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  • 90
    Thermo Fisher turbo dna free kit
    HEATR2 splice mutation results in alteration of the final conserved HEAT repeat and protein instability. (A) Pedigree of related families of UK-Pakistani descent. IV∶4 identifies the proband, also designated by the arrow. Solid symbols (individuals IV∶1, IV∶4 and IV∶10) indicate those affected with PCD. Double lines indicate consanguineous marriages. The individuals labeled <t>DNA</t> signified those that had their DNA included in SNP genotyping. (B) Schematic of HEATR2 transcript showing the transversion mutation ( ENST00000297440:c.2432-1G > C ) affecting the splice acceptor site of the final exon. The mutation results in inactivation of this splice site and utilization of an adjacent downstream cryptic splice acceptor site in exon 13, causing a 2-nucleotide AG deletion in the HEATR2 transcript, resulting in a frameshift in translation ( Figure S2B ). This is predicted to alter the final 44 amino acids of the protein and add an additional 33 amino acids with creation of a novel termination signal at codon 888 in the 3′UTR (See Figure S3A ). This mutation disrupts the final highly conserved HEAT repeat and alters the C-terminus of the ARM-type fold superfamily domain (red). (C) Relative expression levels of HEATR2 transcript by RT-qPCR, when normalized to the reference TBP gene. (D) The PCD transversion mutation ( ENST00000297440:c.2432-1G > C ) does not affect HEATR2 transcript stability or gross splicing as shown by RT-PCR on parental control (C) and patient (P*) <t>cDNA</t> from LCLs. PCR products spanning the gene including the splice acceptor mutation at Exon 11–13 and Exon 12-3′UTR show no obvious alterations in size. Direct sequencing confirmed a 2 base pair deletion consistent with efficient splicing to the cryptic splice acceptor at the start of exon 13 in PCD patients ( Figure S2B ). (E) Western blot analysis on total protein extracts from unrelated control, heterozygous parental and homozygous patient LCLs demonstrates the PCD mutation ( ENST00000297440:c.2432-1G > C ) results in an elongated HEATR2 protein present at reduced levels implying instability. The slight shift in mobility of the protein in the patient is consistent with the predicted 3 kDa size shift due to the amino acid alterations described. β-actin is used as a loading control. (For longer exposure see Figure S3B ). (F) Levels of HEATR2 protein normalized relative to β-actin reveal that parental samples which are heterozygous for the mutation shows a reduction to ≈50% of that of unrelated controls whilst the homozygous patient sample shows a reduction to ≈3% of control levels.
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    Thermo Fisher dna free kit
    Knockout of miR-UL148D impairs the establishment of experimental NR-1 latency in Kasumi-3 cells. (A, C) HCMV genome copies in Kasumi-3 cells (A) and CD34 + HPCs (C) infected with NR-1 or NR-1ΔmiR-UL148D. Total <t>DNA</t> was isolated from the infected cells at various time points after infection, and viral DNA was quantified by qPCR and normalized to cellular GAPDH. (B, D) Representative transcript levels from each class of viral genes in Kasumi-3 cells (B) and HPCs (D) infected with NR-1 or NR-1ΔmiR-UL148D. IE1 (immediately early), UL54 (early lytic transcript), and UL99 (late lytic transcript). Total <t>RNA</t> was isolated from infected cells and assayed by RT-qPCR. Samples were assayed in triplicate, and GAPDH level was used for normalization. (E, F) Restoring miR-UL148D expression via transfection with the miR-UL148D agomir reduced HCMV genome copies (E) and IE1 (F) expression in NR-1ΔmiR-UL148D-infected Kasumi-3 cells. The miR-UL148D agomir was added 24 hours before viral infection, and the culture media was replaced everyday with the addition of fresh agomir. DNA and total RNA were isolated from the Kasumi-3 cells at various time points after infection and quantified by qPCR and RT-qPCR, respectively. Samples were assayed in triplicate, and GAPDH level was used for normalization. Results derived from NR-1-infected Kasumi-3 cells are shown as a control. (G) Both NR-1ΔmiR-UL148D- and NR-1-infected Kasumi-3 cells produced infectious progeny. Infected Kasumi-3 cells harvested 10 days post-infection were stained with a monoclonal antibody against IE1 (clone 1B12, shown in red). GFP (green) and DAPI (blue) served as markers for lytic infection and nuclei, respectively. Infected Kasumi-3 cells were also co-cultured with HFFs. Viral plaque formation in the HFFs (shown by a GFP-positive status) was visualized by fluorescence microscopy. Images were collected using a 40x objective, and representative fields are shown for each infection. Values are shown as the mean ± SEM (n = 3). *, P
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    Thermo Fisher dna free
    Comparison of the expression profiles of selected gene groups in biofilm cells and planktonic cells. Cells were grown as described in the text, and total <t>RNA</t> was extracted from the cells at the five times indicated and used in <t>DNA</t> microarray analyses.
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    Thermo Fisher turbo dna free
    RT-PCR analysis of mutant and wild-type strains. <t>RNA</t> was extracted from cells grown under nitrogen-fixing conditions in AA/8 with 1 μm molybdate and 1 μm vanadate. Specific transcripts were detected with primers designed to amplify part of the genes shown: nif1 genes for the Mo nitrogenase, vnf genes for the V nitrogenase, a vup gene for the V transport system (which is repressed by Mo), and rnpB for a constitutive gene. Lane 1, BP227-43 (point mutation restoring growth of nifH1 mutant); lane 2, BP227-44 (point mutation restoring growth of nifH1 mutant; probable Mo transport mutant); lane 3, BP227-4 ( nifH1 insertion mutation); lane 4, FD (wild-type); lane 5, chromosomal <t>DNA</t> from FD. (Not shown, negative controls verifying that no DNA contaminated the RNA extracts).
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    Thermo Fisher dna free reagent
    A nonsense mutation (in-frame stop codon) was present in the Eh ntr1 gene of genome project strain HM-1:IMSS of Entamoeba and three clinical isolates. (A) The nitroreductases of Entamoeba strains HM-1:IMSS and 200:NIH, as well as those Methanothermobacter thermautotrophicus (Mt) and Archaeoglobus fulgidus (Af), were aligned by using the single-letter code. In this alignment, identical amino acids are marked with an asterisk, while similar amino acids are marked with a colon or a period. Within a putative zero-frame 81-bp intron of EhNTR1 of genome project strain HM-1:IMSS, there was a stop codon (#) where there was an Arg (R) in the wild-type EhNTR1 of 200:NIH. The stop codon, which was also present in the Eh ntr1 genes of 3 of 22 clinical isolates examined (isolates BM1, CM2, and H22), was present in a region that is conserved in bacterial nitroreductases. (B) PCR and RT-PCR with Eh ntr1 gene primers from HM-1:IMSS <t>DNA</t> and <t>RNA,</t> respectively, produced products of the same size, arguing against the presence of an in-frame intron in the Eh ntr1 gene. In contrast, the product obtained by RT-PCR with Eh ntr3 gene primers from HM-1:IMSS RNA was smaller than the product obtained by PCR from DNA, consistent with the presence of an intron in the Eh ntr3 gene of Entamoeba . The sequence of the Eh ntr3 RT-PCR product confirmed the presence of the intron at the position predicted by the genome project (data not shown). (C) Western blotting with a rabbit polyclonal antibody to a recombinant EhNTR1 protein showed that strains 200:NIH and Rahman, which had the wild-type Eh ntr1 gene, both expressed the NTR1 protein. In contrast, strain HM-1:IMSS, which had a nonsense mutation in the Eh ntr1 gene, did not express the NTR1 protein.
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    Thermo Fisher dna free system
    A nonsense mutation (in-frame stop codon) was present in the Eh ntr1 gene of genome project strain HM-1:IMSS of Entamoeba and three clinical isolates. (A) The nitroreductases of Entamoeba strains HM-1:IMSS and 200:NIH, as well as those Methanothermobacter thermautotrophicus (Mt) and Archaeoglobus fulgidus (Af), were aligned by using the single-letter code. In this alignment, identical amino acids are marked with an asterisk, while similar amino acids are marked with a colon or a period. Within a putative zero-frame 81-bp intron of EhNTR1 of genome project strain HM-1:IMSS, there was a stop codon (#) where there was an Arg (R) in the wild-type EhNTR1 of 200:NIH. The stop codon, which was also present in the Eh ntr1 genes of 3 of 22 clinical isolates examined (isolates BM1, CM2, and H22), was present in a region that is conserved in bacterial nitroreductases. (B) PCR and RT-PCR with Eh ntr1 gene primers from HM-1:IMSS <t>DNA</t> and <t>RNA,</t> respectively, produced products of the same size, arguing against the presence of an in-frame intron in the Eh ntr1 gene. In contrast, the product obtained by RT-PCR with Eh ntr3 gene primers from HM-1:IMSS RNA was smaller than the product obtained by PCR from DNA, consistent with the presence of an intron in the Eh ntr3 gene of Entamoeba . The sequence of the Eh ntr3 RT-PCR product confirmed the presence of the intron at the position predicted by the genome project (data not shown). (C) Western blotting with a rabbit polyclonal antibody to a recombinant EhNTR1 protein showed that strains 200:NIH and Rahman, which had the wild-type Eh ntr1 gene, both expressed the NTR1 protein. In contrast, strain HM-1:IMSS, which had a nonsense mutation in the Eh ntr1 gene, did not express the NTR1 protein.
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    Thermo Fisher dna dye picogreen
    Sodium butyrate stimulates mitochondrial biogenesis in PC12-ND6 cells by increasing <t>mt-DNA</t> copy number and expression levels of key regulators of mt-DNA replication. a Representative live-cell confocal micrograph of <t>PicoGreen</t> staining of mt-nucleoids in control and NaBt-treated PC12-ND6 cells transfected with the mito-RFP vector. Scale bar = 5 µm. b Quantification of Mt-DNA copy number by qPCR from control and HDACi-treated PC12-ND6 cells and normalized by ΔΔ C (t). Mean ± SEM ( n = 3; * p
    Dna Dye Picogreen, supplied by Thermo Fisher, 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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    Thermo Fisher dna free enzyme
    Sodium butyrate stimulates mitochondrial biogenesis in PC12-ND6 cells by increasing <t>mt-DNA</t> copy number and expression levels of key regulators of mt-DNA replication. a Representative live-cell confocal micrograph of <t>PicoGreen</t> staining of mt-nucleoids in control and NaBt-treated PC12-ND6 cells transfected with the mito-RFP vector. Scale bar = 5 µm. b Quantification of Mt-DNA copy number by qPCR from control and HDACi-treated PC12-ND6 cells and normalized by ΔΔ C (t). Mean ± SEM ( n = 3; * p
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    Thermo Fisher invitrogen turbo dna free
    Sodium butyrate stimulates mitochondrial biogenesis in PC12-ND6 cells by increasing <t>mt-DNA</t> copy number and expression levels of key regulators of mt-DNA replication. a Representative live-cell confocal micrograph of <t>PicoGreen</t> staining of mt-nucleoids in control and NaBt-treated PC12-ND6 cells transfected with the mito-RFP vector. Scale bar = 5 µm. b Quantification of Mt-DNA copy number by qPCR from control and HDACi-treated PC12-ND6 cells and normalized by ΔΔ C (t). Mean ± SEM ( n = 3; * p
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    Thermo Fisher turbo dna free endonuclease
    Sodium butyrate stimulates mitochondrial biogenesis in PC12-ND6 cells by increasing <t>mt-DNA</t> copy number and expression levels of key regulators of mt-DNA replication. a Representative live-cell confocal micrograph of <t>PicoGreen</t> staining of mt-nucleoids in control and NaBt-treated PC12-ND6 cells transfected with the mito-RFP vector. Scale bar = 5 µm. b Quantification of Mt-DNA copy number by qPCR from control and HDACi-treated PC12-ND6 cells and normalized by ΔΔ C (t). Mean ± SEM ( n = 3; * p
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    Thermo Fisher ambion dna free kit
    Sodium butyrate stimulates mitochondrial biogenesis in PC12-ND6 cells by increasing <t>mt-DNA</t> copy number and expression levels of key regulators of mt-DNA replication. a Representative live-cell confocal micrograph of <t>PicoGreen</t> staining of mt-nucleoids in control and NaBt-treated PC12-ND6 cells transfected with the mito-RFP vector. Scale bar = 5 µm. b Quantification of Mt-DNA copy number by qPCR from control and HDACi-treated PC12-ND6 cells and normalized by ΔΔ C (t). Mean ± SEM ( n = 3; * p
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    Thermo Fisher turbo dna free enzyme
    Sodium butyrate stimulates mitochondrial biogenesis in PC12-ND6 cells by increasing <t>mt-DNA</t> copy number and expression levels of key regulators of mt-DNA replication. a Representative live-cell confocal micrograph of <t>PicoGreen</t> staining of mt-nucleoids in control and NaBt-treated PC12-ND6 cells transfected with the mito-RFP vector. Scale bar = 5 µm. b Quantification of Mt-DNA copy number by qPCR from control and HDACi-treated PC12-ND6 cells and normalized by ΔΔ C (t). Mean ± SEM ( n = 3; * p
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    Thermo Fisher dna free dnase
    Sodium butyrate stimulates mitochondrial biogenesis in PC12-ND6 cells by increasing <t>mt-DNA</t> copy number and expression levels of key regulators of mt-DNA replication. a Representative live-cell confocal micrograph of <t>PicoGreen</t> staining of mt-nucleoids in control and NaBt-treated PC12-ND6 cells transfected with the mito-RFP vector. Scale bar = 5 µm. b Quantification of Mt-DNA copy number by qPCR from control and HDACi-treated PC12-ND6 cells and normalized by ΔΔ C (t). Mean ± SEM ( n = 3; * p
    Dna Free Dnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 418 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher turbo dna free system
    Sodium butyrate stimulates mitochondrial biogenesis in PC12-ND6 cells by increasing <t>mt-DNA</t> copy number and expression levels of key regulators of mt-DNA replication. a Representative live-cell confocal micrograph of <t>PicoGreen</t> staining of mt-nucleoids in control and NaBt-treated PC12-ND6 cells transfected with the mito-RFP vector. Scale bar = 5 µm. b Quantification of Mt-DNA copy number by qPCR from control and HDACi-treated PC12-ND6 cells and normalized by ΔΔ C (t). Mean ± SEM ( n = 3; * p
    Turbo Dna Free System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 164 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher thurbo dna free system
    Sodium butyrate stimulates mitochondrial biogenesis in PC12-ND6 cells by increasing <t>mt-DNA</t> copy number and expression levels of key regulators of mt-DNA replication. a Representative live-cell confocal micrograph of <t>PicoGreen</t> staining of mt-nucleoids in control and NaBt-treated PC12-ND6 cells transfected with the mito-RFP vector. Scale bar = 5 µm. b Quantification of Mt-DNA copy number by qPCR from control and HDACi-treated PC12-ND6 cells and normalized by ΔΔ C (t). Mean ± SEM ( n = 3; * p
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    Thermo Fisher turbo dna free reagent
    <t>DNA</t> and <t>RNA</t> analysis.
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    Thermo Fisher turbo dna free buffer
    <t>DNA</t> and <t>RNA</t> analysis.
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    Thermo Fisher dna free rna
    Analysis of externally regulated expression of transgenic KGF <t>RNA</t> and protein in the lung. Two lines of transgene (Tg)-positive animals were treated with Dox or control solution (5% sucrose) for 2 days. Mice were killed and a part of the lung was used for RNA isolation and another part for protein isolation. ( A ) <t>DNA-free</t> RNA was used for RT-PCR with appropriate primers. In the absence of the external inducer (Dox − ), animals from neither line expressed RNA corresponding to the KGF Tg but expressed transgenic RNA after induction (Dox + ). ( B ) To detect KGF protein expression derived from the Tg, lungs were homogenized in Triton X-100 containing buffer, and the pelleted fraction was solubilized in hot Laemmli buffer and subjected to Western blot analysis. We were unable to detect KGF protein in the Triton X-100-soluble extract, which is in keeping with the propensity of KGF and other members of the fibroblast growth factor family to attach to matrix components. In the Laemmli buffer extract, KGF protein from the Tg could be identified within 48 h of Tg induction. Arrow indicates the KGF protein derived from the Tg that migrates similarly with the standard (recombinant KGF). The faint band in the lane containing extract prepared from the lungs of uninduced animals and non-Tg animals probably corresponds to low levels of endogenous KGF. The level of KGF expression per mouse was estimated to be between 5 and 10 ng. Tg expression in the two lines was confirmed at both RNA and protein levels in two independent experiments using five animals per line per group (±Dox). Shown are data representative of five animals in each group. We have not performed long-term experiments to determine effects of very low, undetectable leaky expression of Tg, if any.
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    Thermo Fisher residual dna
    Analysis of externally regulated expression of transgenic KGF <t>RNA</t> and protein in the lung. Two lines of transgene (Tg)-positive animals were treated with Dox or control solution (5% sucrose) for 2 days. Mice were killed and a part of the lung was used for RNA isolation and another part for protein isolation. ( A ) <t>DNA-free</t> RNA was used for RT-PCR with appropriate primers. In the absence of the external inducer (Dox − ), animals from neither line expressed RNA corresponding to the KGF Tg but expressed transgenic RNA after induction (Dox + ). ( B ) To detect KGF protein expression derived from the Tg, lungs were homogenized in Triton X-100 containing buffer, and the pelleted fraction was solubilized in hot Laemmli buffer and subjected to Western blot analysis. We were unable to detect KGF protein in the Triton X-100-soluble extract, which is in keeping with the propensity of KGF and other members of the fibroblast growth factor family to attach to matrix components. In the Laemmli buffer extract, KGF protein from the Tg could be identified within 48 h of Tg induction. Arrow indicates the KGF protein derived from the Tg that migrates similarly with the standard (recombinant KGF). The faint band in the lane containing extract prepared from the lungs of uninduced animals and non-Tg animals probably corresponds to low levels of endogenous KGF. The level of KGF expression per mouse was estimated to be between 5 and 10 ng. Tg expression in the two lines was confirmed at both RNA and protein levels in two independent experiments using five animals per line per group (±Dox). Shown are data representative of five animals in each group. We have not performed long-term experiments to determine effects of very low, undetectable leaky expression of Tg, if any.
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    Fisher Scientific dna free
    Analysis of externally regulated expression of transgenic KGF <t>RNA</t> and protein in the lung. Two lines of transgene (Tg)-positive animals were treated with Dox or control solution (5% sucrose) for 2 days. Mice were killed and a part of the lung was used for RNA isolation and another part for protein isolation. ( A ) <t>DNA-free</t> RNA was used for RT-PCR with appropriate primers. In the absence of the external inducer (Dox − ), animals from neither line expressed RNA corresponding to the KGF Tg but expressed transgenic RNA after induction (Dox + ). ( B ) To detect KGF protein expression derived from the Tg, lungs were homogenized in Triton X-100 containing buffer, and the pelleted fraction was solubilized in hot Laemmli buffer and subjected to Western blot analysis. We were unable to detect KGF protein in the Triton X-100-soluble extract, which is in keeping with the propensity of KGF and other members of the fibroblast growth factor family to attach to matrix components. In the Laemmli buffer extract, KGF protein from the Tg could be identified within 48 h of Tg induction. Arrow indicates the KGF protein derived from the Tg that migrates similarly with the standard (recombinant KGF). The faint band in the lane containing extract prepared from the lungs of uninduced animals and non-Tg animals probably corresponds to low levels of endogenous KGF. The level of KGF expression per mouse was estimated to be between 5 and 10 ng. Tg expression in the two lines was confirmed at both RNA and protein levels in two independent experiments using five animals per line per group (±Dox). Shown are data representative of five animals in each group. We have not performed long-term experiments to determine effects of very low, undetectable leaky expression of Tg, if any.
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    Analysis of externally regulated expression of transgenic KGF <t>RNA</t> and protein in the lung. Two lines of transgene (Tg)-positive animals were treated with Dox or control solution (5% sucrose) for 2 days. Mice were killed and a part of the lung was used for RNA isolation and another part for protein isolation. ( A ) <t>DNA-free</t> RNA was used for RT-PCR with appropriate primers. In the absence of the external inducer (Dox − ), animals from neither line expressed RNA corresponding to the KGF Tg but expressed transgenic RNA after induction (Dox + ). ( B ) To detect KGF protein expression derived from the Tg, lungs were homogenized in Triton X-100 containing buffer, and the pelleted fraction was solubilized in hot Laemmli buffer and subjected to Western blot analysis. We were unable to detect KGF protein in the Triton X-100-soluble extract, which is in keeping with the propensity of KGF and other members of the fibroblast growth factor family to attach to matrix components. In the Laemmli buffer extract, KGF protein from the Tg could be identified within 48 h of Tg induction. Arrow indicates the KGF protein derived from the Tg that migrates similarly with the standard (recombinant KGF). The faint band in the lane containing extract prepared from the lungs of uninduced animals and non-Tg animals probably corresponds to low levels of endogenous KGF. The level of KGF expression per mouse was estimated to be between 5 and 10 ng. Tg expression in the two lines was confirmed at both RNA and protein levels in two independent experiments using five animals per line per group (±Dox). Shown are data representative of five animals in each group. We have not performed long-term experiments to determine effects of very low, undetectable leaky expression of Tg, if any.
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    Image Search Results


    HEATR2 splice mutation results in alteration of the final conserved HEAT repeat and protein instability. (A) Pedigree of related families of UK-Pakistani descent. IV∶4 identifies the proband, also designated by the arrow. Solid symbols (individuals IV∶1, IV∶4 and IV∶10) indicate those affected with PCD. Double lines indicate consanguineous marriages. The individuals labeled DNA signified those that had their DNA included in SNP genotyping. (B) Schematic of HEATR2 transcript showing the transversion mutation ( ENST00000297440:c.2432-1G > C ) affecting the splice acceptor site of the final exon. The mutation results in inactivation of this splice site and utilization of an adjacent downstream cryptic splice acceptor site in exon 13, causing a 2-nucleotide AG deletion in the HEATR2 transcript, resulting in a frameshift in translation ( Figure S2B ). This is predicted to alter the final 44 amino acids of the protein and add an additional 33 amino acids with creation of a novel termination signal at codon 888 in the 3′UTR (See Figure S3A ). This mutation disrupts the final highly conserved HEAT repeat and alters the C-terminus of the ARM-type fold superfamily domain (red). (C) Relative expression levels of HEATR2 transcript by RT-qPCR, when normalized to the reference TBP gene. (D) The PCD transversion mutation ( ENST00000297440:c.2432-1G > C ) does not affect HEATR2 transcript stability or gross splicing as shown by RT-PCR on parental control (C) and patient (P*) cDNA from LCLs. PCR products spanning the gene including the splice acceptor mutation at Exon 11–13 and Exon 12-3′UTR show no obvious alterations in size. Direct sequencing confirmed a 2 base pair deletion consistent with efficient splicing to the cryptic splice acceptor at the start of exon 13 in PCD patients ( Figure S2B ). (E) Western blot analysis on total protein extracts from unrelated control, heterozygous parental and homozygous patient LCLs demonstrates the PCD mutation ( ENST00000297440:c.2432-1G > C ) results in an elongated HEATR2 protein present at reduced levels implying instability. The slight shift in mobility of the protein in the patient is consistent with the predicted 3 kDa size shift due to the amino acid alterations described. β-actin is used as a loading control. (For longer exposure see Figure S3B ). (F) Levels of HEATR2 protein normalized relative to β-actin reveal that parental samples which are heterozygous for the mutation shows a reduction to ≈50% of that of unrelated controls whilst the homozygous patient sample shows a reduction to ≈3% of control levels.

    Journal: PLoS Genetics

    Article Title: HEATR2 Plays a Conserved Role in Assembly of the Ciliary Motile Apparatus

    doi: 10.1371/journal.pgen.1004577

    Figure Lengend Snippet: HEATR2 splice mutation results in alteration of the final conserved HEAT repeat and protein instability. (A) Pedigree of related families of UK-Pakistani descent. IV∶4 identifies the proband, also designated by the arrow. Solid symbols (individuals IV∶1, IV∶4 and IV∶10) indicate those affected with PCD. Double lines indicate consanguineous marriages. The individuals labeled DNA signified those that had their DNA included in SNP genotyping. (B) Schematic of HEATR2 transcript showing the transversion mutation ( ENST00000297440:c.2432-1G > C ) affecting the splice acceptor site of the final exon. The mutation results in inactivation of this splice site and utilization of an adjacent downstream cryptic splice acceptor site in exon 13, causing a 2-nucleotide AG deletion in the HEATR2 transcript, resulting in a frameshift in translation ( Figure S2B ). This is predicted to alter the final 44 amino acids of the protein and add an additional 33 amino acids with creation of a novel termination signal at codon 888 in the 3′UTR (See Figure S3A ). This mutation disrupts the final highly conserved HEAT repeat and alters the C-terminus of the ARM-type fold superfamily domain (red). (C) Relative expression levels of HEATR2 transcript by RT-qPCR, when normalized to the reference TBP gene. (D) The PCD transversion mutation ( ENST00000297440:c.2432-1G > C ) does not affect HEATR2 transcript stability or gross splicing as shown by RT-PCR on parental control (C) and patient (P*) cDNA from LCLs. PCR products spanning the gene including the splice acceptor mutation at Exon 11–13 and Exon 12-3′UTR show no obvious alterations in size. Direct sequencing confirmed a 2 base pair deletion consistent with efficient splicing to the cryptic splice acceptor at the start of exon 13 in PCD patients ( Figure S2B ). (E) Western blot analysis on total protein extracts from unrelated control, heterozygous parental and homozygous patient LCLs demonstrates the PCD mutation ( ENST00000297440:c.2432-1G > C ) results in an elongated HEATR2 protein present at reduced levels implying instability. The slight shift in mobility of the protein in the patient is consistent with the predicted 3 kDa size shift due to the amino acid alterations described. β-actin is used as a loading control. (For longer exposure see Figure S3B ). (F) Levels of HEATR2 protein normalized relative to β-actin reveal that parental samples which are heterozygous for the mutation shows a reduction to ≈50% of that of unrelated controls whilst the homozygous patient sample shows a reduction to ≈3% of control levels.

    Article Snippet: Total RNA was isolated according to manufacturer's protocol using RNAeasy minicolumns (Qiagen), followed by DNase treatment with Turbo DNA-free kit (Ambion). cDNA was made using First Strand Synthesis of cDNA for RT-PCR (AMV) kit (Roche).

    Techniques: Mutagenesis, Labeling, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Western Blot

    Predicted cis -acting feedback can be mediated by antisense transcription (a) Schematic representation of the model were Tsix acts as the predicted cis -acting repressor: RNA Pol II complexes can bind to the Tsix (blue) and Xist (green) promoters and then move along the gene in a convergent fashion. Mutual repression occurs at three levels: (1) Silencing of the Tsix promoter by Xist RNA, (2) repression of the Xist promoter by antisense transcription and (3) random removal of one Pol II complex, if two antisense Pol II complexes occupy the same DNA element. Black dotted lines indicate interactions removed in the reduced models in (d). Lighter colors and dotted nascent RNA indicate potential interruption of transcription through TI. (b-c) Stochastic simulation of Xist up-regulation for one example parameter set for the model shown in (a), showing three individual cells (b) and a population of 100 cells (c). Light and dark green in (b) represent Xist levels expressed from the two X chromosomes, light and dark grey in (c) represent mono- and bi-allelic Xist expression, as indicated. (d) Testing of model simplifications for the network in (a), where Xist and Tsix interact through one or two of the three repressive mechanisms as indicated. The percentage of parameter sets that can maintain the XaXi state (top) and that can initiate mono-allelic Xist up-regulation (bottom) in a stochastic simulation for each model are shown. Mono-allelic up-regulation was only tested for parameter sets that could maintain the XaXi state (others n.d.). Source data for panel d are available online.

    Journal: Nature structural & molecular biology

    Article Title: A symmetric toggle switch explains the onset of random X inactivation in different mammals

    doi: 10.1038/s41594-019-0214-1

    Figure Lengend Snippet: Predicted cis -acting feedback can be mediated by antisense transcription (a) Schematic representation of the model were Tsix acts as the predicted cis -acting repressor: RNA Pol II complexes can bind to the Tsix (blue) and Xist (green) promoters and then move along the gene in a convergent fashion. Mutual repression occurs at three levels: (1) Silencing of the Tsix promoter by Xist RNA, (2) repression of the Xist promoter by antisense transcription and (3) random removal of one Pol II complex, if two antisense Pol II complexes occupy the same DNA element. Black dotted lines indicate interactions removed in the reduced models in (d). Lighter colors and dotted nascent RNA indicate potential interruption of transcription through TI. (b-c) Stochastic simulation of Xist up-regulation for one example parameter set for the model shown in (a), showing three individual cells (b) and a population of 100 cells (c). Light and dark green in (b) represent Xist levels expressed from the two X chromosomes, light and dark grey in (c) represent mono- and bi-allelic Xist expression, as indicated. (d) Testing of model simplifications for the network in (a), where Xist and Tsix interact through one or two of the three repressive mechanisms as indicated. The percentage of parameter sets that can maintain the XaXi state (top) and that can initiate mono-allelic Xist up-regulation (bottom) in a stochastic simulation for each model are shown. Mono-allelic up-regulation was only tested for parameter sets that could maintain the XaXi state (others n.d.). Source data for panel d are available online.

    Article Snippet: Allele-specific amplicon sequencing RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research) and DNase digest was performed using Turbo DNA free kit (Ambion).

    Techniques: Expressing

    Knockout of miR-UL148D impairs the establishment of experimental NR-1 latency in Kasumi-3 cells. (A, C) HCMV genome copies in Kasumi-3 cells (A) and CD34 + HPCs (C) infected with NR-1 or NR-1ΔmiR-UL148D. Total DNA was isolated from the infected cells at various time points after infection, and viral DNA was quantified by qPCR and normalized to cellular GAPDH. (B, D) Representative transcript levels from each class of viral genes in Kasumi-3 cells (B) and HPCs (D) infected with NR-1 or NR-1ΔmiR-UL148D. IE1 (immediately early), UL54 (early lytic transcript), and UL99 (late lytic transcript). Total RNA was isolated from infected cells and assayed by RT-qPCR. Samples were assayed in triplicate, and GAPDH level was used for normalization. (E, F) Restoring miR-UL148D expression via transfection with the miR-UL148D agomir reduced HCMV genome copies (E) and IE1 (F) expression in NR-1ΔmiR-UL148D-infected Kasumi-3 cells. The miR-UL148D agomir was added 24 hours before viral infection, and the culture media was replaced everyday with the addition of fresh agomir. DNA and total RNA were isolated from the Kasumi-3 cells at various time points after infection and quantified by qPCR and RT-qPCR, respectively. Samples were assayed in triplicate, and GAPDH level was used for normalization. Results derived from NR-1-infected Kasumi-3 cells are shown as a control. (G) Both NR-1ΔmiR-UL148D- and NR-1-infected Kasumi-3 cells produced infectious progeny. Infected Kasumi-3 cells harvested 10 days post-infection were stained with a monoclonal antibody against IE1 (clone 1B12, shown in red). GFP (green) and DAPI (blue) served as markers for lytic infection and nuclei, respectively. Infected Kasumi-3 cells were also co-cultured with HFFs. Viral plaque formation in the HFFs (shown by a GFP-positive status) was visualized by fluorescence microscopy. Images were collected using a 40x objective, and representative fields are shown for each infection. Values are shown as the mean ± SEM (n = 3). *, P

    Journal: PLoS Pathogens

    Article Title: Human Cytomegalovirus miR-UL148D Facilitates Latent Viral Infection by Targeting Host Cell Immediate Early Response Gene 5

    doi: 10.1371/journal.ppat.1006007

    Figure Lengend Snippet: Knockout of miR-UL148D impairs the establishment of experimental NR-1 latency in Kasumi-3 cells. (A, C) HCMV genome copies in Kasumi-3 cells (A) and CD34 + HPCs (C) infected with NR-1 or NR-1ΔmiR-UL148D. Total DNA was isolated from the infected cells at various time points after infection, and viral DNA was quantified by qPCR and normalized to cellular GAPDH. (B, D) Representative transcript levels from each class of viral genes in Kasumi-3 cells (B) and HPCs (D) infected with NR-1 or NR-1ΔmiR-UL148D. IE1 (immediately early), UL54 (early lytic transcript), and UL99 (late lytic transcript). Total RNA was isolated from infected cells and assayed by RT-qPCR. Samples were assayed in triplicate, and GAPDH level was used for normalization. (E, F) Restoring miR-UL148D expression via transfection with the miR-UL148D agomir reduced HCMV genome copies (E) and IE1 (F) expression in NR-1ΔmiR-UL148D-infected Kasumi-3 cells. The miR-UL148D agomir was added 24 hours before viral infection, and the culture media was replaced everyday with the addition of fresh agomir. DNA and total RNA were isolated from the Kasumi-3 cells at various time points after infection and quantified by qPCR and RT-qPCR, respectively. Samples were assayed in triplicate, and GAPDH level was used for normalization. Results derived from NR-1-infected Kasumi-3 cells are shown as a control. (G) Both NR-1ΔmiR-UL148D- and NR-1-infected Kasumi-3 cells produced infectious progeny. Infected Kasumi-3 cells harvested 10 days post-infection were stained with a monoclonal antibody against IE1 (clone 1B12, shown in red). GFP (green) and DAPI (blue) served as markers for lytic infection and nuclei, respectively. Infected Kasumi-3 cells were also co-cultured with HFFs. Viral plaque formation in the HFFs (shown by a GFP-positive status) was visualized by fluorescence microscopy. Images were collected using a 40x objective, and representative fields are shown for each infection. Values are shown as the mean ± SEM (n = 3). *, P

    Article Snippet: The RNA samples were treated with DNase using a DNA-free kit (Ambion, Austin, TX) according to the manufacturer’s instructions.

    Techniques: Knock-Out, Infection, Isolation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Transfection, Derivative Assay, Produced, Staining, Cell Culture, Fluorescence, Microscopy

    MiR-UL148D robustly accumulates in CD34 + progenitor cells during the establishment of experimental HCMV latency. (A) Kasumi-3 and CD34 + HPCs were efficiently infected with the NR-1 strain of HCMV. Kasumi-3 and HPCs were either mock-infected or infected with a GFP-expressing NR-1 strain at the indicated multiplicities of infection (MOIs). Two days later, the cells were analyzed for GFP expression by flow cytometry. An MOI of 5 was used for the following experiment. (B) The maintenance of the NR-1 genome, the suppression of viral IE1 and the presence of latency-associated UL138 over a 10-day time course. DNA and total RNA were isolated from Kasumi-3 cells and HPCs at various time points after infection. Viral genomic DNA was assayed by PCR, and RNA molecules encoding IE1 and UL138 were assayed by RT-PCR. In both cases, gel electrophoresis was used to detect the products of the reactions. (C) Reactivation of NR-1 virus in infected Kasumi-3 cells and HPCs. Kasumi-3 cells and HPCs were latently infected along a 10-day time course. Then, a subset of each cell population was cultured for an additional 2 days under conditions favoring lytic reactivation: Kasumi-3 cells were exposed to TPA, while HPCs were grown in reactivation medium. Following this, total RNA was extracted from the cells, and the ratio of IE1 to UL138 cDNA expression was assessed by qRT-PCR in triplicate. (D) Release of infectious progeny virions in latently infected Kasumi-3 cells and HPCs following reactivation treatment. Latently infected or mock-infected Kasumi-3 cells and HPCs were cultured under conditions favoring lytic reactivation (described above) or control conditions for 6 days, after which the cells were washed with PBS and co-cultured with HFFs for 2 days. Then, the Kasumi-3 cells were removed from the co-cultures, and the HFFs were washed with PBS and cultured for an additional 5 days for fluorescence microscopy analysis of GFP-positive plaques. (E) miR-UL148D showed robust accumulation during the establishment of experimental HCMV latency in Kasumi-3 cells. In total, 20,000 infected cells were harvested for the isolation of total RNA and DNA at each indicted time point along the 10-day time course. Viral DNA was first quantified by qPCR, and then absolute viral genomes copies were calculated by generating a standard curve. HCMV miRNAs were then assayed with a HCMV miRNA probe kit, and their levels were calculated using a standard curve. The HCMV miRNA level per virus was calculated by dividing the amount of each HCMV miRNA by the virus copy number. (F) miR-UL148D accumulated in HPCs latently infected with NR-1. HCMV miRNA levels in NR-1-infected HPCs were determined as described above. Values are shown as the mean ± SEM (n = 3). **, P

    Journal: PLoS Pathogens

    Article Title: Human Cytomegalovirus miR-UL148D Facilitates Latent Viral Infection by Targeting Host Cell Immediate Early Response Gene 5

    doi: 10.1371/journal.ppat.1006007

    Figure Lengend Snippet: MiR-UL148D robustly accumulates in CD34 + progenitor cells during the establishment of experimental HCMV latency. (A) Kasumi-3 and CD34 + HPCs were efficiently infected with the NR-1 strain of HCMV. Kasumi-3 and HPCs were either mock-infected or infected with a GFP-expressing NR-1 strain at the indicated multiplicities of infection (MOIs). Two days later, the cells were analyzed for GFP expression by flow cytometry. An MOI of 5 was used for the following experiment. (B) The maintenance of the NR-1 genome, the suppression of viral IE1 and the presence of latency-associated UL138 over a 10-day time course. DNA and total RNA were isolated from Kasumi-3 cells and HPCs at various time points after infection. Viral genomic DNA was assayed by PCR, and RNA molecules encoding IE1 and UL138 were assayed by RT-PCR. In both cases, gel electrophoresis was used to detect the products of the reactions. (C) Reactivation of NR-1 virus in infected Kasumi-3 cells and HPCs. Kasumi-3 cells and HPCs were latently infected along a 10-day time course. Then, a subset of each cell population was cultured for an additional 2 days under conditions favoring lytic reactivation: Kasumi-3 cells were exposed to TPA, while HPCs were grown in reactivation medium. Following this, total RNA was extracted from the cells, and the ratio of IE1 to UL138 cDNA expression was assessed by qRT-PCR in triplicate. (D) Release of infectious progeny virions in latently infected Kasumi-3 cells and HPCs following reactivation treatment. Latently infected or mock-infected Kasumi-3 cells and HPCs were cultured under conditions favoring lytic reactivation (described above) or control conditions for 6 days, after which the cells were washed with PBS and co-cultured with HFFs for 2 days. Then, the Kasumi-3 cells were removed from the co-cultures, and the HFFs were washed with PBS and cultured for an additional 5 days for fluorescence microscopy analysis of GFP-positive plaques. (E) miR-UL148D showed robust accumulation during the establishment of experimental HCMV latency in Kasumi-3 cells. In total, 20,000 infected cells were harvested for the isolation of total RNA and DNA at each indicted time point along the 10-day time course. Viral DNA was first quantified by qPCR, and then absolute viral genomes copies were calculated by generating a standard curve. HCMV miRNAs were then assayed with a HCMV miRNA probe kit, and their levels were calculated using a standard curve. The HCMV miRNA level per virus was calculated by dividing the amount of each HCMV miRNA by the virus copy number. (F) miR-UL148D accumulated in HPCs latently infected with NR-1. HCMV miRNA levels in NR-1-infected HPCs were determined as described above. Values are shown as the mean ± SEM (n = 3). **, P

    Article Snippet: The RNA samples were treated with DNase using a DNA-free kit (Ambion, Austin, TX) according to the manufacturer’s instructions.

    Techniques: Infection, Expressing, Flow Cytometry, Cytometry, Isolation, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Cell Culture, Quantitative RT-PCR, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction

    CDC25 expression is essential for silencing IE1 transcription. (A, B) Stable overexpression of IER5 induced viral lytic gene transcription in NR-1-infected Kasumi-3 cells. IER5 was stably overexpressed in Kasumi-3 cells infected with LV-IER5. An empty-backbone lentivirus was used as a control. The cells were then infected with NR-1 virus for a 10-day time course. The infected cells were analyzed for IER5 and CDC25B protein expression (A) and IE1 (immediately early), UL54 (early lytic transcript), and UL99 (late lytic transcript) mRNA expression (B). (C, D) Stable overexpression of CDC25B suppressed viral lytic gene transcription in NR-1ΔmiR-UL148D-infected Kasumi-3 cells. CDC25B was also stably overexpressed in Kasumi-3 cells infected with LV-CDC25B. An empty-backbone lentivirus was used as a control. The cells were then infected with NR-1 or NR-1ΔmiR-UL148D virus for a 10-day time course. IER5 and CDC25B protein expression (C) and IE1, UL54, and UL99 mRNA expression (D) were then measured in the Kasumi-3 cells. (E) Pharmaceutical inhibition of CDC25B efficiently induced viral lytic gene transcription in NR-1-infected cells. After infection with NR-1, Kasumi-3 cells were incubated with NSC663284, a specific CDC25B inhibitor, at a concentration of 5 μM for 48 hours. Then, the treated cells were further cultured in fresh media for 5 days. The DNA content in the treated cells was analyzed 2 dpi. Total RNA was extracted for RT-qPCR analysis of IE1, UL54 and UL99 expression at the indicated time points along a 7-day time course. Values are shown as the mean ± SEM (n = 3). **, P

    Journal: PLoS Pathogens

    Article Title: Human Cytomegalovirus miR-UL148D Facilitates Latent Viral Infection by Targeting Host Cell Immediate Early Response Gene 5

    doi: 10.1371/journal.ppat.1006007

    Figure Lengend Snippet: CDC25 expression is essential for silencing IE1 transcription. (A, B) Stable overexpression of IER5 induced viral lytic gene transcription in NR-1-infected Kasumi-3 cells. IER5 was stably overexpressed in Kasumi-3 cells infected with LV-IER5. An empty-backbone lentivirus was used as a control. The cells were then infected with NR-1 virus for a 10-day time course. The infected cells were analyzed for IER5 and CDC25B protein expression (A) and IE1 (immediately early), UL54 (early lytic transcript), and UL99 (late lytic transcript) mRNA expression (B). (C, D) Stable overexpression of CDC25B suppressed viral lytic gene transcription in NR-1ΔmiR-UL148D-infected Kasumi-3 cells. CDC25B was also stably overexpressed in Kasumi-3 cells infected with LV-CDC25B. An empty-backbone lentivirus was used as a control. The cells were then infected with NR-1 or NR-1ΔmiR-UL148D virus for a 10-day time course. IER5 and CDC25B protein expression (C) and IE1, UL54, and UL99 mRNA expression (D) were then measured in the Kasumi-3 cells. (E) Pharmaceutical inhibition of CDC25B efficiently induced viral lytic gene transcription in NR-1-infected cells. After infection with NR-1, Kasumi-3 cells were incubated with NSC663284, a specific CDC25B inhibitor, at a concentration of 5 μM for 48 hours. Then, the treated cells were further cultured in fresh media for 5 days. The DNA content in the treated cells was analyzed 2 dpi. Total RNA was extracted for RT-qPCR analysis of IE1, UL54 and UL99 expression at the indicated time points along a 7-day time course. Values are shown as the mean ± SEM (n = 3). **, P

    Article Snippet: The RNA samples were treated with DNase using a DNA-free kit (Ambion, Austin, TX) according to the manufacturer’s instructions.

    Techniques: Expressing, Over Expression, Infection, Stable Transfection, Inhibition, Incubation, Concentration Assay, Cell Culture, Quantitative RT-PCR

    5′ RACE confirms the location of transcription intiation in HeT-A. The two panels show ethidium bromide stained agarose gels of 5′ RACE reactions using the HeT-A outer and inner primers HR1 and HR2. The sources of RNA were Oregon-R adults or third instar larvae, as indicated. Lanes 1 and 2 correspond to the experimental and control lanes, respectively. The migration of DNA standards (in bp) is indicated to the left of each panel. Below the panels is a sequence corresponding to the largest cloned RACE product obtained from adult RNA. Positions 1–92 and 111–137 are 91/92 and 27/27 matches, respectively, to positions 6999–7090 and 7136–7162 in HeT-A 23Zn (accession no. U06920). Filled circles below two bases indicate the previously identified transcription initiation sites ( 18 ). Lines ending in squares indicate the sites corresponding to three RACE products obtained from adult RNA and a line ending in a circle indicates the site corresponding to the major RACE product from L3 RNA.

    Journal: Nucleic Acids Research

    Article Title: Identification of multiple transcription initiation, polyadenylation, and splice sites in the Drosophila melanogaster TART family of telomeric retrotransposons

    doi: 10.1093/nar/gkl709

    Figure Lengend Snippet: 5′ RACE confirms the location of transcription intiation in HeT-A. The two panels show ethidium bromide stained agarose gels of 5′ RACE reactions using the HeT-A outer and inner primers HR1 and HR2. The sources of RNA were Oregon-R adults or third instar larvae, as indicated. Lanes 1 and 2 correspond to the experimental and control lanes, respectively. The migration of DNA standards (in bp) is indicated to the left of each panel. Below the panels is a sequence corresponding to the largest cloned RACE product obtained from adult RNA. Positions 1–92 and 111–137 are 91/92 and 27/27 matches, respectively, to positions 6999–7090 and 7136–7162 in HeT-A 23Zn (accession no. U06920). Filled circles below two bases indicate the previously identified transcription initiation sites ( 18 ). Lines ending in squares indicate the sites corresponding to three RACE products obtained from adult RNA and a line ending in a circle indicates the site corresponding to the major RACE product from L3 RNA.

    Article Snippet: Samples were then heated at 95–96°C for 6 min (to denature any DNA:RNA hybrids) and digested with 2 U of DNase I (DNA-free kit, Ambion) at 37°C for 1 h. DNase I was removed using DNase I inactivation reagent (DNA-free kit, Ambion) following the manufacturer's recommendations.

    Techniques: Staining, Migration, Sequencing, Clone Assay

    Representative 5′ and 3′ RACE reactions. Each panel is an ethidium bromide stained agarose gel of RACE reactions. Above each panel is a 5′ end or 3′ end designation (5a–5e or 3a–3e, respectively) used for discussion purposes. Lanes 1 and 2 are the experimental and control lanes, respectively. The sources of RNA for the reactions shown were Oregon-R adults (5a, 5c, 5d, 3c, 3d and 3e), S2 cells (5b and 3b), Mk-G(II)12 adults (5e) or Mk-G(II)12 third instar larvae (3a). The TART primers used for the reactions shown were as follows (both outer and inner primers are listed for reactions in which two rounds of PCR with nested primers were used): 5a: TR1 + TR2; 5b: TR6 + TR7; 5c: TCR1 + TCR2; 5d: TAB1; 5e: ADR1 + ADR2; 3a: TA53 + TA54; 3b: TA51 + TA52; 3c: TA3; 3d: TR8 + TR9; and 3e: TA31. White boxes indicate products (which in some cases are very faint) that either corresponded to the major 5′ end used for sequence comparisons (5a) or that met our criteria for representing polyadenylation sites (3a, 3c, 3d and 3e), as determined by sequencing of cloned products (see Materials and Methods). The migration of DNA standards (in bp) is indicated to the left of each panel.

    Journal: Nucleic Acids Research

    Article Title: Identification of multiple transcription initiation, polyadenylation, and splice sites in the Drosophila melanogaster TART family of telomeric retrotransposons

    doi: 10.1093/nar/gkl709

    Figure Lengend Snippet: Representative 5′ and 3′ RACE reactions. Each panel is an ethidium bromide stained agarose gel of RACE reactions. Above each panel is a 5′ end or 3′ end designation (5a–5e or 3a–3e, respectively) used for discussion purposes. Lanes 1 and 2 are the experimental and control lanes, respectively. The sources of RNA for the reactions shown were Oregon-R adults (5a, 5c, 5d, 3c, 3d and 3e), S2 cells (5b and 3b), Mk-G(II)12 adults (5e) or Mk-G(II)12 third instar larvae (3a). The TART primers used for the reactions shown were as follows (both outer and inner primers are listed for reactions in which two rounds of PCR with nested primers were used): 5a: TR1 + TR2; 5b: TR6 + TR7; 5c: TCR1 + TCR2; 5d: TAB1; 5e: ADR1 + ADR2; 3a: TA53 + TA54; 3b: TA51 + TA52; 3c: TA3; 3d: TR8 + TR9; and 3e: TA31. White boxes indicate products (which in some cases are very faint) that either corresponded to the major 5′ end used for sequence comparisons (5a) or that met our criteria for representing polyadenylation sites (3a, 3c, 3d and 3e), as determined by sequencing of cloned products (see Materials and Methods). The migration of DNA standards (in bp) is indicated to the left of each panel.

    Article Snippet: Samples were then heated at 95–96°C for 6 min (to denature any DNA:RNA hybrids) and digested with 2 U of DNase I (DNA-free kit, Ambion) at 37°C for 1 h. DNase I was removed using DNase I inactivation reagent (DNA-free kit, Ambion) following the manufacturer's recommendations.

    Techniques: Staining, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing, Clone Assay, Migration

    Aag2-AF10 and Aag2-AF12 cell lines harbour barely detectable levels of phasi charoen-like virus. (A) Short PCR amplicons spanning the three PCLV genome segments (L, M, S) amplified from RNA or genomic DNA isolated from parental Aag2 cells, either with or without a reverse transcription step (RT). Purified nucleic acids were treated with RNase or DNase prior to PCR. MDCK cell RNA, the cellular Rps7 gene/mRNA and the RNA virus NDV, which was spiked into cells immediately prior to RNA extraction, serve as controls. (B) Detection of the PCLV S segment and CFAV by RT-PCR in Aag2-derived clonal cell lines. Cellular Rps7 mRNA serves as a loading control. (C) Detection of the PCLV L, M and S genome (-ssRNA) and antigenome (+ssRNA) segments in select Aag2-derived clonal cell lines by sense-specific RT-PCR. Rps7 mRNA serves as a loading control. (D) PCLV L segment RT-qPCR ΔΔC t (normalised to Rps7 mRNA) for select Aag2-derived clonal cell lines expressed relative to parental Aag2 cell line at (i) early passages (Aag2-AF10, passage 2; Aag2-AF12, passage 3) and (ii) later (‘medium’) passages (Aag2-AF10, passage 8; Aag2-AF12, passage 12). (E) RT-qPCR quantification of viral RNA copies for PCLV L segment (i) and CFAV (ii) at late passages in Aag2 (parental), Aag2-AF5, Aag2-AF10 and Aag2-AF10 cells. Starting passages (“~P15”) were passage 15 (parental Aag2), 12 (Aag2-AF5), 14 (Aag2-AF10) and 13 (Aag2-AF12). Data points represent three independently passaged lines. Error bars represent standard deviation. ** P

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Aedes aegypti (Aag2)-derived clonal mosquito cell lines reveal the effects of pre-existing persistent infection with the insect-specific bunyavirus Phasi Charoen-like virus on arbovirus replication

    doi: 10.1371/journal.pntd.0007346

    Figure Lengend Snippet: Aag2-AF10 and Aag2-AF12 cell lines harbour barely detectable levels of phasi charoen-like virus. (A) Short PCR amplicons spanning the three PCLV genome segments (L, M, S) amplified from RNA or genomic DNA isolated from parental Aag2 cells, either with or without a reverse transcription step (RT). Purified nucleic acids were treated with RNase or DNase prior to PCR. MDCK cell RNA, the cellular Rps7 gene/mRNA and the RNA virus NDV, which was spiked into cells immediately prior to RNA extraction, serve as controls. (B) Detection of the PCLV S segment and CFAV by RT-PCR in Aag2-derived clonal cell lines. Cellular Rps7 mRNA serves as a loading control. (C) Detection of the PCLV L, M and S genome (-ssRNA) and antigenome (+ssRNA) segments in select Aag2-derived clonal cell lines by sense-specific RT-PCR. Rps7 mRNA serves as a loading control. (D) PCLV L segment RT-qPCR ΔΔC t (normalised to Rps7 mRNA) for select Aag2-derived clonal cell lines expressed relative to parental Aag2 cell line at (i) early passages (Aag2-AF10, passage 2; Aag2-AF12, passage 3) and (ii) later (‘medium’) passages (Aag2-AF10, passage 8; Aag2-AF12, passage 12). (E) RT-qPCR quantification of viral RNA copies for PCLV L segment (i) and CFAV (ii) at late passages in Aag2 (parental), Aag2-AF5, Aag2-AF10 and Aag2-AF10 cells. Starting passages (“~P15”) were passage 15 (parental Aag2), 12 (Aag2-AF5), 14 (Aag2-AF10) and 13 (Aag2-AF12). Data points represent three independently passaged lines. Error bars represent standard deviation. ** P

    Article Snippet: Nucleic acids were treated with DNase for 40 min at 37°C using the DNA-free DNA Removal Kit (ThermoFisher Scientific) or with RNase A (Sigma-Aldrich) for 1 h at 37°C as per manufacturers’ instructions.

    Techniques: Polymerase Chain Reaction, Amplification, Isolation, Purification, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Quantitative RT-PCR, Standard Deviation

    Comparison of the expression profiles of selected gene groups in biofilm cells and planktonic cells. Cells were grown as described in the text, and total RNA was extracted from the cells at the five times indicated and used in DNA microarray analyses.

    Journal:

    Article Title: Differential Gene Expression Profiling of Staphylococcus aureus Cultivated under Biofilm and Planktonic Conditions

    doi: 10.1128/AEM.71.5.2663-2676.2005

    Figure Lengend Snippet: Comparison of the expression profiles of selected gene groups in biofilm cells and planktonic cells. Cells were grown as described in the text, and total RNA was extracted from the cells at the five times indicated and used in DNA microarray analyses.

    Article Snippet: Contaminating DNA in the RNA preparations was removed using “DNA-free” (Ambion).

    Techniques: Expressing, Microarray

    The clp genes are expressed during RB growth. (A) Gene maps of the clp genes in the Chlamydia trachomatis ). The clp genes are circled. The clpP2 gene is ct706 . (B to E) Temporal expression of the clp genes. Quantitative reverse transcription-PCR (RT-qPCR) analysis of the clp genes from two independent time course experiments of a C. trachomatis serovar L2 infection of HEp2 cells was performed. Total RNA and DNA were collected at the indicated times postinfection and processed as described in Materials and Methods. Equivalent amounts of cDNA were used for each assay and analyzed in triplicate. Results are reported as a ratio of cDNA to genomic DNA (gDNA). Standard deviations for each were typically less than 10% of the sample. Note that some transcripts were not detectable at 1 hpi. Western blotting was performed on whole-cell lysates of total protein from a time course of Chlamydia sp.-infected cells, separated by SDS-PAGE, and transferred to a nitrocellulose membrane for blotting. All four of the genes analyzed appear to be expressed mid-developmental cycle. (F) Major outer membrane protein (MOMP) was blotted as a control for chlamydial development over the course of infection. Ab, antibody.

    Journal: Journal of Bacteriology

    Article Title: Initial Characterization of the Two ClpP Paralogs of Chlamydia trachomatis Suggests Unique Functionality for Each

    doi: 10.1128/JB.00635-18

    Figure Lengend Snippet: The clp genes are expressed during RB growth. (A) Gene maps of the clp genes in the Chlamydia trachomatis ). The clp genes are circled. The clpP2 gene is ct706 . (B to E) Temporal expression of the clp genes. Quantitative reverse transcription-PCR (RT-qPCR) analysis of the clp genes from two independent time course experiments of a C. trachomatis serovar L2 infection of HEp2 cells was performed. Total RNA and DNA were collected at the indicated times postinfection and processed as described in Materials and Methods. Equivalent amounts of cDNA were used for each assay and analyzed in triplicate. Results are reported as a ratio of cDNA to genomic DNA (gDNA). Standard deviations for each were typically less than 10% of the sample. Note that some transcripts were not detectable at 1 hpi. Western blotting was performed on whole-cell lysates of total protein from a time course of Chlamydia sp.-infected cells, separated by SDS-PAGE, and transferred to a nitrocellulose membrane for blotting. All four of the genes analyzed appear to be expressed mid-developmental cycle. (F) Major outer membrane protein (MOMP) was blotted as a control for chlamydial development over the course of infection. Ab, antibody.

    Article Snippet: DNA was removed from total RNA by rigorous DNA-free treatment (Thermo Fisher) before 1 µg was reverse transcribed with Superscript III reverse transcriptase (RT; Thermo Fisher).

    Techniques: Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Western Blot, SDS Page

    RT-PCR analysis of mutant and wild-type strains. RNA was extracted from cells grown under nitrogen-fixing conditions in AA/8 with 1 μm molybdate and 1 μm vanadate. Specific transcripts were detected with primers designed to amplify part of the genes shown: nif1 genes for the Mo nitrogenase, vnf genes for the V nitrogenase, a vup gene for the V transport system (which is repressed by Mo), and rnpB for a constitutive gene. Lane 1, BP227-43 (point mutation restoring growth of nifH1 mutant); lane 2, BP227-44 (point mutation restoring growth of nifH1 mutant; probable Mo transport mutant); lane 3, BP227-4 ( nifH1 insertion mutation); lane 4, FD (wild-type); lane 5, chromosomal DNA from FD. (Not shown, negative controls verifying that no DNA contaminated the RNA extracts).

    Journal: Journal of Bacteriology

    Article Title: Cross-Functionality of Nitrogenase Components NifH1 and VnfH in Anabaena variabilis

    doi: 10.1128/JB.00618-06

    Figure Lengend Snippet: RT-PCR analysis of mutant and wild-type strains. RNA was extracted from cells grown under nitrogen-fixing conditions in AA/8 with 1 μm molybdate and 1 μm vanadate. Specific transcripts were detected with primers designed to amplify part of the genes shown: nif1 genes for the Mo nitrogenase, vnf genes for the V nitrogenase, a vup gene for the V transport system (which is repressed by Mo), and rnpB for a constitutive gene. Lane 1, BP227-43 (point mutation restoring growth of nifH1 mutant); lane 2, BP227-44 (point mutation restoring growth of nifH1 mutant; probable Mo transport mutant); lane 3, BP227-4 ( nifH1 insertion mutation); lane 4, FD (wild-type); lane 5, chromosomal DNA from FD. (Not shown, negative controls verifying that no DNA contaminated the RNA extracts).

    Article Snippet: The RNA was further treated to remove DNA by the Turbo DNA-free procedure (Ambion).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Mutagenesis

    A nonsense mutation (in-frame stop codon) was present in the Eh ntr1 gene of genome project strain HM-1:IMSS of Entamoeba and three clinical isolates. (A) The nitroreductases of Entamoeba strains HM-1:IMSS and 200:NIH, as well as those Methanothermobacter thermautotrophicus (Mt) and Archaeoglobus fulgidus (Af), were aligned by using the single-letter code. In this alignment, identical amino acids are marked with an asterisk, while similar amino acids are marked with a colon or a period. Within a putative zero-frame 81-bp intron of EhNTR1 of genome project strain HM-1:IMSS, there was a stop codon (#) where there was an Arg (R) in the wild-type EhNTR1 of 200:NIH. The stop codon, which was also present in the Eh ntr1 genes of 3 of 22 clinical isolates examined (isolates BM1, CM2, and H22), was present in a region that is conserved in bacterial nitroreductases. (B) PCR and RT-PCR with Eh ntr1 gene primers from HM-1:IMSS DNA and RNA, respectively, produced products of the same size, arguing against the presence of an in-frame intron in the Eh ntr1 gene. In contrast, the product obtained by RT-PCR with Eh ntr3 gene primers from HM-1:IMSS RNA was smaller than the product obtained by PCR from DNA, consistent with the presence of an intron in the Eh ntr3 gene of Entamoeba . The sequence of the Eh ntr3 RT-PCR product confirmed the presence of the intron at the position predicted by the genome project (data not shown). (C) Western blotting with a rabbit polyclonal antibody to a recombinant EhNTR1 protein showed that strains 200:NIH and Rahman, which had the wild-type Eh ntr1 gene, both expressed the NTR1 protein. In contrast, strain HM-1:IMSS, which had a nonsense mutation in the Eh ntr1 gene, did not express the NTR1 protein.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Giardia, Entamoeba, and Trichomonas Enzymes Activate Metronidazole (Nitroreductases) and Inactivate Metronidazole (Nitroimidazole Reductases) ▿ Enzymes Activate Metronidazole (Nitroreductases) and Inactivate Metronidazole (Nitroimidazole Reductases) ▿ †

    doi: 10.1128/AAC.00909-08

    Figure Lengend Snippet: A nonsense mutation (in-frame stop codon) was present in the Eh ntr1 gene of genome project strain HM-1:IMSS of Entamoeba and three clinical isolates. (A) The nitroreductases of Entamoeba strains HM-1:IMSS and 200:NIH, as well as those Methanothermobacter thermautotrophicus (Mt) and Archaeoglobus fulgidus (Af), were aligned by using the single-letter code. In this alignment, identical amino acids are marked with an asterisk, while similar amino acids are marked with a colon or a period. Within a putative zero-frame 81-bp intron of EhNTR1 of genome project strain HM-1:IMSS, there was a stop codon (#) where there was an Arg (R) in the wild-type EhNTR1 of 200:NIH. The stop codon, which was also present in the Eh ntr1 genes of 3 of 22 clinical isolates examined (isolates BM1, CM2, and H22), was present in a region that is conserved in bacterial nitroreductases. (B) PCR and RT-PCR with Eh ntr1 gene primers from HM-1:IMSS DNA and RNA, respectively, produced products of the same size, arguing against the presence of an in-frame intron in the Eh ntr1 gene. In contrast, the product obtained by RT-PCR with Eh ntr3 gene primers from HM-1:IMSS RNA was smaller than the product obtained by PCR from DNA, consistent with the presence of an intron in the Eh ntr3 gene of Entamoeba . The sequence of the Eh ntr3 RT-PCR product confirmed the presence of the intron at the position predicted by the genome project (data not shown). (C) Western blotting with a rabbit polyclonal antibody to a recombinant EhNTR1 protein showed that strains 200:NIH and Rahman, which had the wild-type Eh ntr1 gene, both expressed the NTR1 protein. In contrast, strain HM-1:IMSS, which had a nonsense mutation in the Eh ntr1 gene, did not express the NTR1 protein.

    Article Snippet: Total RNA was isolated from mid-log-phase Entamoeba and Trichomonas with the Trizol reagent (Invitrogen), and the RNA was treated with a DNA-free reagent (Ambion), according to the manufacturer's instructions.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Produced, Sequencing, Western Blot, Recombinant

    Absolute and relative (malignant/normal) values of biochemical parameters in normal and malignant tissues of liver, kidney, colon, and lung. A. and B. RNA content. C. and D. DNA content. Data are expressed as in Figure 1 .

    Journal: PLoS ONE

    Article Title: Biological Stoichiometry in Human Cancer

    doi: 10.1371/journal.pone.0001028

    Figure Lengend Snippet: Absolute and relative (malignant/normal) values of biochemical parameters in normal and malignant tissues of liver, kidney, colon, and lung. A. and B. RNA content. C. and D. DNA content. Data are expressed as in Figure 1 .

    Article Snippet: The final RNA product was treated with RNAse-free DNAse using DNA-free reagents (Ambion).

    Techniques:

    Sodium butyrate stimulates mitochondrial biogenesis in PC12-ND6 cells by increasing mt-DNA copy number and expression levels of key regulators of mt-DNA replication. a Representative live-cell confocal micrograph of PicoGreen staining of mt-nucleoids in control and NaBt-treated PC12-ND6 cells transfected with the mito-RFP vector. Scale bar = 5 µm. b Quantification of Mt-DNA copy number by qPCR from control and HDACi-treated PC12-ND6 cells and normalized by ΔΔ C (t). Mean ± SEM ( n = 3; * p

    Journal: Cell Death & Disease

    Article Title: Epigenetic modifiers promote mitochondrial biogenesis and oxidative metabolism leading to enhanced differentiation of neuroprogenitor cells

    doi: 10.1038/s41419-018-0396-1

    Figure Lengend Snippet: Sodium butyrate stimulates mitochondrial biogenesis in PC12-ND6 cells by increasing mt-DNA copy number and expression levels of key regulators of mt-DNA replication. a Representative live-cell confocal micrograph of PicoGreen staining of mt-nucleoids in control and NaBt-treated PC12-ND6 cells transfected with the mito-RFP vector. Scale bar = 5 µm. b Quantification of Mt-DNA copy number by qPCR from control and HDACi-treated PC12-ND6 cells and normalized by ΔΔ C (t). Mean ± SEM ( n = 3; * p

    Article Snippet: Untreated and treated PC12-ND6 cells were incubated in 1 ml of serum-free medium containing 3 µl of the fluorescent DNA dye PicoGreen® (Invitrogen) for 1 h at 37 °C.

    Techniques: Expressing, Staining, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction

    DNA and RNA analysis.

    Journal: Journal of Virology

    Article Title: Murine Cytomegalovirus Protein pM92 Is a Conserved Regulator of Viral Late Gene Expression

    doi: 10.1128/JVI.02684-13

    Figure Lengend Snippet: DNA and RNA analysis.

    Article Snippet: Total RNA was extracted by TRIzol reagent (Invitrogen) and treated with Turbo DNA-free reagents (Ambion) to remove contaminating DNA.

    Techniques:

    Analysis of externally regulated expression of transgenic KGF RNA and protein in the lung. Two lines of transgene (Tg)-positive animals were treated with Dox or control solution (5% sucrose) for 2 days. Mice were killed and a part of the lung was used for RNA isolation and another part for protein isolation. ( A ) DNA-free RNA was used for RT-PCR with appropriate primers. In the absence of the external inducer (Dox − ), animals from neither line expressed RNA corresponding to the KGF Tg but expressed transgenic RNA after induction (Dox + ). ( B ) To detect KGF protein expression derived from the Tg, lungs were homogenized in Triton X-100 containing buffer, and the pelleted fraction was solubilized in hot Laemmli buffer and subjected to Western blot analysis. We were unable to detect KGF protein in the Triton X-100-soluble extract, which is in keeping with the propensity of KGF and other members of the fibroblast growth factor family to attach to matrix components. In the Laemmli buffer extract, KGF protein from the Tg could be identified within 48 h of Tg induction. Arrow indicates the KGF protein derived from the Tg that migrates similarly with the standard (recombinant KGF). The faint band in the lane containing extract prepared from the lungs of uninduced animals and non-Tg animals probably corresponds to low levels of endogenous KGF. The level of KGF expression per mouse was estimated to be between 5 and 10 ng. Tg expression in the two lines was confirmed at both RNA and protein levels in two independent experiments using five animals per line per group (±Dox). Shown are data representative of five animals in each group. We have not performed long-term experiments to determine effects of very low, undetectable leaky expression of Tg, if any.

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

    Article Title: Inducible expression of keratinocyte growth factor (KGF) in mice inhibits lung epithelial cell death induced by hyperoxia

    doi: 10.1073/pnas.1031851100

    Figure Lengend Snippet: Analysis of externally regulated expression of transgenic KGF RNA and protein in the lung. Two lines of transgene (Tg)-positive animals were treated with Dox or control solution (5% sucrose) for 2 days. Mice were killed and a part of the lung was used for RNA isolation and another part for protein isolation. ( A ) DNA-free RNA was used for RT-PCR with appropriate primers. In the absence of the external inducer (Dox − ), animals from neither line expressed RNA corresponding to the KGF Tg but expressed transgenic RNA after induction (Dox + ). ( B ) To detect KGF protein expression derived from the Tg, lungs were homogenized in Triton X-100 containing buffer, and the pelleted fraction was solubilized in hot Laemmli buffer and subjected to Western blot analysis. We were unable to detect KGF protein in the Triton X-100-soluble extract, which is in keeping with the propensity of KGF and other members of the fibroblast growth factor family to attach to matrix components. In the Laemmli buffer extract, KGF protein from the Tg could be identified within 48 h of Tg induction. Arrow indicates the KGF protein derived from the Tg that migrates similarly with the standard (recombinant KGF). The faint band in the lane containing extract prepared from the lungs of uninduced animals and non-Tg animals probably corresponds to low levels of endogenous KGF. The level of KGF expression per mouse was estimated to be between 5 and 10 ng. Tg expression in the two lines was confirmed at both RNA and protein levels in two independent experiments using five animals per line per group (±Dox). Shown are data representative of five animals in each group. We have not performed long-term experiments to determine effects of very low, undetectable leaky expression of Tg, if any.

    Article Snippet: Reverse transcription was performed with 3 μg of DNA-free RNA (Superscript II, Life Technologies) according to manufacturer's protocol using the supplied random hexamers.

    Techniques: Expressing, Transgenic Assay, Mouse Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Western Blot, Recombinant

    FRG1 regulates the stability of the Rbfox1 mRNA. (a) ChIP of FRG1 in the promoter region of the Rbfox1 gene. (b) The distribution of RNA pol II on C2C12- EV and C2C12- FRG1 cells in the promoter region of the Rbfox1 gene. (c) Real-time RT-PCR of the kinetics of Rbfox1 , c-Myc and Gapdh expression after 8 hours of ActD (Actinomycin D) treatment on C2C12- EV and C2C12- FRG1 cells. (d) RNA-IP experiment on samples from (c) using anti-FRG1 antibodies or control IgG antibodies. Immunoprecipitated material was analyzed by real-time RT-PCR, normalized versus the relative input and plotted as fold enrichment versus the IgG. RT-minus control experiments showed the absence of DNA contamination (data not shown).

    Journal: PLoS Genetics

    Article Title: Rbfox1 Downregulation and Altered Calpain 3 Splicing by FRG1 in a Mouse Model of Facioscapulohumeral Muscular Dystrophy (FSHD)

    doi: 10.1371/journal.pgen.1003186

    Figure Lengend Snippet: FRG1 regulates the stability of the Rbfox1 mRNA. (a) ChIP of FRG1 in the promoter region of the Rbfox1 gene. (b) The distribution of RNA pol II on C2C12- EV and C2C12- FRG1 cells in the promoter region of the Rbfox1 gene. (c) Real-time RT-PCR of the kinetics of Rbfox1 , c-Myc and Gapdh expression after 8 hours of ActD (Actinomycin D) treatment on C2C12- EV and C2C12- FRG1 cells. (d) RNA-IP experiment on samples from (c) using anti-FRG1 antibodies or control IgG antibodies. Immunoprecipitated material was analyzed by real-time RT-PCR, normalized versus the relative input and plotted as fold enrichment versus the IgG. RT-minus control experiments showed the absence of DNA contamination (data not shown).

    Article Snippet: Concentration, DNA-free RNA purity and integrity was determined by Nanodrop and by Agilent 2100 Bioanalyzer.

    Techniques: Chromatin Immunoprecipitation, Quantitative RT-PCR, Expressing, Immunoprecipitation

    DNase I footprinting analysis of PdxR binding to the mutant pdxRS regulatory regions pdxRSp1 (A), pdxRSp2 (B, D), and pdxRSp4 (C, D) DNA fragments, radioactively labeled on the bottom (A, B, C) or top (D) strand, were incubated with increasing concentrations of purified PdxR and in the absence or presence of 100 μM PLP. PdxR monomer concentrations used (nM) are indicated below each lane. The corresponding A + G sequencing ladder is shown in the left and right lanes. The protected areas are indicated by vertical lines.

    Journal: Molecular microbiology

    Article Title: Role of PdxR in the activation of vitamin B6 biosynthesis in Listeria monocytogenes

    doi: 10.1111/mmi.12618

    Figure Lengend Snippet: DNase I footprinting analysis of PdxR binding to the mutant pdxRS regulatory regions pdxRSp1 (A), pdxRSp2 (B, D), and pdxRSp4 (C, D) DNA fragments, radioactively labeled on the bottom (A, B, C) or top (D) strand, were incubated with increasing concentrations of purified PdxR and in the absence or presence of 100 μM PLP. PdxR monomer concentrations used (nM) are indicated below each lane. The corresponding A + G sequencing ladder is shown in the left and right lanes. The protected areas are indicated by vertical lines.

    Article Snippet: For real-time RT-PCR experiments, purified RNA (~10 μg) was further treated with Turbo DNA- free DNase I (Ambion) according to the manufacturer’s instructions.

    Techniques: Footprinting, Binding Assay, Mutagenesis, Labeling, Incubation, Purification, Plasmid Purification, Sequencing