ESCs revealed TetR-MAX-mediated local activation of the PCGF6-PRC1 pathway. ChIP experiments were performed at least in biological duplicate with error bars showing SEM. DOI: http://dx.doi.org/10.7554/eLife.21064.011

    Article Snippet: Real-time qPCR was performed using the Brilliant III SYBR Green QPCR Master Mix (Agilent).

    Techniques: Sequencing, Mutagenesis, Transfection, Expressing, Immunoprecipitation, Binding Assay, Standard Deviation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Activation Assay

    The role of MAX/MGA in recruiting PCGF6-PRC1 to its target genes. ( A ) The expression of FLAG-tagged exogenous PCGF6 in wild type (WT) and Eed -KO ESCs. Expression levels of FLAG-tagged PCGF6 and endogenous LAMIN B protein in wild type and Eed -KO ESCs mock or FLAG-PCGF6 construct transfected were tested by immunoblotting. ( B ) Local levels of FLAG at the indicated promoter regions in wild type or Eed -KO ESCs expressing FLAG-tagged PCGF6 were determined by ChIP-qPCR analysis. Underlined genes are canonical PRC1 targets. The relative amount of ChIPed DNA is depicted as a percentage of input DNA. Error bars represent standard deviation determined from at least three independent experiments. ( C ) ChIP-qPCR showing a marginal binding of canonical PRC1 (cPRC1; PCGF2) to PCGF6-bound genes with high H3K27me3 (PCGF6 +H3K27me3+/hi) and those with low H3K27me3 (PCGF6 +H3K27me3-/lo) in wild type (WT) ESCs. Eed -KO ESCs were used to confirm whether H3K27me3-dependent recruitment of cPRC1 is active at indicated gene locus. NC: negative control. ( D ) Top four de novo motif recognition hits for genes bound by PCGF6, RING1B, CBX7 (NCBI GEO accession number GSM1041373), MAX (NCBI GEO accession number GSM1171650) or MYC (NCBI GEO accession number GSM1171648) in wild type ESCs. ( E ) Expression levels of Max and Mga in untreated ESCs or those treated with control, Max siRNA or Mga siRNA revealed by RT-qPCR analysis (left). Transcription levels were normalized to a Gapdh control and are depicted as fold change relative to untreated ESCs. Error bars represent standard deviation determined from at least three independent experiments. Expression levels of FLAG-PCGF6, MAX, LAMIN B and RING1B in whole cell lysates of untreated and siRNA-treated ESCs are also shown (right). ( F ) Expression levels of the indicated genes in untreated ESCs or those treated either with control, Max siRNA or Mga siRNA. Underlined genes are canonical PRC1 targets. Expression levels were normalized to a Gapdh control and are depicted as fold change relative to mock ESCs (No treatment). Error bars represent standard deviation determined from at least three independent experiments. ( G ) Gene ontology (GO) term analysis showing that genes related to meiotic process are highly overrepresented among PCGF6+RING1B+MAX+MYC- genes. Genes were classified based on binding by PCGF6, RING1B, MAX and/or MYC and the enrichment of respective GO terms in each subset of genes was determined. The p -values for the significance of over-representation against total genes are shown along the x-axis. ( H ) Changes in local PCGF6 and MAX deposition at selected PCGF6/MAX co-bound gene that do not become de-repressed in Max -KO ESCs were determined by ChIP-qPCR using Max -KO ESCs stably expressing both an HA-tagged Pcgf6 and a Flag-tagged Max (WT or Δb) expression vectors, and are shown as described in Figure 2C . DOI: http://dx.doi.org/10.7554/eLife.21064.010

    Journal: eLife

    Article Title: PCGF6-PRC1 suppresses premature differentiation of mouse embryonic stem cells by regulating germ cell-related genes

    doi: 10.7554/eLife.21064

    Figure Lengend Snippet: The role of MAX/MGA in recruiting PCGF6-PRC1 to its target genes. ( A ) The expression of FLAG-tagged exogenous PCGF6 in wild type (WT) and Eed -KO ESCs. Expression levels of FLAG-tagged PCGF6 and endogenous LAMIN B protein in wild type and Eed -KO ESCs mock or FLAG-PCGF6 construct transfected were tested by immunoblotting. ( B ) Local levels of FLAG at the indicated promoter regions in wild type or Eed -KO ESCs expressing FLAG-tagged PCGF6 were determined by ChIP-qPCR analysis. Underlined genes are canonical PRC1 targets. The relative amount of ChIPed DNA is depicted as a percentage of input DNA. Error bars represent standard deviation determined from at least three independent experiments. ( C ) ChIP-qPCR showing a marginal binding of canonical PRC1 (cPRC1; PCGF2) to PCGF6-bound genes with high H3K27me3 (PCGF6 +H3K27me3+/hi) and those with low H3K27me3 (PCGF6 +H3K27me3-/lo) in wild type (WT) ESCs. Eed -KO ESCs were used to confirm whether H3K27me3-dependent recruitment of cPRC1 is active at indicated gene locus. NC: negative control. ( D ) Top four de novo motif recognition hits for genes bound by PCGF6, RING1B, CBX7 (NCBI GEO accession number GSM1041373), MAX (NCBI GEO accession number GSM1171650) or MYC (NCBI GEO accession number GSM1171648) in wild type ESCs. ( E ) Expression levels of Max and Mga in untreated ESCs or those treated with control, Max siRNA or Mga siRNA revealed by RT-qPCR analysis (left). Transcription levels were normalized to a Gapdh control and are depicted as fold change relative to untreated ESCs. Error bars represent standard deviation determined from at least three independent experiments. Expression levels of FLAG-PCGF6, MAX, LAMIN B and RING1B in whole cell lysates of untreated and siRNA-treated ESCs are also shown (right). ( F ) Expression levels of the indicated genes in untreated ESCs or those treated either with control, Max siRNA or Mga siRNA. Underlined genes are canonical PRC1 targets. Expression levels were normalized to a Gapdh control and are depicted as fold change relative to mock ESCs (No treatment). Error bars represent standard deviation determined from at least three independent experiments. ( G ) Gene ontology (GO) term analysis showing that genes related to meiotic process are highly overrepresented among PCGF6+RING1B+MAX+MYC- genes. Genes were classified based on binding by PCGF6, RING1B, MAX and/or MYC and the enrichment of respective GO terms in each subset of genes was determined. The p -values for the significance of over-representation against total genes are shown along the x-axis. ( H ) Changes in local PCGF6 and MAX deposition at selected PCGF6/MAX co-bound gene that do not become de-repressed in Max -KO ESCs were determined by ChIP-qPCR using Max -KO ESCs stably expressing both an HA-tagged Pcgf6 and a Flag-tagged Max (WT or Δb) expression vectors, and are shown as described in Figure 2C . DOI: http://dx.doi.org/10.7554/eLife.21064.010

    Article Snippet: Real-time qPCR was performed using the Brilliant III SYBR Green QPCR Master Mix (Agilent).

    Techniques: Expressing, Construct, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation, Binding Assay, Negative Control, Quantitative RT-PCR, Stable Transfection

    Loss of function of aggregated p53. (A) The level of LacZ mRNA was measured in cells with or without seeds using reverse transcription followed by real-time PCR analysis. Two independent reference genes, CDC19 and TAF10 , were used for normalization and fold change in the mRNA levels. The data represent the averages of three independent real-time PCR analyses. The error bars indicate standard deviations from the mean. (B) (Top) ChIP of p53 from cells with or without seeds. Yeast cells were analyzed by PCR and visualized by agarose gel electrophoresis. Differences in amplification were observed in input, IP, and mock IP. TAF10 was used as a nonbinding control. (Bottom) Graphical representation of chromatin immunoprecipitation of p53 displaying percent enrichment/input by real-time qPCR analysis. The antibody used for ChIP was anti-p53 D0-1 (5 μg). The data represent averages of the results of three independent real-time analyses. (C) Immunofluorescence study of chromatin spread of yeast cells with or without seeds. The cells without seeds, but not those with seeds, displayed p53 in a majority of the spread as chromatin bound. Scale bar, ∼3 μm. Statistical analysis of the total percent spread was calculated as represented graphically below.

    Journal: Molecular and Cellular Biology

    Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

    doi: 10.1128/MCB.00118-17

    Figure Lengend Snippet: Loss of function of aggregated p53. (A) The level of LacZ mRNA was measured in cells with or without seeds using reverse transcription followed by real-time PCR analysis. Two independent reference genes, CDC19 and TAF10 , were used for normalization and fold change in the mRNA levels. The data represent the averages of three independent real-time PCR analyses. The error bars indicate standard deviations from the mean. (B) (Top) ChIP of p53 from cells with or without seeds. Yeast cells were analyzed by PCR and visualized by agarose gel electrophoresis. Differences in amplification were observed in input, IP, and mock IP. TAF10 was used as a nonbinding control. (Bottom) Graphical representation of chromatin immunoprecipitation of p53 displaying percent enrichment/input by real-time qPCR analysis. The antibody used for ChIP was anti-p53 D0-1 (5 μg). The data represent averages of the results of three independent real-time analyses. (C) Immunofluorescence study of chromatin spread of yeast cells with or without seeds. The cells without seeds, but not those with seeds, displayed p53 in a majority of the spread as chromatin bound. Scale bar, ∼3 μm. Statistical analysis of the total percent spread was calculated as represented graphically below.

    Article Snippet: Brilliant-III UltraFast SYBR green qPCR master mix (Agilent Technologies) was used for this, according to the manufacturer's protocol.

    Techniques: Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Immunofluorescence

    Effect of P8 fibril on GFP-tagged functional p53. (A) Immunofluorescence assay to visualize p53-GFP in yeast cells with or without seeds. Aggregates of p53 are seen as green cytoplasmic focus structures specifically in the cells harboring the seeds. However, in cells without any seeds, p53-GFP was colocalized with DAPI. Scale bar, ∼5 μm. (B) Graphical representation of ChIP of p53 displaying percent enrichment/input at the p53 binding element by real-time qPCR analysis. The antibody used for ChIP was anti-p53 D0-1 (5 μg). The data represent the averages of the results of three independent real-time analyses. The error bars indicate standard deviations from the mean.

    Journal: Molecular and Cellular Biology

    Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

    doi: 10.1128/MCB.00118-17

    Figure Lengend Snippet: Effect of P8 fibril on GFP-tagged functional p53. (A) Immunofluorescence assay to visualize p53-GFP in yeast cells with or without seeds. Aggregates of p53 are seen as green cytoplasmic focus structures specifically in the cells harboring the seeds. However, in cells without any seeds, p53-GFP was colocalized with DAPI. Scale bar, ∼5 μm. (B) Graphical representation of ChIP of p53 displaying percent enrichment/input at the p53 binding element by real-time qPCR analysis. The antibody used for ChIP was anti-p53 D0-1 (5 μg). The data represent the averages of the results of three independent real-time analyses. The error bars indicate standard deviations from the mean.

    Article Snippet: Brilliant-III UltraFast SYBR green qPCR master mix (Agilent Technologies) was used for this, according to the manufacturer's protocol.

    Techniques: Functional Assay, Immunofluorescence, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction

    bZIP and inositol pyrophosphate kinase (DINS) domains of Kcs1 are required for INO1 transcription. A, diagram of the bZIP and the DINS functional domains of Kcs1 indicating site mutations disrupting individual domains. B, serial 10-fold dilutions of kcs1 Δ cells carrying either empty vector (pURA3), mutated bZIP domain (p kcs1 L1L2→AA ), mutated DINS domain (p kcs1 SLL→AAA ), or WT KCS1 were spotted on I− or I+ plates. Plates were incubated at 30 °C for 3 days. The figure shown is representative of three experiments. C, cells harboring the empty vector (pEV), WT KCS1, or mutated KCS1 were cultured in I+ to the mid-logarithmic phase ( A 550 of 0.5), pelleted, washed with prewarmed I+ or I−, and resuspended in fresh prewarmed I+ or I−. After the shift, cells were grown for 2 h. INO1 mRNA was quantified using RT-qPCR as described under “Experimental Procedures.” The data shown in C are the average of three experiments ± S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of Inositol Metabolism Is Fine-tuned by Inositol Pyrophosphates in Saccharomyces cerevisiae * * ♦

    doi: 10.1074/jbc.M113.493353

    Figure Lengend Snippet: bZIP and inositol pyrophosphate kinase (DINS) domains of Kcs1 are required for INO1 transcription. A, diagram of the bZIP and the DINS functional domains of Kcs1 indicating site mutations disrupting individual domains. B, serial 10-fold dilutions of kcs1 Δ cells carrying either empty vector (pURA3), mutated bZIP domain (p kcs1 L1L2→AA ), mutated DINS domain (p kcs1 SLL→AAA ), or WT KCS1 were spotted on I− or I+ plates. Plates were incubated at 30 °C for 3 days. The figure shown is representative of three experiments. C, cells harboring the empty vector (pEV), WT KCS1, or mutated KCS1 were cultured in I+ to the mid-logarithmic phase ( A 550 of 0.5), pelleted, washed with prewarmed I+ or I−, and resuspended in fresh prewarmed I+ or I−. After the shift, cells were grown for 2 h. INO1 mRNA was quantified using RT-qPCR as described under “Experimental Procedures.” The data shown in C are the average of three experiments ± S.D.

    Article Snippet: RT-qPCRs were performed in a 20-μl volume using Brilliant III Ultra-Faster SYBR Green qPCR master mix (Agilent Technologies, Santa Clara, CA).

    Techniques: Functional Assay, Plasmid Preparation, Incubation, Cell Culture, Quantitative RT-PCR

    peTrpL and Tc are involved in the differential posttranscriptional regulation of smeABR. (A) qRT-PCR analysis of changes in the smeR levels 10 min after IPTG addition to two parallel 2011 Δ trpL (pSRKGm-peTrpL) cultures. Tc (1.5 μg/ml) was added together with IPTG to one of the cultures (indicated). (B) RT-PCR analysis with a forward primer located in smeB and reverse primer located in smeR . The PCR template input is indicated at the top. (C) Changes in the smeA levels 10 and 20 min after IPTG addition. See also descriptions for panel A. (D and E) mRNA stability determination by qRT-PCR using smeR and smeA specific primer pairs. To 2011 Δ trpL (pSRKGm-peTrpL) cultures, IPTG and/or Tc (1.5 μg/ml) was added and 10 min thereafter, rifampin was added. The relative mRNA level values after stop of transcription by rifampin were determined and plotted against the time. The calculated half-lives are indicated. (F and G) Northern blot analysis of RNA from the experiments described for panels D and E. RNAs detected by the used probes are indicated on the left side. 16S rRNA was used as a loading control. The conditions used and time after rifampin addition are given at the top, the calculated half-lives at the bottom (see also Fig. S1 in the supplemental material). Ten minutes after IPTG and Tc addition (0 min in respect to rifampin addition), the tricistronic smeABR mRNA was detected with the smeR -specific probe (internally radiolabeled 128-nt in vitro transcript) but not with the smeA - and smeB -directed probes (radiolabeled DNA probes generated by random priming). This could be explained by the higher sensitivity and stronger binding of the RNA probe. In all graphs, data from three independent cultures are presented as means ± standard deviations.

    Journal: mBio

    Article Title: The Leader Peptide peTrpL Forms Antibiotic-Containing Ribonucleoprotein Complexes for Posttranscriptional Regulation of Multiresistance Genes

    doi: 10.1128/mBio.01027-20

    Figure Lengend Snippet: peTrpL and Tc are involved in the differential posttranscriptional regulation of smeABR. (A) qRT-PCR analysis of changes in the smeR levels 10 min after IPTG addition to two parallel 2011 Δ trpL (pSRKGm-peTrpL) cultures. Tc (1.5 μg/ml) was added together with IPTG to one of the cultures (indicated). (B) RT-PCR analysis with a forward primer located in smeB and reverse primer located in smeR . The PCR template input is indicated at the top. (C) Changes in the smeA levels 10 and 20 min after IPTG addition. See also descriptions for panel A. (D and E) mRNA stability determination by qRT-PCR using smeR and smeA specific primer pairs. To 2011 Δ trpL (pSRKGm-peTrpL) cultures, IPTG and/or Tc (1.5 μg/ml) was added and 10 min thereafter, rifampin was added. The relative mRNA level values after stop of transcription by rifampin were determined and plotted against the time. The calculated half-lives are indicated. (F and G) Northern blot analysis of RNA from the experiments described for panels D and E. RNAs detected by the used probes are indicated on the left side. 16S rRNA was used as a loading control. The conditions used and time after rifampin addition are given at the top, the calculated half-lives at the bottom (see also Fig. S1 in the supplemental material). Ten minutes after IPTG and Tc addition (0 min in respect to rifampin addition), the tricistronic smeABR mRNA was detected with the smeR -specific probe (internally radiolabeled 128-nt in vitro transcript) but not with the smeA - and smeB -directed probes (radiolabeled DNA probes generated by random priming). This could be explained by the higher sensitivity and stronger binding of the RNA probe. In all graphs, data from three independent cultures are presented as means ± standard deviations.

    Article Snippet: Relative steady-state levels of specific RNAs by real-time qRT-PCR were analyzed using the Brilliant III Ultra Fast SYBR green QRT-PCR master mix (Agilent, Waldbronn, Germany).

    Techniques: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Northern Blot, In Vitro, Generated, Binding Assay

    CoIP with 3×FLAG-peTrpL identifies smeR mRNA and its asRNA as Tc-dependent peTrpL targets. (A) Growth of strains 2011 (pSRKGm-peTrpL) and 2011 (pSRKGm-3×FLAG-peTrpL) (top), and 2011 Δ trpL (pSRKGm-3×FLAG-peTrpL) (bottom) in microtiter plates. IPTG presence and peptide products are indicated on the left. For other descriptions, see the legend for Fig. 1E . (B) Integrated Genome Browser view of the smeABR locus with mapped cDNA reads of the RNA-seq analysis of RNA, which was coimmunoprecipitated from strain 2011 (pSRKGm-3×FLAG-peTrpL, pRK4352) 10 min after peptide induction. Mock CoIP, strain 2011 (pSRKGm-peTrpL, pRK4352) was used. Tc was present in the growth medium (20 μg/ml) and in the CoIP washing buffer (2 μg/ml). Shown is representative data from one of three independent experiments. (C) qRT-PCR analysis showing the enrichment of smeR mRNA and as- smeR RNA in CoIPs with 3×FLAG-peTrpL or 3×FLAG peptide. Presence of Tc (2 μg/ml) in the washing buffer is indicated. Two-plasmid strains containing pRK4352 and one of the plasmids pSRKGm-3×FLAG-peTrpL or pSRKGm-3×FLAG was used. (D) qRT-PCR analysis showing the enrichment of the indicated RNAs in CoIPs with S. meliloti 2011 producing 3×FLAG-peTrpL (WT), 3×FLAG-peTrpL-W10A (W10A), or 3×FLAG-peTrpL-W12A (W12A) in the absence of plasmid pRK4352; 1.5 μg/ml Tc was present in the washing buffer. se, sense RNA; as, asRNA. The graphs show data from three independent cultures (mean ± standard deviation). RNA enrichment was calculated in comparison to the mock CoIP.

    Journal: mBio

    Article Title: The Leader Peptide peTrpL Forms Antibiotic-Containing Ribonucleoprotein Complexes for Posttranscriptional Regulation of Multiresistance Genes

    doi: 10.1128/mBio.01027-20

    Figure Lengend Snippet: CoIP with 3×FLAG-peTrpL identifies smeR mRNA and its asRNA as Tc-dependent peTrpL targets. (A) Growth of strains 2011 (pSRKGm-peTrpL) and 2011 (pSRKGm-3×FLAG-peTrpL) (top), and 2011 Δ trpL (pSRKGm-3×FLAG-peTrpL) (bottom) in microtiter plates. IPTG presence and peptide products are indicated on the left. For other descriptions, see the legend for Fig. 1E . (B) Integrated Genome Browser view of the smeABR locus with mapped cDNA reads of the RNA-seq analysis of RNA, which was coimmunoprecipitated from strain 2011 (pSRKGm-3×FLAG-peTrpL, pRK4352) 10 min after peptide induction. Mock CoIP, strain 2011 (pSRKGm-peTrpL, pRK4352) was used. Tc was present in the growth medium (20 μg/ml) and in the CoIP washing buffer (2 μg/ml). Shown is representative data from one of three independent experiments. (C) qRT-PCR analysis showing the enrichment of smeR mRNA and as- smeR RNA in CoIPs with 3×FLAG-peTrpL or 3×FLAG peptide. Presence of Tc (2 μg/ml) in the washing buffer is indicated. Two-plasmid strains containing pRK4352 and one of the plasmids pSRKGm-3×FLAG-peTrpL or pSRKGm-3×FLAG was used. (D) qRT-PCR analysis showing the enrichment of the indicated RNAs in CoIPs with S. meliloti 2011 producing 3×FLAG-peTrpL (WT), 3×FLAG-peTrpL-W10A (W10A), or 3×FLAG-peTrpL-W12A (W12A) in the absence of plasmid pRK4352; 1.5 μg/ml Tc was present in the washing buffer. se, sense RNA; as, asRNA. The graphs show data from three independent cultures (mean ± standard deviation). RNA enrichment was calculated in comparison to the mock CoIP.

    Article Snippet: Relative steady-state levels of specific RNAs by real-time qRT-PCR were analyzed using the Brilliant III Ultra Fast SYBR green QRT-PCR master mix (Agilent, Waldbronn, Germany).

    Techniques: Co-Immunoprecipitation Assay, RNA Sequencing Assay, Quantitative RT-PCR, Plasmid Preparation, Standard Deviation

    Conservation of peTrpL function in resistance. (A) qRT-PCR analysis of the expression of smeR homologs and trpD upon overproduction of the respective peTrpL homologs in A. tumefaciens and B. japonicum . Data from three independent cultures are presented as means ± standard deviations. (B) Growth of the indicated A. tumefaciens strain in microtiter plates. Presence of IPTG in the medium and the Tc concentrations used are indicated. A representative plate is shown. (C) Growth curves of B. japonicum containing the indicated plasmids (pRJ-MCS, empty vector). Medium supplemented with 100 μg/ml Tc was used. Data from three independent cultures are presented as mean ± standard deviations (smaller than the symbols in the graph).

    Journal: mBio

    Article Title: The Leader Peptide peTrpL Forms Antibiotic-Containing Ribonucleoprotein Complexes for Posttranscriptional Regulation of Multiresistance Genes

    doi: 10.1128/mBio.01027-20

    Figure Lengend Snippet: Conservation of peTrpL function in resistance. (A) qRT-PCR analysis of the expression of smeR homologs and trpD upon overproduction of the respective peTrpL homologs in A. tumefaciens and B. japonicum . Data from three independent cultures are presented as means ± standard deviations. (B) Growth of the indicated A. tumefaciens strain in microtiter plates. Presence of IPTG in the medium and the Tc concentrations used are indicated. A representative plate is shown. (C) Growth curves of B. japonicum containing the indicated plasmids (pRJ-MCS, empty vector). Medium supplemented with 100 μg/ml Tc was used. Data from three independent cultures are presented as mean ± standard deviations (smaller than the symbols in the graph).

    Article Snippet: Relative steady-state levels of specific RNAs by real-time qRT-PCR were analyzed using the Brilliant III Ultra Fast SYBR green QRT-PCR master mix (Agilent, Waldbronn, Germany).

    Techniques: Quantitative RT-PCR, Expressing, Plasmid Preparation

    The as- smeR RNA is induced by substrates of the SmeAB efflux pump. (A) Kinetics of changes in the level of smeR mRNA at 1, 3, 5, and 10 min after addition of IPTG and 1.5 μg/ml Tc to cultures of S. meliloti 2011 Δ trpL harboring one of the following plasmids: pSRKGm-peTrpL (WT), pSRK-Gm-3.UAG (3.UAG), pSRKGm-peTrpL-W10A (W10A), or pSRKGm-peTrpL-W12A (W12A), as determined by qRT-PCR. Changes were calculated in comparison to the EVC. (B) Kinetics of changes in the level of as- smeR RNA. See also the description for panel A. (C) Kinetics of changes in the level of smeR mRNA and as- smeR RNA at 1, 3, 5, and 10 min after addition of 1.5 μg/ml Tc to cultures of strains 2011 and 2011 Δ trpL , as determined by qRT-PCR. Changes were calculated in comparison to the cultures to which the solvent ethanol was added instead of Tc. (D to F) qRT-PCR analysis of reporter egfp mRNA reflecting P as promoter activity. (D) Changes in the egfp level upon addition of 20 μg/ml Tc to 2011 (pSUP-PasRegfp) cultures for the indicated time (min Tc). The cultures harboring the chromosomally integrated plasmid, which confers resistance to Tc, were incubated overnight in medium without Tc. No plasmid loss was detected by qPCR. (E) Changes in the egfp level 3 min after addition of Tc to 2011 (pSUP-PasRegfp) cultures. Used Tc concentrations are indicated. (F) Changes in the egfp level 3 min after addition of the indicated antibiotics and flavonoids at subinhibitory concentrations to 2011 (pSUP-PasRegfp) cultures. In all graphs, data from three independent cultures are presented as means ± standard deviations.

    Journal: mBio

    Article Title: The Leader Peptide peTrpL Forms Antibiotic-Containing Ribonucleoprotein Complexes for Posttranscriptional Regulation of Multiresistance Genes

    doi: 10.1128/mBio.01027-20

    Figure Lengend Snippet: The as- smeR RNA is induced by substrates of the SmeAB efflux pump. (A) Kinetics of changes in the level of smeR mRNA at 1, 3, 5, and 10 min after addition of IPTG and 1.5 μg/ml Tc to cultures of S. meliloti 2011 Δ trpL harboring one of the following plasmids: pSRKGm-peTrpL (WT), pSRK-Gm-3.UAG (3.UAG), pSRKGm-peTrpL-W10A (W10A), or pSRKGm-peTrpL-W12A (W12A), as determined by qRT-PCR. Changes were calculated in comparison to the EVC. (B) Kinetics of changes in the level of as- smeR RNA. See also the description for panel A. (C) Kinetics of changes in the level of smeR mRNA and as- smeR RNA at 1, 3, 5, and 10 min after addition of 1.5 μg/ml Tc to cultures of strains 2011 and 2011 Δ trpL , as determined by qRT-PCR. Changes were calculated in comparison to the cultures to which the solvent ethanol was added instead of Tc. (D to F) qRT-PCR analysis of reporter egfp mRNA reflecting P as promoter activity. (D) Changes in the egfp level upon addition of 20 μg/ml Tc to 2011 (pSUP-PasRegfp) cultures for the indicated time (min Tc). The cultures harboring the chromosomally integrated plasmid, which confers resistance to Tc, were incubated overnight in medium without Tc. No plasmid loss was detected by qPCR. (E) Changes in the egfp level 3 min after addition of Tc to 2011 (pSUP-PasRegfp) cultures. Used Tc concentrations are indicated. (F) Changes in the egfp level 3 min after addition of the indicated antibiotics and flavonoids at subinhibitory concentrations to 2011 (pSUP-PasRegfp) cultures. In all graphs, data from three independent cultures are presented as means ± standard deviations.

    Article Snippet: Relative steady-state levels of specific RNAs by real-time qRT-PCR were analyzed using the Brilliant III Ultra Fast SYBR green QRT-PCR master mix (Agilent, Waldbronn, Germany).

    Techniques: Quantitative RT-PCR, Activity Assay, Plasmid Preparation, Incubation, Real-time Polymerase Chain Reaction

    peTrpL increases multidrug resistance and forms antibiotic-dependent ribonucleoprotein (ARNP) complexes. (A) Growth of strain 2011 Δ trpL (pSRKGm-peTrpL) in microtiter plates. The increasing concentrations of the antibiotics and flavonoids are given at the top (μg/ml). The antimicrobial compounds are indicated on the right and IPTG presence on the left. Shown are representative plates with final growth. (B) qRT-PCR analysis of changes in the smeR levels 10 min after addition of the indicated antibiotics and flavonoids (used at subinhibitory concentrations) to cultures of strains 2011 Δ trpL and 2011. trpC , control mRNA. (C) qRT-PCR analysis of enrichment of the indicated RNAs by CoIP with 3×FLAG-peTrpL in comparison to the mock CoIP. Antibiotics and flavonoids (used at subinhibitory concentrations), which were added together with IPTG to cultures of S. meliloti 2011 containing either pSRKGm-3×FLAG-peTrpL or pSRKGm-peTrpL (mock CoIP), are indicated at the bottom. Presence (+A) or absence (−A) of the antibiotics or flavonoids in the washing buffer are indicated at the top. RNA enrichment was calculated in comparison to the mock CoIP. In all graphs, data from three independent cultures are presented as means ± standard deviations.

    Journal: mBio

    Article Title: The Leader Peptide peTrpL Forms Antibiotic-Containing Ribonucleoprotein Complexes for Posttranscriptional Regulation of Multiresistance Genes

    doi: 10.1128/mBio.01027-20

    Figure Lengend Snippet: peTrpL increases multidrug resistance and forms antibiotic-dependent ribonucleoprotein (ARNP) complexes. (A) Growth of strain 2011 Δ trpL (pSRKGm-peTrpL) in microtiter plates. The increasing concentrations of the antibiotics and flavonoids are given at the top (μg/ml). The antimicrobial compounds are indicated on the right and IPTG presence on the left. Shown are representative plates with final growth. (B) qRT-PCR analysis of changes in the smeR levels 10 min after addition of the indicated antibiotics and flavonoids (used at subinhibitory concentrations) to cultures of strains 2011 Δ trpL and 2011. trpC , control mRNA. (C) qRT-PCR analysis of enrichment of the indicated RNAs by CoIP with 3×FLAG-peTrpL in comparison to the mock CoIP. Antibiotics and flavonoids (used at subinhibitory concentrations), which were added together with IPTG to cultures of S. meliloti 2011 containing either pSRKGm-3×FLAG-peTrpL or pSRKGm-peTrpL (mock CoIP), are indicated at the bottom. Presence (+A) or absence (−A) of the antibiotics or flavonoids in the washing buffer are indicated at the top. RNA enrichment was calculated in comparison to the mock CoIP. In all graphs, data from three independent cultures are presented as means ± standard deviations.

    Article Snippet: Relative steady-state levels of specific RNAs by real-time qRT-PCR were analyzed using the Brilliant III Ultra Fast SYBR green QRT-PCR master mix (Agilent, Waldbronn, Germany).

    Techniques: Quantitative RT-PCR, Co-Immunoprecipitation Assay

    The frmRAB operon is induced after exposure of E . coli MG 1655 to concentrations of TMAO ≥ 5 m M . Anaerobic batch cultures were exposed to different concentrations (0–40 m M ) of TMAO . After 30 min, total RNA was isolated for qRT‐PCR of the frmR mRNA . The data shown and the mean and standard deviation for the fold increase relative to the 0 m M TMAO culture and are typical of three independent experiments.

    Journal: Environmental Microbiology

    Article Title: Adaptation of anaerobic cultures of Escherichia coliK‐12 in response to environmental trimethylamine‐ N‐oxide

    doi: 10.1111/1462-2920.12726

    Figure Lengend Snippet: The frmRAB operon is induced after exposure of E . coli MG 1655 to concentrations of TMAO ≥ 5 m M . Anaerobic batch cultures were exposed to different concentrations (0–40 m M ) of TMAO . After 30 min, total RNA was isolated for qRT‐PCR of the frmR mRNA . The data shown and the mean and standard deviation for the fold increase relative to the 0 m M TMAO culture and are typical of three independent experiments.

    Article Snippet: The amounts of frmR mRNA in samples of total RNA (100 ng; isolated as described above) after exposure of these cultures to different concentrations of TMAO for 30 min were determined on an RT‐PCR plate in a Mx3005P Thermocycler (Agilent Technologies) using Brilliant III Ultra‐Fast SYBR Green qRT‐PCR Master Mix kit (Agilent Technologies) according to the manufacturer's instructions.

    Techniques: Isolation, Quantitative RT-PCR, Standard Deviation

    Induced expression levels of genes CSP1 and CSP2 during chlamydospore formation. C. dubliniensis Wü284, C. albicans SC5314 and the C. albicans nrg1 Δ mutant were grown for 28 h in Staib medium and YPD medium, respectively, before total RNA was isolated. (A) qRT-PCR measurements detected a strong upregulation of cdCSP1 and cdCSP2 gene expression levels in C. dubliniensis during growth in Staib versus YPD medium. (B) Similarly, the C. albicans homologues caCSP1 and caCSP2 were found to be upregulated in the chlamydospore producing C. albicans nrg1 Δ mutant stronger than in C. albicans wild-type yeast cells. The results are the means ±SD from three biological replicates, ‘*’ indicates that the detected differences were significant ( P

    Journal: PLoS ONE

    Article Title: Global Transcriptome Sequencing Identifies Chlamydospore Specific Markers in Candida albicans and Candida dubliniensis

    doi: 10.1371/journal.pone.0061940

    Figure Lengend Snippet: Induced expression levels of genes CSP1 and CSP2 during chlamydospore formation. C. dubliniensis Wü284, C. albicans SC5314 and the C. albicans nrg1 Δ mutant were grown for 28 h in Staib medium and YPD medium, respectively, before total RNA was isolated. (A) qRT-PCR measurements detected a strong upregulation of cdCSP1 and cdCSP2 gene expression levels in C. dubliniensis during growth in Staib versus YPD medium. (B) Similarly, the C. albicans homologues caCSP1 and caCSP2 were found to be upregulated in the chlamydospore producing C. albicans nrg1 Δ mutant stronger than in C. albicans wild-type yeast cells. The results are the means ±SD from three biological replicates, ‘*’ indicates that the detected differences were significant ( P

    Article Snippet: Quantitative real-time (q)RT-PCR One hundred ng of total RNA were used to perform qRT-PCR with a one step approach using the Brilliant III SYBR Green Ultra-Fast QRT PCR master mix kit (Agilent Technologies, La Jolla, USA).

    Techniques: Expressing, Mutagenesis, Isolation, Quantitative RT-PCR

    MiR-specific qPCR on synthetic templates with DNA primers . A The effect of primer concentration on Cq value of ssc-let-7d and ssc-miR-26a miR-specific qPCR assays. Real-time PCR assays were performed in parallel at three different concentrations (125, 250 and 500 nM) of the forward and of the reverse primers. B Amplification curves of an eight log 10 dilution series of a synthetic ssc-let-7d template in the ssc-let-7d miR-specific qPCR assays. All samples contained a final concentration of 0.2 ng/μl salmon sperm DNA. C Extrapolation of Cq as function of the log 10 of the number of templates for the same experiment as in B was a straight line (R 2 = 0.9993) with slope of -3.341 (PCR efficiency = 99%) over eight log 10 dilution of the template. D Melting curve analysis of the same experiment. No template control is labeled ntc. Melting curve analysis was performed from 60°C to 99°C.

    Journal: BMC Biotechnology

    Article Title: Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

    doi: 10.1186/1472-6750-11-70

    Figure Lengend Snippet: MiR-specific qPCR on synthetic templates with DNA primers . A The effect of primer concentration on Cq value of ssc-let-7d and ssc-miR-26a miR-specific qPCR assays. Real-time PCR assays were performed in parallel at three different concentrations (125, 250 and 500 nM) of the forward and of the reverse primers. B Amplification curves of an eight log 10 dilution series of a synthetic ssc-let-7d template in the ssc-let-7d miR-specific qPCR assays. All samples contained a final concentration of 0.2 ng/μl salmon sperm DNA. C Extrapolation of Cq as function of the log 10 of the number of templates for the same experiment as in B was a straight line (R 2 = 0.9993) with slope of -3.341 (PCR efficiency = 99%) over eight log 10 dilution of the template. D Melting curve analysis of the same experiment. No template control is labeled ntc. Melting curve analysis was performed from 60°C to 99°C.

    Article Snippet: To investigate the effect of different PCR master mixes on the performance of miR-specific qPCR with DNA primers we compared the amplification of synthetic templates with the QuantiFast SYBR Green PCR master mix (Qiagen, Germany) and the Brilliant III Ultra-Fast QPCR Master Mix (Agilent, USA).

    Techniques: Real-time Polymerase Chain Reaction, Concentration Assay, Amplification, Polymerase Chain Reaction, Labeling

    MiR-specific qPCR in different qPCR master mixes . A Comparison of amplification curves of a synthetic ssc-let-7d template in the ssc-let-7d miR-specific qPCR assay in QuantiFast and in Brilliant III qPCR Master mixes. B Melting curve analysis of the same experiment. No template control is labeled ntc. Melting curve analysis was performed from 60°C to 99°C. No change in fluorescence (dF/dT = 0) was observed above 80°C and this part of the curves was omitted from the figure. C Extrapolation of Cq as function of the log 10 of the number of templates for the same experiment as in A was a straight line (R 2 indicated on figure) and for both master mixes the PCR efficiency was 99% as calculated from the slope of the regression line.

    Journal: BMC Biotechnology

    Article Title: Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

    doi: 10.1186/1472-6750-11-70

    Figure Lengend Snippet: MiR-specific qPCR in different qPCR master mixes . A Comparison of amplification curves of a synthetic ssc-let-7d template in the ssc-let-7d miR-specific qPCR assay in QuantiFast and in Brilliant III qPCR Master mixes. B Melting curve analysis of the same experiment. No template control is labeled ntc. Melting curve analysis was performed from 60°C to 99°C. No change in fluorescence (dF/dT = 0) was observed above 80°C and this part of the curves was omitted from the figure. C Extrapolation of Cq as function of the log 10 of the number of templates for the same experiment as in A was a straight line (R 2 indicated on figure) and for both master mixes the PCR efficiency was 99% as calculated from the slope of the regression line.

    Article Snippet: To investigate the effect of different PCR master mixes on the performance of miR-specific qPCR with DNA primers we compared the amplification of synthetic templates with the QuantiFast SYBR Green PCR master mix (Qiagen, Germany) and the Brilliant III Ultra-Fast QPCR Master Mix (Agilent, USA).

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Labeling, Fluorescence, Polymerase Chain Reaction

    Discrimination between miRNAs with single nucleotide differences . A Position of the single nucleotide mismatches relative to the PCR primers for the ssc-let-7a, ssc-miR-23a, ssc-miR-125b and ssc-miR-150 qPCR assays. The ssc-miR-23b sequence used for mismatch discrimination was taken from miRBase and is different from the ssc-miR-23b sequence found in uterus and used for designing the ssc-miR-23b qPCR primers (Table 1). B Discrimination between closely related miRNA templates for miR-specific qPCR assays with DNA primers. Mismatches in the miRNA compared to the PCR primers are underlined. The data represents the results of three to four measurements. C Amplification curves of ssc-let-7a and ssc-let-7e synthetic template in the ssc-let-7a miR-specific qPCR assays. All samples including the no template control (ntc) contained a final concentration of 0.2 ng/μl salmon sperm DNA.

    Journal: BMC Biotechnology

    Article Title: Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

    doi: 10.1186/1472-6750-11-70

    Figure Lengend Snippet: Discrimination between miRNAs with single nucleotide differences . A Position of the single nucleotide mismatches relative to the PCR primers for the ssc-let-7a, ssc-miR-23a, ssc-miR-125b and ssc-miR-150 qPCR assays. The ssc-miR-23b sequence used for mismatch discrimination was taken from miRBase and is different from the ssc-miR-23b sequence found in uterus and used for designing the ssc-miR-23b qPCR primers (Table 1). B Discrimination between closely related miRNA templates for miR-specific qPCR assays with DNA primers. Mismatches in the miRNA compared to the PCR primers are underlined. The data represents the results of three to four measurements. C Amplification curves of ssc-let-7a and ssc-let-7e synthetic template in the ssc-let-7a miR-specific qPCR assays. All samples including the no template control (ntc) contained a final concentration of 0.2 ng/μl salmon sperm DNA.

    Article Snippet: To investigate the effect of different PCR master mixes on the performance of miR-specific qPCR with DNA primers we compared the amplification of synthetic templates with the QuantiFast SYBR Green PCR master mix (Qiagen, Germany) and the Brilliant III Ultra-Fast QPCR Master Mix (Agilent, USA).

    Techniques: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Sequencing, Amplification, Concentration Assay

    TGA2 controls the UV-B-induced expression of GSTU7, GSTU8 and GSTU25 genes, via recruitment to their promoters. Wild type (WT), tga256 triple mutant ( tga256 ) and complemented plants ( tga256 /TGA2 line #1) were irradiated with UV-B for 5 hours (+). Untreated plants were used as control (-). (A) The expression of GSTU7 , GSTU8 , and GSTU25 genes was evaluated by RT-qPCR. Data is presented as mean values of GSTU gene expression relative to the expression of the housekeeping YLS8 gene. Error bars represent standard deviation from three biological replicates (4-5 seedlings each). Different letters above bars indicate significant differences (ANOVA/Fisher’s LSD test, p

    Journal: bioRxiv

    Article Title: Transcription factor TGA2 is essential for UV-B stress tolerance controlling oxidative stress in Arabidopsis

    doi: 10.1101/2020.05.24.113589

    Figure Lengend Snippet: TGA2 controls the UV-B-induced expression of GSTU7, GSTU8 and GSTU25 genes, via recruitment to their promoters. Wild type (WT), tga256 triple mutant ( tga256 ) and complemented plants ( tga256 /TGA2 line #1) were irradiated with UV-B for 5 hours (+). Untreated plants were used as control (-). (A) The expression of GSTU7 , GSTU8 , and GSTU25 genes was evaluated by RT-qPCR. Data is presented as mean values of GSTU gene expression relative to the expression of the housekeeping YLS8 gene. Error bars represent standard deviation from three biological replicates (4-5 seedlings each). Different letters above bars indicate significant differences (ANOVA/Fisher’s LSD test, p

    Article Snippet: RT-qPCR analysis Total RNA od whole seedlings was obtained from frozen samples using the TRIzol® Reagent (Invitrogen). cDNA was synthesized with the ImProm II Kit (Promega). qPCR was performed using the Brilliant III Ultra-Fast SYBR® Green QPCR Master mix reagents (Agilent Technologies) on a AriaMx realtime PCR system equipment.

    Techniques: Expressing, Mutagenesis, Irradiation, Quantitative RT-PCR, Standard Deviation