nicotinamide adenine dinucleotide nad  (New England Biolabs)


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    Name:
    beta Nicotinamide adenine dinucleotide NAD
    Description:
    beta Nicotinamide adenine dinucleotide NAD 0 2 ml
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    b9007s
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    New England Biolabs nicotinamide adenine dinucleotide nad
    beta Nicotinamide adenine dinucleotide NAD
    beta Nicotinamide adenine dinucleotide NAD 0 2 ml
    https://www.bioz.com/result/nicotinamide adenine dinucleotide nad/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nicotinamide adenine dinucleotide nad - by Bioz Stars, 2020-09
    96/100 stars

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    1) Product Images from "Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions"

    Article Title: Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw998

    ATP-hydrolysis by RecA under the conditions for the ligation enhancement. Radioactive [α- 33 P]ATP (1.3 mM) was incubated with the indicated amounts of RecA for 30 min with RecA in the complete system, except for the amounts of DNA and the absence of DNA ligase and NAD. After the reaction, the amounts of hydrolyzed ATP were analyzed. The averages of more than three independent experiments were plotted with standard deviations. The small extent of ATP-hydrolysis observed in the absence of DNA was probably caused by an impurity in the RecA preparations used in these experiments, since the values varied from 0 to a few nmol among RecA preparations. Closed symbols, reactions in the basic reaction mixture (with 3.8% (w/v) PEG), open symbols, omit PEG. •, ∘ (circles), linear dsDNA with 5′ four nucleotide overhangs generated by Hin dIII; ▪, □ (squares), linear dsDNA with 3′ four nucleotide overhangs generated by Pst I; ▴, Δ (triangles), linear dsDNA with blunt ends generated by Hin cII; ♦, ◊ (diamonds), ssDNA; ▿ (reversed triangles), ssDNA without PEG and in the presence of 13 mM MgCl 2 (the standard reaction mixtures for homologous joint formation by RecA: ( 31 )); ▹ (sideways triangles), without DNA (with 1.3 mM MgCl 2 , without PEG).
    Figure Legend Snippet: ATP-hydrolysis by RecA under the conditions for the ligation enhancement. Radioactive [α- 33 P]ATP (1.3 mM) was incubated with the indicated amounts of RecA for 30 min with RecA in the complete system, except for the amounts of DNA and the absence of DNA ligase and NAD. After the reaction, the amounts of hydrolyzed ATP were analyzed. The averages of more than three independent experiments were plotted with standard deviations. The small extent of ATP-hydrolysis observed in the absence of DNA was probably caused by an impurity in the RecA preparations used in these experiments, since the values varied from 0 to a few nmol among RecA preparations. Closed symbols, reactions in the basic reaction mixture (with 3.8% (w/v) PEG), open symbols, omit PEG. •, ∘ (circles), linear dsDNA with 5′ four nucleotide overhangs generated by Hin dIII; ▪, □ (squares), linear dsDNA with 3′ four nucleotide overhangs generated by Pst I; ▴, Δ (triangles), linear dsDNA with blunt ends generated by Hin cII; ♦, ◊ (diamonds), ssDNA; ▿ (reversed triangles), ssDNA without PEG and in the presence of 13 mM MgCl 2 (the standard reaction mixtures for homologous joint formation by RecA: ( 31 )); ▹ (sideways triangles), without DNA (with 1.3 mM MgCl 2 , without PEG).

    Techniques Used: Ligation, Incubation, Generated

    Related Articles

    Concentration Assay:

    Article Title: High-Throughput Generation of In Silico Derived Synthetic Antibodies via One-step Enzymatic DNA Assembly of Fragments.
    Article Snippet: .. Phage-display technology offers robust methods for isolating antibody (Ab) molecules with specificity for different target antigens. .. Phage-display technology offers robust methods for isolating antibody (Ab) molecules with specificity for different target antigens.

    Incubation:

    Article Title: A Serine-Threonine Kinase (StkP) Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells In Vitro
    Article Snippet: .. Finally 2μl of 1mM NAD+ (β-Nicotinamide adenine dinucleotide, NEB) and 4μl E .coli DNA ligase (NEB) was added, incubated at 16°C overnight and then stored at 4°C until transformation. ..

    other:

    Article Title: Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions
    Article Snippet: Basic reaction mixture The basic reaction mixture contained 30 mM Tris-HCl (pH 7.5), 7 mM MgCl2 , 3.8% (w/v) polyethylene glycol 6000 (PEG), 0.1 mM nicotinamide adenine dinucleotide (NAD), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1.8 mM dithiothreitol (DTT) and 88 μg/ml bovine serum albumin ((BSA); New England Biolabs and Roche, molecular biology grade).

    Polymerase Chain Reaction:

    Article Title: Genetically controlled membrane synthesis in liposomes
    Article Snippet: .. 100 ng of backbone and an equimolar amount of the egfp-lactC2 PCR fragment were mixed in a solution containing 100 mM Tris-HCl, 50 mM MgCl2 , 0.2 mM each dNTP, 10 mM dithiothreitol (DTT), 5% w/v PEG-8000, 1 mM nicotinamide adenine dinucleotide, 5.33 U mL−1 T5 Exonuclease, 33.3 U mL−1 Phusion polymerase and 5.33 U mL−1 Taq-ligase in a final volume of 20 µL. .. The assembly reaction was incubated at 50 °C for 60 min. Then, 20 U µL−1 of DpnI restriction enzyme (New England Biolabs, USA) were added to digest possible methylated DNA left and the mixture was incubated for an additional 15 min at 37 °C.

    Transformation Assay:

    Article Title: A Serine-Threonine Kinase (StkP) Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells In Vitro
    Article Snippet: .. Finally 2μl of 1mM NAD+ (β-Nicotinamide adenine dinucleotide, NEB) and 4μl E .coli DNA ligase (NEB) was added, incubated at 16°C overnight and then stored at 4°C until transformation. ..

    Crocin Bleaching Assay:

    Article Title: Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions
    Article Snippet: .. To make the linear cassette, 25 fmoles of each fragment were combined in a 20 ul isothermal assembly reaction with 2× CBA isothermal assembly mastermix (320 μl of 5 × ISO Reaction Buffer [25% polyethylene glycol 8000, 500 mM tris-HCl (pH 8.0), 50 mM MgCl2 , 50 mM dithiothreitol (DTT), 1.0 μM each dNTPs, 5 μM nicotinamide adenine dinucleotide, 6.4 μl of T5 Exonuclease (1 U/ml) (Epicentre), 20 μl of Phusion polymerase (2 U/ml) [New England Biolabs (NEB)], 80 μl of Taq ligase (40 U/ml) (NEB), and 374 μl of deionized H2 O . .. The cassette was amplified using 1ul of the isothermal assembly reaction, with primers 15165 and 15169 (0.4 μM) in a PCR reaction with 2× Phusion HF mastermix, (100 ul, 25 cycle, 60 °C melting temperature, 6 minute extension) and purified using the Qiagen Qiaquick PCR purification kit.

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    New England Biolabs dpn ii
    Enrichment of 3T3-HOX11 DPs by <t>cDNA</t> RDA. (A) Ethidium-stained agarose gel electrophoresis of starting cDNA <t>Dpn</t> II fragment representations and various RDA enriched populations obtained by mutual subtraction of NIH 3T3 cells (3T3) with 3T3-HOX11 (left lanes show products obtained with NIH 3T3 cells as the tester and right lanes show products obtained with 3T3-HOX11 cells as the tester). Tester representation (R; unselected cDNA) and DP1, DP2, DP3, and DP4 are shown. The arrow indicates an enriched HOX11 Dpn II fragment identified by Southern filter hybridization with a HOX11 -specific probe. (B) Southern filter hybridizations (with Slim1 , Aldh1 , HOX11 , and β- actin probes as indicated) of the agarose gel-fractionated RDA DPs shown in panel A. Fragment sizes are indicated. (C) Northern filter hybridization of 10 μg of total RNA prepared from untransfected NIH 3T3 cells and three independent 3T3-HOX11 clones and hybridized with HOX11 , Slim1 , Aldh1 , and ATP synthase probes (as indicated). 3T3-HOX11 clones 5 and 18 were found to express HOX11 protein, while clone 11 did not (data not shown). Hybridization of the filter with ATP synthase was used to assess the quality of the RNA transferred.
    Dpn Ii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nad rna
    <t>NAD</t> + -capped RNAs are present in plant tissues/organs. ( A ) Quantitation of NAD + /NADH levels in <t>RNA.</t> ( A , Left ) NAD + /NADH concentrations in negative (5′ pppRNA) and positive (5′ NAD + -RNA) controls that were generated through in vitro transcription. ( A , Right ) NAD + /NADH concentrations in cellular RNAs from 12-d-old seedlings and inflorescences. Concentrations of NAD + /NADH were calculated by dividing the detected value of NAD + /NADH by the amount of RNAs analyzed. Three independent replicates were performed. Error bars represent SD. **** P
    Nad Rna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dpn i
    Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with <t>Dpn</t> I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).
    Dpn I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enrichment of 3T3-HOX11 DPs by cDNA RDA. (A) Ethidium-stained agarose gel electrophoresis of starting cDNA Dpn II fragment representations and various RDA enriched populations obtained by mutual subtraction of NIH 3T3 cells (3T3) with 3T3-HOX11 (left lanes show products obtained with NIH 3T3 cells as the tester and right lanes show products obtained with 3T3-HOX11 cells as the tester). Tester representation (R; unselected cDNA) and DP1, DP2, DP3, and DP4 are shown. The arrow indicates an enriched HOX11 Dpn II fragment identified by Southern filter hybridization with a HOX11 -specific probe. (B) Southern filter hybridizations (with Slim1 , Aldh1 , HOX11 , and β- actin probes as indicated) of the agarose gel-fractionated RDA DPs shown in panel A. Fragment sizes are indicated. (C) Northern filter hybridization of 10 μg of total RNA prepared from untransfected NIH 3T3 cells and three independent 3T3-HOX11 clones and hybridized with HOX11 , Slim1 , Aldh1 , and ATP synthase probes (as indicated). 3T3-HOX11 clones 5 and 18 were found to express HOX11 protein, while clone 11 did not (data not shown). Hybridization of the filter with ATP synthase was used to assess the quality of the RNA transferred.

    Journal: Molecular and Cellular Biology

    Article Title: The T-Cell Oncogenic Protein HOX11 Activates Aldh1 Expression in NIH 3T3 Cells but Represses Its Expression in Mouse Spleen Development

    doi:

    Figure Lengend Snippet: Enrichment of 3T3-HOX11 DPs by cDNA RDA. (A) Ethidium-stained agarose gel electrophoresis of starting cDNA Dpn II fragment representations and various RDA enriched populations obtained by mutual subtraction of NIH 3T3 cells (3T3) with 3T3-HOX11 (left lanes show products obtained with NIH 3T3 cells as the tester and right lanes show products obtained with 3T3-HOX11 cells as the tester). Tester representation (R; unselected cDNA) and DP1, DP2, DP3, and DP4 are shown. The arrow indicates an enriched HOX11 Dpn II fragment identified by Southern filter hybridization with a HOX11 -specific probe. (B) Southern filter hybridizations (with Slim1 , Aldh1 , HOX11 , and β- actin probes as indicated) of the agarose gel-fractionated RDA DPs shown in panel A. Fragment sizes are indicated. (C) Northern filter hybridization of 10 μg of total RNA prepared from untransfected NIH 3T3 cells and three independent 3T3-HOX11 clones and hybridized with HOX11 , Slim1 , Aldh1 , and ATP synthase probes (as indicated). 3T3-HOX11 clones 5 and 18 were found to express HOX11 protein, while clone 11 did not (data not shown). Hybridization of the filter with ATP synthase was used to assess the quality of the RNA transferred.

    Article Snippet: Double-stranded cDNA (approximately 2 μg) was digested with Dpn II (NEB), extracted with phenol-chloroform, and ethanol precipitated.

    Techniques: Staining, Agarose Gel Electrophoresis, Hybridization, Northern Blot, Clone Assay

    NAD + -capped RNAs are present in plant tissues/organs. ( A ) Quantitation of NAD + /NADH levels in RNA. ( A , Left ) NAD + /NADH concentrations in negative (5′ pppRNA) and positive (5′ NAD + -RNA) controls that were generated through in vitro transcription. ( A , Right ) NAD + /NADH concentrations in cellular RNAs from 12-d-old seedlings and inflorescences. Concentrations of NAD + /NADH were calculated by dividing the detected value of NAD + /NADH by the amount of RNAs analyzed. Three independent replicates were performed. Error bars represent SD. **** P

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

    Article Title: NAD+-capped RNAs are widespread in the Arabidopsis transcriptome and can probably be translated

    doi: 10.1073/pnas.1903682116

    Figure Lengend Snippet: NAD + -capped RNAs are present in plant tissues/organs. ( A ) Quantitation of NAD + /NADH levels in RNA. ( A , Left ) NAD + /NADH concentrations in negative (5′ pppRNA) and positive (5′ NAD + -RNA) controls that were generated through in vitro transcription. ( A , Right ) NAD + /NADH concentrations in cellular RNAs from 12-d-old seedlings and inflorescences. Concentrations of NAD + /NADH were calculated by dividing the detected value of NAD + /NADH by the amount of RNAs analyzed. Three independent replicates were performed. Error bars represent SD. **** P

    Article Snippet: In vitro transcription reactions contained 5 µg of dsDNA templates; 1X T7 polymerase buffer (New England Biolabs); 10 mM cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP); 10 mM NAD+ (for 5′ NAD+ -RNA) or 10 mM ATP (for 5′ pppRNA); and T7 RNAP (New England Biolabs) and were allowed to proceed at 37 °C overnight.

    Techniques: Quantitation Assay, Generated, In Vitro

    NAD-RNAs are poly(A) RNAs. ( A ) RNA gel blot assay to detect NAD-RNAs and non–NAD-RNAs derived from RBCS1B/2B/3B genes. NAD-RNAs (labeled as “N”) were isolated by NAD capture with streptavidin beads, while RNAs in the supernatant were considered as non–NAD-RNAs (indicated as “n”). One-fifth of non–NAD-RNAs in the supernatant were resolved in an agarose gel together with all captured NAD-RNAs. The intensities of bands were measured by ImageJ. The bar plot on the Right shows the relative signal intensities (N vs. n) from 5 independent replicates (after correcting for differences in loading). Error bars represent SD. Dots indicate the values of the 5 replicates. ( B ) Quantification of transcript levels in ADPRC+ vs. ADPRC− samples from seedlings by qRT-PCR. From left to right are 6 nuclear genes, 6 mitochondrial genes, 2 chloroplast genes, 2 rRNAs, and 2 snoRNAs. ( C ) Detection of NAD + -capped poly(A) RNAs by qRT-PCR. After NAD capture from total RNAs, NAD-RNAs were eluted from beads, reverse transcribed by either oligo (dT) (“OligodT ADPRC+/−”) or random primers (“Random ADPRC+/−”), and then detected with gene-specific primers. For B and C , qRT-PCR values were normalized to those of the ADPRC− samples, which were arbitrarily set to 1. Two biological replicates were performed, each with 3 technical replicates. Error bars represent SD. ( D ) Comparison of signal intensity (ADPRC+/ADPRC− ratio) in NAD captureSeq from total RNAs and that from poly(A) RNAs.

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

    Article Title: NAD+-capped RNAs are widespread in the Arabidopsis transcriptome and can probably be translated

    doi: 10.1073/pnas.1903682116

    Figure Lengend Snippet: NAD-RNAs are poly(A) RNAs. ( A ) RNA gel blot assay to detect NAD-RNAs and non–NAD-RNAs derived from RBCS1B/2B/3B genes. NAD-RNAs (labeled as “N”) were isolated by NAD capture with streptavidin beads, while RNAs in the supernatant were considered as non–NAD-RNAs (indicated as “n”). One-fifth of non–NAD-RNAs in the supernatant were resolved in an agarose gel together with all captured NAD-RNAs. The intensities of bands were measured by ImageJ. The bar plot on the Right shows the relative signal intensities (N vs. n) from 5 independent replicates (after correcting for differences in loading). Error bars represent SD. Dots indicate the values of the 5 replicates. ( B ) Quantification of transcript levels in ADPRC+ vs. ADPRC− samples from seedlings by qRT-PCR. From left to right are 6 nuclear genes, 6 mitochondrial genes, 2 chloroplast genes, 2 rRNAs, and 2 snoRNAs. ( C ) Detection of NAD + -capped poly(A) RNAs by qRT-PCR. After NAD capture from total RNAs, NAD-RNAs were eluted from beads, reverse transcribed by either oligo (dT) (“OligodT ADPRC+/−”) or random primers (“Random ADPRC+/−”), and then detected with gene-specific primers. For B and C , qRT-PCR values were normalized to those of the ADPRC− samples, which were arbitrarily set to 1. Two biological replicates were performed, each with 3 technical replicates. Error bars represent SD. ( D ) Comparison of signal intensity (ADPRC+/ADPRC− ratio) in NAD captureSeq from total RNAs and that from poly(A) RNAs.

    Article Snippet: In vitro transcription reactions contained 5 µg of dsDNA templates; 1X T7 polymerase buffer (New England Biolabs); 10 mM cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP); 10 mM NAD+ (for 5′ NAD+ -RNA) or 10 mM ATP (for 5′ pppRNA); and T7 RNAP (New England Biolabs) and were allowed to proceed at 37 °C overnight.

    Techniques: Western Blot, Derivative Assay, Labeling, Isolation, Agarose Gel Electrophoresis, Quantitative RT-PCR

    Puromycin treatment leads to the dissociation of NAD-RNAs from polysomes. ( A ) RNA gel blot assays showing the enrichment of NAD-RNAs in the polysomal fraction. NAD capture was performed with total RNAs or polysomal RNAs, and NAD-RNAs (on beads; labeled as “N”) and non–NAD-RNAs (in supernatant; labeled as “n”; one-fifth of supernatant RNAs were loaded) were resolved in a gel. Hybridization was performed with a probe common to RBCS1B/2B/3B genes. Signal intensity of the bands was measured by ImageJ. Levels of non–NAD-RNAs in total extract and polysomes were arbitrarily set as 1, and the relative abundance of NAD-RNAs is indicated by the numbers. The numbers reflected the N/n ratio without correction for the differences in loading. Note that the “ADPRC−” samples (lanes 4 and 7) had little background, which demonstrated the specificity of NAD capture. Two biological replicates were performed and gave similar results. ( B ) Quantification of transcript levels in ADPRC+ vs. ADPRC− samples by qRT-PCR. NAD capture was performed with total or polysomal RNAs. qRT-PCR values were normalized to those of the ADPRC− samples, which were arbitrarily set to 1. Two biological replicates, each with 3 technical replicates, were performed. Error bars represent SD. *** P

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

    Article Title: NAD+-capped RNAs are widespread in the Arabidopsis transcriptome and can probably be translated

    doi: 10.1073/pnas.1903682116

    Figure Lengend Snippet: Puromycin treatment leads to the dissociation of NAD-RNAs from polysomes. ( A ) RNA gel blot assays showing the enrichment of NAD-RNAs in the polysomal fraction. NAD capture was performed with total RNAs or polysomal RNAs, and NAD-RNAs (on beads; labeled as “N”) and non–NAD-RNAs (in supernatant; labeled as “n”; one-fifth of supernatant RNAs were loaded) were resolved in a gel. Hybridization was performed with a probe common to RBCS1B/2B/3B genes. Signal intensity of the bands was measured by ImageJ. Levels of non–NAD-RNAs in total extract and polysomes were arbitrarily set as 1, and the relative abundance of NAD-RNAs is indicated by the numbers. The numbers reflected the N/n ratio without correction for the differences in loading. Note that the “ADPRC−” samples (lanes 4 and 7) had little background, which demonstrated the specificity of NAD capture. Two biological replicates were performed and gave similar results. ( B ) Quantification of transcript levels in ADPRC+ vs. ADPRC− samples by qRT-PCR. NAD capture was performed with total or polysomal RNAs. qRT-PCR values were normalized to those of the ADPRC− samples, which were arbitrarily set to 1. Two biological replicates, each with 3 technical replicates, were performed. Error bars represent SD. *** P

    Article Snippet: In vitro transcription reactions contained 5 µg of dsDNA templates; 1X T7 polymerase buffer (New England Biolabs); 10 mM cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP); 10 mM NAD+ (for 5′ NAD+ -RNA) or 10 mM ATP (for 5′ pppRNA); and T7 RNAP (New England Biolabs) and were allowed to proceed at 37 °C overnight.

    Techniques: Western Blot, Labeling, Hybridization, Quantitative RT-PCR

    NAD-RNAs are enriched on polysomes. ( A and B ) Scatter plots showing transcript abundance in ADPRC+ vs. ADPRC− samples in NAD captureSeq from total RNAs ( A ) and polysomal RNAs ( B ). Green dots indicate NAD-RNAs; black dots indicate non–NAD-RNAs. “All” represents all expressed genes; “NAD” represents genes producing NAD-RNAs. ( C ) Comparison of signal/noise ratios (ADPRC+/ADPRC− ratios) between total RNA and polysomal RNA NAD captureSeq. Genes that were found to produce NAD-RNAs in either total RNAs or polysomal RNAs were included. ( D ) Comparison of mRNA abundance between total RNA and polysomal RNA samples by mRNA-seq. Blue dots represent PEGs, and green dots show PDGs. ( E and F ) Comparison of signal/noise ratios (ADPRC+/ADPRC− ratios) between total RNA and polysomal RNA NAD captureSeq for PEGs ( E ) and PDGs ( F ).

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

    Article Title: NAD+-capped RNAs are widespread in the Arabidopsis transcriptome and can probably be translated

    doi: 10.1073/pnas.1903682116

    Figure Lengend Snippet: NAD-RNAs are enriched on polysomes. ( A and B ) Scatter plots showing transcript abundance in ADPRC+ vs. ADPRC− samples in NAD captureSeq from total RNAs ( A ) and polysomal RNAs ( B ). Green dots indicate NAD-RNAs; black dots indicate non–NAD-RNAs. “All” represents all expressed genes; “NAD” represents genes producing NAD-RNAs. ( C ) Comparison of signal/noise ratios (ADPRC+/ADPRC− ratios) between total RNA and polysomal RNA NAD captureSeq. Genes that were found to produce NAD-RNAs in either total RNAs or polysomal RNAs were included. ( D ) Comparison of mRNA abundance between total RNA and polysomal RNA samples by mRNA-seq. Blue dots represent PEGs, and green dots show PDGs. ( E and F ) Comparison of signal/noise ratios (ADPRC+/ADPRC− ratios) between total RNA and polysomal RNA NAD captureSeq for PEGs ( E ) and PDGs ( F ).

    Article Snippet: In vitro transcription reactions contained 5 µg of dsDNA templates; 1X T7 polymerase buffer (New England Biolabs); 10 mM cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP); 10 mM NAD+ (for 5′ NAD+ -RNA) or 10 mM ATP (for 5′ pppRNA); and T7 RNAP (New England Biolabs) and were allowed to proceed at 37 °C overnight.

    Techniques:

    Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with Dpn I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).

    Journal: Frontiers in Public Health

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    doi: 10.3389/fpubh.2016.00131

    Figure Lengend Snippet: Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with Dpn I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).

    Article Snippet: Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites.

    Techniques: Mutagenesis, Methylation, Quantitative RT-PCR, Expressing, Marker, Negative Control

    Phenotypic influence of Dam on P fimbriae . (A) PCR screening for pap EF in UPEC strains cC119 (lane 4), CFT073 (lane 5), and cU155 (lane 6). The 100-bp molecular weight marker (Invitrogen), negative control and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 1 and 2, respectively. (B) PCR screening for pap I– pap B intergenic regulatory region in UPEC strains from UPEC strains cC119 (lane 2), CFT073 (lane 3), and cU155 (lane 4). The 1-kb plus molecular marker (Invitrogen, CA, USA), negative control, and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 2 and 5, respectively. (C) Schematic representation of pSAMS1 recombinant plasmid containing cloned pap IB insert within pCRII–TOPOII vector. (D) Dam methylation patterns for pap I-B regulatory region. Sau 3AI (lane 2), Mbo I (lane 3), and Dpn I (lane 4) digests of pSAMS2 isolated from cC119 are shown. MW represents the 1 kb Plus molecular marker (Invitrogen). An undigested pap IB fragment (lane 5) is also represented. (E) Semi-quantitative (sq) RT-PCR for pap I expression in cC119 (lane 1), cC119 Δ dam (lane 2), CFT073 wild-type (lane 3) and CFT073 Δ dam (lane 4). The 1 kb Plus molecular marker (Invitrogen) and amplified chromosomal DNA for UPEC strains cC119 and CFT073 are shown in lanes MW, 5 and 6, respectively.

    Journal: Frontiers in Public Health

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    doi: 10.3389/fpubh.2016.00131

    Figure Lengend Snippet: Phenotypic influence of Dam on P fimbriae . (A) PCR screening for pap EF in UPEC strains cC119 (lane 4), CFT073 (lane 5), and cU155 (lane 6). The 100-bp molecular weight marker (Invitrogen), negative control and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 1 and 2, respectively. (B) PCR screening for pap I– pap B intergenic regulatory region in UPEC strains from UPEC strains cC119 (lane 2), CFT073 (lane 3), and cU155 (lane 4). The 1-kb plus molecular marker (Invitrogen, CA, USA), negative control, and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 2 and 5, respectively. (C) Schematic representation of pSAMS1 recombinant plasmid containing cloned pap IB insert within pCRII–TOPOII vector. (D) Dam methylation patterns for pap I-B regulatory region. Sau 3AI (lane 2), Mbo I (lane 3), and Dpn I (lane 4) digests of pSAMS2 isolated from cC119 are shown. MW represents the 1 kb Plus molecular marker (Invitrogen). An undigested pap IB fragment (lane 5) is also represented. (E) Semi-quantitative (sq) RT-PCR for pap I expression in cC119 (lane 1), cC119 Δ dam (lane 2), CFT073 wild-type (lane 3) and CFT073 Δ dam (lane 4). The 1 kb Plus molecular marker (Invitrogen) and amplified chromosomal DNA for UPEC strains cC119 and CFT073 are shown in lanes MW, 5 and 6, respectively.

    Article Snippet: Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites.

    Techniques: Polymerase Chain Reaction, Molecular Weight, Marker, Negative Control, Positive Control, Recombinant, Plasmid Preparation, Clone Assay, Methylation, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification

    Genotypic and growth characteristics displayed by parental and dam- mutant strains of UPEC . (A) Schematic diagram of gene disruption strategy for chromosomal insertion of chloramphenicol resistance gene from pKD3 into dam gene within UPEC chromosome subsequent to λ red recombineering with pKM208. (B) Amplified dam fragment from wild type UPEC strains CFT073 (lane 1) and cured parental strains C119 (lane 2) to produce 1071 bp amplicon. MW is 1 kb DNA ladder (Bioneer Corporation, Republic of Korea) and −ve is negative control. (C) PCR screening of UPEC candidates for dam mutation observed as 1323 bp products using primers UR427 and UR428. MW is a 1 kb Plus DNA ladder (Invitrogen, USA). (D) Dam methylation pattern in UPEC CFT073 wild type (lanes 1, 2, 8, 9, 14, 15), C119 wild type (lanes 3, 4, 10, 11, 16, 17), and E. coli K-12 substrain MG1655 (5, 12, 18) strains subsequent to digestion with Mbo I, Sau 3AI, and Dpn I. The negative control (7, 13, 19) and 1 kb Plus DNA ladder (MW) are also shown. (E) Dam methylation pattern in UPEC dam mutants CFT073 (lanes 1, 2, 3, 8, 9, 10, 15, 16, 17) and C119 wild-type (lanes 4, 5, 6, 11, 12, 13, 18, 19) subsequent to digestion with Sau 3AI, Mbo I, and Dpn I. The negative control (lanes 7, 14) and 1 kb Plus DNA ladder (MW) are also shown. (F) Growth curve (CFU/milliliter versus time) for UPEC strains CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam .

    Journal: Frontiers in Public Health

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    doi: 10.3389/fpubh.2016.00131

    Figure Lengend Snippet: Genotypic and growth characteristics displayed by parental and dam- mutant strains of UPEC . (A) Schematic diagram of gene disruption strategy for chromosomal insertion of chloramphenicol resistance gene from pKD3 into dam gene within UPEC chromosome subsequent to λ red recombineering with pKM208. (B) Amplified dam fragment from wild type UPEC strains CFT073 (lane 1) and cured parental strains C119 (lane 2) to produce 1071 bp amplicon. MW is 1 kb DNA ladder (Bioneer Corporation, Republic of Korea) and −ve is negative control. (C) PCR screening of UPEC candidates for dam mutation observed as 1323 bp products using primers UR427 and UR428. MW is a 1 kb Plus DNA ladder (Invitrogen, USA). (D) Dam methylation pattern in UPEC CFT073 wild type (lanes 1, 2, 8, 9, 14, 15), C119 wild type (lanes 3, 4, 10, 11, 16, 17), and E. coli K-12 substrain MG1655 (5, 12, 18) strains subsequent to digestion with Mbo I, Sau 3AI, and Dpn I. The negative control (7, 13, 19) and 1 kb Plus DNA ladder (MW) are also shown. (E) Dam methylation pattern in UPEC dam mutants CFT073 (lanes 1, 2, 3, 8, 9, 10, 15, 16, 17) and C119 wild-type (lanes 4, 5, 6, 11, 12, 13, 18, 19) subsequent to digestion with Sau 3AI, Mbo I, and Dpn I. The negative control (lanes 7, 14) and 1 kb Plus DNA ladder (MW) are also shown. (F) Growth curve (CFU/milliliter versus time) for UPEC strains CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam .

    Article Snippet: Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites.

    Techniques: Mutagenesis, Amplification, Negative Control, Polymerase Chain Reaction, Methylation