rna  (Worthington Biochemical)


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    Name:
    Ribonucleic Acid
    Description:
    Primarily ribosomal RNA Suitable substrate for ribonuclease assays
    Catalog Number:
    LS003451
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    Baker's Yeast
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    Cas Number:
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    Structured Review

    Worthington Biochemical rna
    Mechanism of FXN activation by repeat-targeted duplex RNAs. ( a ) RIP examining the association of Ago2 with FXN pre-mRNA after treatment with 50 nM duplex <t>RNA</t> and analysis by real-time <t>PCR.</t> An arrow marks the PCR product of FXN pre-mRNA, which was confirmed by sequencing ( Supplementary Fig. 7 ) ( b ) Anti-GAA duplex RNA with central mismatches (siGAA 9,10 mm with mismatches on both strands; 25 nM) activates FXN expression at a level similar to the analogous fully complementary duplex RNA ( n =3). siExon3 is a positive control for transfection efficiency targeting exon 3 of FXN . ( c ) ChIP for RNAP2 using four different primer sets ( n =4). ( d ) ChIP for transcription-associated histone modification markers H3K4me3, H3K9me2, H3K9me3, H3K9Ac, H3K27me3 and H4Ac ( n =4–8). ( e ) FXN mRNA stability assay. Cells were transfected with duplex RNAs siGAA or CM at 25 nM ( n =3). Actinomycin D (5 μg ml −1 ) was added with fresh media 3 days after transfection and cells were collected at the indicated time points. HPRT expression was measured for normalization. All experiments were performed in GM03816 patient-derived cells. All data are presented as mean±STDEV. * P
    Primarily ribosomal RNA Suitable substrate for ribonuclease assays
    https://www.bioz.com/result/rna/product/Worthington Biochemical
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rna - by Bioz Stars, 2021-04
    96/100 stars

    Images

    1) Product Images from "Activating frataxin expression by repeat-targeted nucleic acids"

    Article Title: Activating frataxin expression by repeat-targeted nucleic acids

    Journal: Nature Communications

    doi: 10.1038/ncomms10606

    Mechanism of FXN activation by repeat-targeted duplex RNAs. ( a ) RIP examining the association of Ago2 with FXN pre-mRNA after treatment with 50 nM duplex RNA and analysis by real-time PCR. An arrow marks the PCR product of FXN pre-mRNA, which was confirmed by sequencing ( Supplementary Fig. 7 ) ( b ) Anti-GAA duplex RNA with central mismatches (siGAA 9,10 mm with mismatches on both strands; 25 nM) activates FXN expression at a level similar to the analogous fully complementary duplex RNA ( n =3). siExon3 is a positive control for transfection efficiency targeting exon 3 of FXN . ( c ) ChIP for RNAP2 using four different primer sets ( n =4). ( d ) ChIP for transcription-associated histone modification markers H3K4me3, H3K9me2, H3K9me3, H3K9Ac, H3K27me3 and H4Ac ( n =4–8). ( e ) FXN mRNA stability assay. Cells were transfected with duplex RNAs siGAA or CM at 25 nM ( n =3). Actinomycin D (5 μg ml −1 ) was added with fresh media 3 days after transfection and cells were collected at the indicated time points. HPRT expression was measured for normalization. All experiments were performed in GM03816 patient-derived cells. All data are presented as mean±STDEV. * P
    Figure Legend Snippet: Mechanism of FXN activation by repeat-targeted duplex RNAs. ( a ) RIP examining the association of Ago2 with FXN pre-mRNA after treatment with 50 nM duplex RNA and analysis by real-time PCR. An arrow marks the PCR product of FXN pre-mRNA, which was confirmed by sequencing ( Supplementary Fig. 7 ) ( b ) Anti-GAA duplex RNA with central mismatches (siGAA 9,10 mm with mismatches on both strands; 25 nM) activates FXN expression at a level similar to the analogous fully complementary duplex RNA ( n =3). siExon3 is a positive control for transfection efficiency targeting exon 3 of FXN . ( c ) ChIP for RNAP2 using four different primer sets ( n =4). ( d ) ChIP for transcription-associated histone modification markers H3K4me3, H3K9me2, H3K9me3, H3K9Ac, H3K27me3 and H4Ac ( n =4–8). ( e ) FXN mRNA stability assay. Cells were transfected with duplex RNAs siGAA or CM at 25 nM ( n =3). Actinomycin D (5 μg ml −1 ) was added with fresh media 3 days after transfection and cells were collected at the indicated time points. HPRT expression was measured for normalization. All experiments were performed in GM03816 patient-derived cells. All data are presented as mean±STDEV. * P

    Techniques Used: Activation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Expressing, Positive Control, Transfection, Chromatin Immunoprecipitation, Modification, Stability Assay, Derivative Assay

    2) Product Images from "Recognition of 5?-terminal TAR structure in human immunodeficiency virus-1 mRNA by eukaryotic translation initiation factor 2"

    Article Title: Recognition of 5?-terminal TAR structure in human immunodeficiency virus-1 mRNA by eukaryotic translation initiation factor 2

    Journal: Nucleic Acids Research

    doi:

    Role of TAR and the AUG initiation codon context in binding of eIF2. Uniformly 32 P-labeled 77 nt TAR T7 transcript (0.08 pmol, 1.25 × 10 5 c.p.m./pmol) was incubated without eIF2 or with 0.12 pmol of eIF2, in the absence or presence of 0.25–2 pmol of unlabeled TAR, TAR–LUC or LUC T7 RNA transcript as shown schematically in ( C ). Electrophoresis on a native gel was used to separate free TAR RNA from complex. The autoradiogram shows free and bound RNA ( A ). Bound RNA is quantitated in ( B ).
    Figure Legend Snippet: Role of TAR and the AUG initiation codon context in binding of eIF2. Uniformly 32 P-labeled 77 nt TAR T7 transcript (0.08 pmol, 1.25 × 10 5 c.p.m./pmol) was incubated without eIF2 or with 0.12 pmol of eIF2, in the absence or presence of 0.25–2 pmol of unlabeled TAR, TAR–LUC or LUC T7 RNA transcript as shown schematically in ( C ). Electrophoresis on a native gel was used to separate free TAR RNA from complex. The autoradiogram shows free and bound RNA ( A ). Bound RNA is quantitated in ( B ).

    Techniques Used: Binding Assay, Labeling, Incubation, Electrophoresis

    Complex formation between HIV-1 TAR and eIF2. ( A ) Uniformly 32 P-labeled 77 nt T7 transcript (0.08 pmol, 1.25 × 10 5 c.p.m./pmol) containing TAR abutted by plasmid-derived nucleotides underlined in ( C ) was incubated without eIF2 or with 0.07, 0.2 or 0.3 pmol of eIF2. The reaction mixture was subjected to electrophoresis on a native gel to separate free TAR RNA from complexes C1 and C2. The autoradiogram is shown. ( B ) In a separate experiment, TAR RNA (0.1 pmol) and, where shown, 0.25 pmol of eIF2 were incubated in the absence or presence of unlabeled TAR RNA (0.5, 1, 2 or 4 pmol) (TAR competitor). Bovine serum albumin (BSA) served as protein control. Analysis was done as in (A).
    Figure Legend Snippet: Complex formation between HIV-1 TAR and eIF2. ( A ) Uniformly 32 P-labeled 77 nt T7 transcript (0.08 pmol, 1.25 × 10 5 c.p.m./pmol) containing TAR abutted by plasmid-derived nucleotides underlined in ( C ) was incubated without eIF2 or with 0.07, 0.2 or 0.3 pmol of eIF2. The reaction mixture was subjected to electrophoresis on a native gel to separate free TAR RNA from complexes C1 and C2. The autoradiogram is shown. ( B ) In a separate experiment, TAR RNA (0.1 pmol) and, where shown, 0.25 pmol of eIF2 were incubated in the absence or presence of unlabeled TAR RNA (0.5, 1, 2 or 4 pmol) (TAR competitor). Bovine serum albumin (BSA) served as protein control. Analysis was done as in (A).

    Techniques Used: Labeling, Plasmid Preparation, Derivative Assay, Incubation, Electrophoresis

    HIV-1 TAR and the AUG initiation codon each contribute to affinity for eIF2. ( A ) Uniformly 32 P-labeled human IFN-γ mRNA 5′-terminal 203 nt transcript (0.08 pmol, 1.25 × 10 5 c.p.m./pmol) was incubated without eIF2 or with 0.3 pmol of eIF2, in the absence or presence of unlabeled HIV-1 T7 transcripts (0.1–1 pmol) (comp RNA) as shown schematically in (C). The autoradiogram shows free and bound RNA (A). Bound RNA is quantitated in ( B ). Relative affinities for eIF2 are summarized in ( C ).
    Figure Legend Snippet: HIV-1 TAR and the AUG initiation codon each contribute to affinity for eIF2. ( A ) Uniformly 32 P-labeled human IFN-γ mRNA 5′-terminal 203 nt transcript (0.08 pmol, 1.25 × 10 5 c.p.m./pmol) was incubated without eIF2 or with 0.3 pmol of eIF2, in the absence or presence of unlabeled HIV-1 T7 transcripts (0.1–1 pmol) (comp RNA) as shown schematically in (C). The autoradiogram shows free and bound RNA (A). Bound RNA is quantitated in ( B ). Relative affinities for eIF2 are summarized in ( C ).

    Techniques Used: Labeling, Incubation

    A 3 nt change in TAR strongly reduces its affinity for eIF2. Uniformly 32 P-labeled 77 nt TAR T7 transcript (0.08 pmol, 1.25 × 10 5 c.p.m./pmol) was incubated without eIF2 or with 0.14 pmol of eIF2, in the absence or presence of 0.1–4 pmol of wild-type (wt) or mutant TAR T7 RNA transcript shown schematically in ( D ) (mutations are denoted in bold). Electrophoresis on a native gel was used to separate free TAR RNA from complexes C1 and C2 ( A ). C2 and C1 are quantitated in ( B ) and ( C ), respectively. In ( E ), activation of PKR by the indicated concentrations of dsRNA, wt or M5 TAR transcript was assayed by phosphorylation of the PKR (68 kDa) and eIF2α (38 kDa) bands. The autoradiogram is shown.
    Figure Legend Snippet: A 3 nt change in TAR strongly reduces its affinity for eIF2. Uniformly 32 P-labeled 77 nt TAR T7 transcript (0.08 pmol, 1.25 × 10 5 c.p.m./pmol) was incubated without eIF2 or with 0.14 pmol of eIF2, in the absence or presence of 0.1–4 pmol of wild-type (wt) or mutant TAR T7 RNA transcript shown schematically in ( D ) (mutations are denoted in bold). Electrophoresis on a native gel was used to separate free TAR RNA from complexes C1 and C2 ( A ). C2 and C1 are quantitated in ( B ) and ( C ), respectively. In ( E ), activation of PKR by the indicated concentrations of dsRNA, wt or M5 TAR transcript was assayed by phosphorylation of the PKR (68 kDa) and eIF2α (38 kDa) bands. The autoradiogram is shown.

    Techniques Used: Labeling, Incubation, Mutagenesis, Electrophoresis, Activation Assay

    Effect of lower stem mutations in TAR on affinity for eIF2. Uniformly 32 P-labeled human IFN-γ mRNA 5′-terminal 203 nt transcript (0.08 pmol, 1.25 × 10 5 c.p.m./pmol) was incubated without eIF2 or with 0.09 pmol of eIF2, in the absence or presence of 0.01–1 pmol of unlabeled 80 nt wild-type TAR SP6 transcript (TAR) or mutant forms as shown schematically in ( C ). The autoradiogram of a native gel shows free and bound RNA ( A ). Bound RNA is quantitated in ( B ).
    Figure Legend Snippet: Effect of lower stem mutations in TAR on affinity for eIF2. Uniformly 32 P-labeled human IFN-γ mRNA 5′-terminal 203 nt transcript (0.08 pmol, 1.25 × 10 5 c.p.m./pmol) was incubated without eIF2 or with 0.09 pmol of eIF2, in the absence or presence of 0.01–1 pmol of unlabeled 80 nt wild-type TAR SP6 transcript (TAR) or mutant forms as shown schematically in ( C ). The autoradiogram of a native gel shows free and bound RNA ( A ). Bound RNA is quantitated in ( B ).

    Techniques Used: Labeling, Incubation, Mutagenesis

    3) Product Images from "The A946T variant IFIH1 RNA sensor mediates an interferon program that limits viral infection but increases the risk for autoimmunity"

    Article Title: The A946T variant IFIH1 RNA sensor mediates an interferon program that limits viral infection but increases the risk for autoimmunity

    Journal: Nature immunology

    doi: 10.1038/ni.3766

    mIFIH1 R mediates increased sensitivity to self-RNA ligands (a-e) Control or ADAR- null HEK293T cells were generated by lenti-CRISPR technology. Cell lines were transfected with 1 μg of mIFIH1 risk or non-risk constructs shown in Fig 2 . Cells were analyzed by flow cytometry at 27 hours post-transfection for (a) geometric mean fluorescent intensity (MFI) and (b) percent GFP expression and combined data from four biological replicates are shown. (c-e) Quantitative RT-PCR for IFNB1 mRNA expression in control vs. ADAR- null cells transfected with mIFIH1 NR construct. (c) Representative data from one experiment. (d) Combined data from four biological replicates. (e) Relative levels of IFNB1 mRNA expression in mIFIH1 NR vs. mIFIH1 R transfected ADAR- null cells showing combined data from four biological replicates. Results were normalized by the Livak method as in Fig 2d . Statistical analysis performed using a two-tail student T test. Each data point represents one biological replicate. Error bars represent ± SEM. *p
    Figure Legend Snippet: mIFIH1 R mediates increased sensitivity to self-RNA ligands (a-e) Control or ADAR- null HEK293T cells were generated by lenti-CRISPR technology. Cell lines were transfected with 1 μg of mIFIH1 risk or non-risk constructs shown in Fig 2 . Cells were analyzed by flow cytometry at 27 hours post-transfection for (a) geometric mean fluorescent intensity (MFI) and (b) percent GFP expression and combined data from four biological replicates are shown. (c-e) Quantitative RT-PCR for IFNB1 mRNA expression in control vs. ADAR- null cells transfected with mIFIH1 NR construct. (c) Representative data from one experiment. (d) Combined data from four biological replicates. (e) Relative levels of IFNB1 mRNA expression in mIFIH1 NR vs. mIFIH1 R transfected ADAR- null cells showing combined data from four biological replicates. Results were normalized by the Livak method as in Fig 2d . Statistical analysis performed using a two-tail student T test. Each data point represents one biological replicate. Error bars represent ± SEM. *p

    Techniques Used: Generated, CRISPR, Transfection, Construct, Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: The A946T variant IFIH1 RNA sensor mediates an interferon program that limits viral infection but increases the risk for autoimmunity
    Article Snippet: Analyses of mouse tissues Murine serum was tested by ELISA for reactivity to calf thymus dsDNA (Sigma-Aldrich), and sm-RNP (ATR1-10; Arotech Diagnostics Limited) which has previously been described . .. To obtain RNA samples for quantitative PCR, murine spleens were digested with collagenase Type 4 (Worthington Biochemical), followed by calcium chelation, RBC lysis and made into single cell suspensions. .. Insulin was detected by immunohistochemistry in paraffin embedded pancreas tissue sections using a Leica Bond MAX Automated Immunostainer (Leica Microsystems) and guinea pig anti-insulin (Dako-A0564) primary antibody with rabbit anti-guinea pig (Abcam) and goat anti-rabbit poly-HRP (Leica Microsystems) secondary antibodies.

    Lysis:

    Article Title: The A946T variant IFIH1 RNA sensor mediates an interferon program that limits viral infection but increases the risk for autoimmunity
    Article Snippet: Analyses of mouse tissues Murine serum was tested by ELISA for reactivity to calf thymus dsDNA (Sigma-Aldrich), and sm-RNP (ATR1-10; Arotech Diagnostics Limited) which has previously been described . .. To obtain RNA samples for quantitative PCR, murine spleens were digested with collagenase Type 4 (Worthington Biochemical), followed by calcium chelation, RBC lysis and made into single cell suspensions. .. Insulin was detected by immunohistochemistry in paraffin embedded pancreas tissue sections using a Leica Bond MAX Automated Immunostainer (Leica Microsystems) and guinea pig anti-insulin (Dako-A0564) primary antibody with rabbit anti-guinea pig (Abcam) and goat anti-rabbit poly-HRP (Leica Microsystems) secondary antibodies.

    Mutagenesis:

    Article Title: Role for Bovine Viral Diarrhea Virus Erns Glycoprotein in the Control of Activation of Beta Interferon by Double-Stranded RNA
    Article Snippet: The glycoproteins in cell culture medium were further incubated at 37°C for 18 h, and the RNase activity was determined as described previously ( ) with slight modification. .. Briefly, the assay mixture (25 μl in total) contained 0.2 μg of wild-type or mutant Erns , 12.5 μg of yeast 16-23S RNA (Worthington), and 40 U of RNase inhibitor (RNasin; Promega) in 20 mM sodium acetate buffer (pH 4.5). .. The mixture was incubated at 37°C for 45 min, and the enzyme reaction was stopped by acid precipitation with 5 μl of 25% (vol/vol) HClO4 containing 0.75% (wt/vol) uranyl acetate.

    Labeling:

    Article Title: Recognition of 5?-terminal TAR structure in human immunodeficiency virus-1 mRNA by eukaryotic translation initiation factor 2
    Article Snippet: 5′-End-labeled 77 nt TAR T7 transcript was generated from unlabeled transcript (60 pmol) by dephosphorylation with calf alkaline phosphatase, incubation with 70 µCi of [γ-32 P]ATP (3 Ci/µmol) and T4 polynucleotide kinase, 6% polyacrylamide/ 8 M urea gel electrophoresis and elution of labeled product. .. Labeled RNA (0.3 pmol) was incubated without or with eIF2 (0.12 pmol) in binding buffer for 15 min at 30°C followed by incubation for 10 min on ice and then digested for 30 min at 30°C with 0.01 U of RNase T1 (Worthington), phenol extracted and ethanol precipitated. .. The RNA was dissolved in loading buffer and analyzed on an 8% polyacrylamide sequencing gel.

    Incubation:

    Article Title: Recognition of 5?-terminal TAR structure in human immunodeficiency virus-1 mRNA by eukaryotic translation initiation factor 2
    Article Snippet: 5′-End-labeled 77 nt TAR T7 transcript was generated from unlabeled transcript (60 pmol) by dephosphorylation with calf alkaline phosphatase, incubation with 70 µCi of [γ-32 P]ATP (3 Ci/µmol) and T4 polynucleotide kinase, 6% polyacrylamide/ 8 M urea gel electrophoresis and elution of labeled product. .. Labeled RNA (0.3 pmol) was incubated without or with eIF2 (0.12 pmol) in binding buffer for 15 min at 30°C followed by incubation for 10 min on ice and then digested for 30 min at 30°C with 0.01 U of RNase T1 (Worthington), phenol extracted and ethanol precipitated. .. The RNA was dissolved in loading buffer and analyzed on an 8% polyacrylamide sequencing gel.

    Binding Assay:

    Article Title: Recognition of 5?-terminal TAR structure in human immunodeficiency virus-1 mRNA by eukaryotic translation initiation factor 2
    Article Snippet: 5′-End-labeled 77 nt TAR T7 transcript was generated from unlabeled transcript (60 pmol) by dephosphorylation with calf alkaline phosphatase, incubation with 70 µCi of [γ-32 P]ATP (3 Ci/µmol) and T4 polynucleotide kinase, 6% polyacrylamide/ 8 M urea gel electrophoresis and elution of labeled product. .. Labeled RNA (0.3 pmol) was incubated without or with eIF2 (0.12 pmol) in binding buffer for 15 min at 30°C followed by incubation for 10 min on ice and then digested for 30 min at 30°C with 0.01 U of RNase T1 (Worthington), phenol extracted and ethanol precipitated. .. The RNA was dissolved in loading buffer and analyzed on an 8% polyacrylamide sequencing gel.

    Positive Control:

    Article Title: Contrasting Sex-and Caste-Dependent piRNA Profiles in the Transposon Depleted Haplodiploid Honeybee Apis mellifera
    Article Snippet: .. Enriched small RNA samples, as well as synthetic piRNAs (positive control, , online), were enzymatically digested with RNase T2 (#LS01501, Worthington Biochemical Corporation). ..

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    • rna  (Worthington Biochemical)
    96
    Worthington Biochemical rna
    mIFIH1 R mediates increased sensitivity to <t>self-RNA</t> ligands (a-e) Control or ADAR- null HEK293T cells were generated by lenti-CRISPR technology. Cell lines were transfected with 1 μg of mIFIH1 risk or non-risk constructs shown in Fig 2 . Cells were analyzed by flow cytometry at 27 hours post-transfection for (a) geometric mean fluorescent intensity (MFI) and (b) percent GFP expression and combined data from four biological replicates are shown. (c-e) Quantitative <t>RT-PCR</t> for IFNB1 mRNA expression in control vs. ADAR- null cells transfected with mIFIH1 NR construct. (c) Representative data from one experiment. (d) Combined data from four biological replicates. (e) Relative levels of IFNB1 mRNA expression in mIFIH1 NR vs. mIFIH1 R transfected ADAR- null cells showing combined data from four biological replicates. Results were normalized by the Livak method as in Fig 2d . Statistical analysis performed using a two-tail student T test. Each data point represents one biological replicate. Error bars represent ± SEM. *p
    Rna, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna/product/Worthington Biochemical
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rna - by Bioz Stars, 2021-04
    96/100 stars
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    7sk rna  (Worthington Biochemical)
    86
    Worthington Biochemical 7sk rna
    Most cellular <t>7SK</t> <t>RNA</t> including those bound to P‐TEFb are pseudouridylated at U250
    7sk Rna, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/7sk rna/product/Worthington Biochemical
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    7sk rna - by Bioz Stars, 2021-04
    86/100 stars
    		  Buy from Supplier

    Image Search Results


    mIFIH1 R mediates increased sensitivity to self-RNA ligands (a-e) Control or ADAR- null HEK293T cells were generated by lenti-CRISPR technology. Cell lines were transfected with 1 μg of mIFIH1 risk or non-risk constructs shown in Fig 2 . Cells were analyzed by flow cytometry at 27 hours post-transfection for (a) geometric mean fluorescent intensity (MFI) and (b) percent GFP expression and combined data from four biological replicates are shown. (c-e) Quantitative RT-PCR for IFNB1 mRNA expression in control vs. ADAR- null cells transfected with mIFIH1 NR construct. (c) Representative data from one experiment. (d) Combined data from four biological replicates. (e) Relative levels of IFNB1 mRNA expression in mIFIH1 NR vs. mIFIH1 R transfected ADAR- null cells showing combined data from four biological replicates. Results were normalized by the Livak method as in Fig 2d . Statistical analysis performed using a two-tail student T test. Each data point represents one biological replicate. Error bars represent ± SEM. *p

    Journal: Nature immunology

    Article Title: The A946T variant IFIH1 RNA sensor mediates an interferon program that limits viral infection but increases the risk for autoimmunity

    doi: 10.1038/ni.3766

    Figure Lengend Snippet: mIFIH1 R mediates increased sensitivity to self-RNA ligands (a-e) Control or ADAR- null HEK293T cells were generated by lenti-CRISPR technology. Cell lines were transfected with 1 μg of mIFIH1 risk or non-risk constructs shown in Fig 2 . Cells were analyzed by flow cytometry at 27 hours post-transfection for (a) geometric mean fluorescent intensity (MFI) and (b) percent GFP expression and combined data from four biological replicates are shown. (c-e) Quantitative RT-PCR for IFNB1 mRNA expression in control vs. ADAR- null cells transfected with mIFIH1 NR construct. (c) Representative data from one experiment. (d) Combined data from four biological replicates. (e) Relative levels of IFNB1 mRNA expression in mIFIH1 NR vs. mIFIH1 R transfected ADAR- null cells showing combined data from four biological replicates. Results were normalized by the Livak method as in Fig 2d . Statistical analysis performed using a two-tail student T test. Each data point represents one biological replicate. Error bars represent ± SEM. *p

    Article Snippet: To obtain RNA samples for quantitative PCR, murine spleens were digested with collagenase Type 4 (Worthington Biochemical), followed by calcium chelation, RBC lysis and made into single cell suspensions.

    Techniques: Generated, CRISPR, Transfection, Construct, Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR

    In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: In Vitro, Binding Assay, Sequencing, Incubation, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Activity Assay, Produced

    Branched RNA–cDNA. ( A ) The 580 nt DGR RNA was biotinylated at its 3′ end and used as a template for reverse-transcription by bRT-Avd, after which biotinylated RNA was captured with streptavidin beads, and the presence of TR- cDNA was detected by PCR using the indicated primers. ( B ) The 580 nt DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The 580 nt DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( C ) Hybrid dA56 580 nt DGR RNA containing deoxyadenosine at Sp 56 (indicated with H at 2′ position) and hybrid d56 580 nt DGR RNA containing adenosine at Sp 56 (indicated with OH at 2′). Both molecules terminate at Sp 140 and have a dideoxynucleotide at the 3′ end (indicated with H at 3′). ( D ) Radiolabeled products resulting from bRT-Avd activity for 12 h with 580 nt DGR RNA, hybrid 580 nt dA56, or hybrid 580 nt A56 DGR RNA as template. Products were untreated (U) or RNase-treated (+R), and resolved by denaturing PAGE. Separate samples of dA56 and A56 were 5′ 32 P-labeled for visualization of input templates (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Branched RNA–cDNA. ( A ) The 580 nt DGR RNA was biotinylated at its 3′ end and used as a template for reverse-transcription by bRT-Avd, after which biotinylated RNA was captured with streptavidin beads, and the presence of TR- cDNA was detected by PCR using the indicated primers. ( B ) The 580 nt DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The 580 nt DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( C ) Hybrid dA56 580 nt DGR RNA containing deoxyadenosine at Sp 56 (indicated with H at 2′ position) and hybrid d56 580 nt DGR RNA containing adenosine at Sp 56 (indicated with OH at 2′). Both molecules terminate at Sp 140 and have a dideoxynucleotide at the 3′ end (indicated with H at 3′). ( D ) Radiolabeled products resulting from bRT-Avd activity for 12 h with 580 nt DGR RNA, hybrid 580 nt dA56, or hybrid 580 nt A56 DGR RNA as template. Products were untreated (U) or RNase-treated (+R), and resolved by denaturing PAGE. Separate samples of dA56 and A56 were 5′ 32 P-labeled for visualization of input templates (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: Polymerase Chain Reaction, Purification, Agarose Gel Electrophoresis, Activity Assay, Polyacrylamide Gel Electrophoresis, Labeling

    Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Incubation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    TR -Sp interactions. ( A ) Complementarity between TR (blue) and Sp (purple) segments. Potential basepairs are numbered (wobble in red). ( B ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2h with the WT or mutated 580 nt DGR RNA as template. Products were treated with RNase, and resolved by denaturing PAGE. Numbers over lane labels correspond to the basepair tested by the mutation, with ‘R’ referring to restoration of the basepair. Sp Mut4 corresponds to Sp 55-CAGC substituted with 55-GUCG.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: TR -Sp interactions. ( A ) Complementarity between TR (blue) and Sp (purple) segments. Potential basepairs are numbered (wobble in red). ( B ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2h with the WT or mutated 580 nt DGR RNA as template. Products were treated with RNase, and resolved by denaturing PAGE. Numbers over lane labels correspond to the basepair tested by the mutation, with ‘R’ referring to restoration of the basepair. Sp Mut4 corresponds to Sp 55-CAGC substituted with 55-GUCG.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis, Mutagenesis

    Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Amplification, Produced, Sequencing, Activity Assay, Polyacrylamide Gel Electrophoresis

    Specificity to DGR RNA. ( A ) Top, schematic of DGR RNA template and primer P G117 . Bottom, radiolabeled products resulting from bRT-Avd activity for 2 h with intact 580 nt DGR RNA or DGR RNA truncated at Sp A56 as template. Reverse transcription reactions were carried out in the absence (-P) or presence of primer P G117 . Reaction products were untreated (U), treated with RNase (+R), or treated with DNase (+D), and resolved by denaturing PAGE. The blue line indicates ODN-primed cDNA products. The red dot indicates ODN-primed 120 nt cDNA (cDNA + 20 nt primer for a 140 nt band). ( B ) Protection of internally-labeled 580 nt DGR RNA from RNase by bRT, Avd, or bRT-Avd, with products resolved by 15% denaturing PAGE. The protected band (P) is indicated. ( C ) RNase protection by Avd, as in panel B, carried out on internally-labeled wild-type 580 nt DGR RNA or 580 nt DGR RNA with scrambled (Sc) Sp sequences, with the first lane in each pair untreated and the second RNase-treated. Products were resolved by denaturing PAGE. The protected band (P) is indicated. ( D ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the WT 580 nt DGR RNA or DGR RNA containing scrambled (Sc) Sp sequences as template. The last lane shows the activity of Avd alone for 2 h with the WT 580 nt DGR RNA as template. Products were treated with RNase, and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs indicated. ( E ) Model of processive polymerization of adenine-mutagenized cDNA by bRT-Avd/RNA ribonucleoprotein particle. The 2′-OH of Sp 56 serves as the priming site and forms a 2′-5′ phosphodiester bond with the cDNA. The first nucleotide reverse transcribed is TR 117. Adenines in TR are unfaithfully reverse transcribed by bRT-Avd (represented by ‘N’). The RNP promotes processive polymerization, which terminates at one of two stops in the DGR RNA.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Specificity to DGR RNA. ( A ) Top, schematic of DGR RNA template and primer P G117 . Bottom, radiolabeled products resulting from bRT-Avd activity for 2 h with intact 580 nt DGR RNA or DGR RNA truncated at Sp A56 as template. Reverse transcription reactions were carried out in the absence (-P) or presence of primer P G117 . Reaction products were untreated (U), treated with RNase (+R), or treated with DNase (+D), and resolved by denaturing PAGE. The blue line indicates ODN-primed cDNA products. The red dot indicates ODN-primed 120 nt cDNA (cDNA + 20 nt primer for a 140 nt band). ( B ) Protection of internally-labeled 580 nt DGR RNA from RNase by bRT, Avd, or bRT-Avd, with products resolved by 15% denaturing PAGE. The protected band (P) is indicated. ( C ) RNase protection by Avd, as in panel B, carried out on internally-labeled wild-type 580 nt DGR RNA or 580 nt DGR RNA with scrambled (Sc) Sp sequences, with the first lane in each pair untreated and the second RNase-treated. Products were resolved by denaturing PAGE. The protected band (P) is indicated. ( D ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the WT 580 nt DGR RNA or DGR RNA containing scrambled (Sc) Sp sequences as template. The last lane shows the activity of Avd alone for 2 h with the WT 580 nt DGR RNA as template. Products were treated with RNase, and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs indicated. ( E ) Model of processive polymerization of adenine-mutagenized cDNA by bRT-Avd/RNA ribonucleoprotein particle. The 2′-OH of Sp 56 serves as the priming site and forms a 2′-5′ phosphodiester bond with the cDNA. The first nucleotide reverse transcribed is TR 117. Adenines in TR are unfaithfully reverse transcribed by bRT-Avd (represented by ‘N’). The RNP promotes processive polymerization, which terminates at one of two stops in the DGR RNA.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis, Labeling

    In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: In Vitro, Binding Assay, Sequencing, Incubation, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Activity Assay, Produced

    Branched RNA–cDNA. ( A ) The 580 nt DGR RNA was biotinylated at its 3′ end and used as a template for reverse-transcription by bRT-Avd, after which biotinylated RNA was captured with streptavidin beads, and the presence of TR- cDNA was detected by PCR using the indicated primers. ( B ) The 580 nt DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The 580 nt DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( C ) Hybrid dA56 580 nt DGR RNA containing deoxyadenosine at Sp 56 (indicated with H at 2′ position) and hybrid d56 580 nt DGR RNA containing adenosine at Sp 56 (indicated with OH at 2′). Both molecules terminate at Sp 140 and have a dideoxynucleotide at the 3′ end (indicated with H at 3′). ( D ) Radiolabeled products resulting from bRT-Avd activity for 12 h with 580 nt DGR RNA, hybrid 580 nt dA56, or hybrid 580 nt A56 DGR RNA as template. Products were untreated (U) or RNase-treated (+R), and resolved by denaturing PAGE. Separate samples of dA56 and A56 were 5′ 32 P-labeled for visualization of input templates (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Branched RNA–cDNA. ( A ) The 580 nt DGR RNA was biotinylated at its 3′ end and used as a template for reverse-transcription by bRT-Avd, after which biotinylated RNA was captured with streptavidin beads, and the presence of TR- cDNA was detected by PCR using the indicated primers. ( B ) The 580 nt DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The 580 nt DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( C ) Hybrid dA56 580 nt DGR RNA containing deoxyadenosine at Sp 56 (indicated with H at 2′ position) and hybrid d56 580 nt DGR RNA containing adenosine at Sp 56 (indicated with OH at 2′). Both molecules terminate at Sp 140 and have a dideoxynucleotide at the 3′ end (indicated with H at 3′). ( D ) Radiolabeled products resulting from bRT-Avd activity for 12 h with 580 nt DGR RNA, hybrid 580 nt dA56, or hybrid 580 nt A56 DGR RNA as template. Products were untreated (U) or RNase-treated (+R), and resolved by denaturing PAGE. Separate samples of dA56 and A56 were 5′ 32 P-labeled for visualization of input templates (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: Polymerase Chain Reaction, Purification, Agarose Gel Electrophoresis, Activity Assay, Polyacrylamide Gel Electrophoresis, Labeling

    Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Incubation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    TR -Sp interactions. ( A ) Complementarity between TR (blue) and Sp (purple) segments. Potential basepairs are numbered (wobble in red). ( B ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2h with the WT or mutated 580 nt DGR RNA as template. Products were treated with RNase, and resolved by denaturing PAGE. Numbers over lane labels correspond to the basepair tested by the mutation, with ‘R’ referring to restoration of the basepair. Sp Mut4 corresponds to Sp 55-CAGC substituted with 55-GUCG.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: TR -Sp interactions. ( A ) Complementarity between TR (blue) and Sp (purple) segments. Potential basepairs are numbered (wobble in red). ( B ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2h with the WT or mutated 580 nt DGR RNA as template. Products were treated with RNase, and resolved by denaturing PAGE. Numbers over lane labels correspond to the basepair tested by the mutation, with ‘R’ referring to restoration of the basepair. Sp Mut4 corresponds to Sp 55-CAGC substituted with 55-GUCG.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis, Mutagenesis

    Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Amplification, Produced, Sequencing, Activity Assay, Polyacrylamide Gel Electrophoresis

    Specificity to DGR RNA. ( A ) Top, schematic of DGR RNA template and primer P G117 . Bottom, radiolabeled products resulting from bRT-Avd activity for 2 h with intact 580 nt DGR RNA or DGR RNA truncated at Sp A56 as template. Reverse transcription reactions were carried out in the absence (-P) or presence of primer P G117 . Reaction products were untreated (U), treated with RNase (+R), or treated with DNase (+D), and resolved by denaturing PAGE. The blue line indicates ODN-primed cDNA products. The red dot indicates ODN-primed 120 nt cDNA (cDNA + 20 nt primer for a 140 nt band). ( B ) Protection of internally-labeled 580 nt DGR RNA from RNase by bRT, Avd, or bRT-Avd, with products resolved by 15% denaturing PAGE. The protected band (P) is indicated. ( C ) RNase protection by Avd, as in panel B, carried out on internally-labeled wild-type 580 nt DGR RNA or 580 nt DGR RNA with scrambled (Sc) Sp sequences, with the first lane in each pair untreated and the second RNase-treated. Products were resolved by denaturing PAGE. The protected band (P) is indicated. ( D ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the WT 580 nt DGR RNA or DGR RNA containing scrambled (Sc) Sp sequences as template. The last lane shows the activity of Avd alone for 2 h with the WT 580 nt DGR RNA as template. Products were treated with RNase, and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs indicated. ( E ) Model of processive polymerization of adenine-mutagenized cDNA by bRT-Avd/RNA ribonucleoprotein particle. The 2′-OH of Sp 56 serves as the priming site and forms a 2′-5′ phosphodiester bond with the cDNA. The first nucleotide reverse transcribed is TR 117. Adenines in TR are unfaithfully reverse transcribed by bRT-Avd (represented by ‘N’). The RNP promotes processive polymerization, which terminates at one of two stops in the DGR RNA.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Specificity to DGR RNA. ( A ) Top, schematic of DGR RNA template and primer P G117 . Bottom, radiolabeled products resulting from bRT-Avd activity for 2 h with intact 580 nt DGR RNA or DGR RNA truncated at Sp A56 as template. Reverse transcription reactions were carried out in the absence (-P) or presence of primer P G117 . Reaction products were untreated (U), treated with RNase (+R), or treated with DNase (+D), and resolved by denaturing PAGE. The blue line indicates ODN-primed cDNA products. The red dot indicates ODN-primed 120 nt cDNA (cDNA + 20 nt primer for a 140 nt band). ( B ) Protection of internally-labeled 580 nt DGR RNA from RNase by bRT, Avd, or bRT-Avd, with products resolved by 15% denaturing PAGE. The protected band (P) is indicated. ( C ) RNase protection by Avd, as in panel B, carried out on internally-labeled wild-type 580 nt DGR RNA or 580 nt DGR RNA with scrambled (Sc) Sp sequences, with the first lane in each pair untreated and the second RNase-treated. Products were resolved by denaturing PAGE. The protected band (P) is indicated. ( D ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the WT 580 nt DGR RNA or DGR RNA containing scrambled (Sc) Sp sequences as template. The last lane shows the activity of Avd alone for 2 h with the WT 580 nt DGR RNA as template. Products were treated with RNase, and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs indicated. ( E ) Model of processive polymerization of adenine-mutagenized cDNA by bRT-Avd/RNA ribonucleoprotein particle. The 2′-OH of Sp 56 serves as the priming site and forms a 2′-5′ phosphodiester bond with the cDNA. The first nucleotide reverse transcribed is TR 117. Adenines in TR are unfaithfully reverse transcribed by bRT-Avd (represented by ‘N’). The RNP promotes processive polymerization, which terminates at one of two stops in the DGR RNA.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis, Labeling

    Most cellular 7SK RNA including those bound to P‐TEFb are pseudouridylated at U250

    Journal: EMBO Reports

    Article Title: Pseudouridylation of 7 SK sn RNA promotes 7 SK sn RNP formation to suppress HIV‐1 transcription and escape from latency

    doi: 10.15252/embr.201642682

    Figure Lengend Snippet: Most cellular 7SK RNA including those bound to P‐TEFb are pseudouridylated at U250

    Article Snippet: For 2D‐TLC, 7SK RNA isolated from CDK9‐Flag immunoprecipitates or in vitro transcribed was digested to completion with 0.5–1 U of RNase T2 (Worthington) in 50 mM ammonium acetate, 0.05% SDS, and 1 mM EDTA.

    Techniques:

    Mutations at or around U250 in 7SK RNA prevent endogenous CDK9 from binding

    Journal: EMBO Reports

    Article Title: Pseudouridylation of 7 SK sn RNA promotes 7 SK sn RNP formation to suppress HIV‐1 transcription and escape from latency

    doi: 10.15252/embr.201642682

    Figure Lengend Snippet: Mutations at or around U250 in 7SK RNA prevent endogenous CDK9 from binding

    Article Snippet: For 2D‐TLC, 7SK RNA isolated from CDK9‐Flag immunoprecipitates or in vitro transcribed was digested to completion with 0.5–1 U of RNase T2 (Worthington) in 50 mM ammonium acetate, 0.05% SDS, and 1 mM EDTA.

    Techniques: Binding Assay

    Drug‐induced 7SK RNP destruction does not change Ψ250 level in 7SK RNA

    Journal: EMBO Reports

    Article Title: Pseudouridylation of 7 SK sn RNA promotes 7 SK sn RNP formation to suppress HIV‐1 transcription and escape from latency

    doi: 10.15252/embr.201642682

    Figure Lengend Snippet: Drug‐induced 7SK RNP destruction does not change Ψ250 level in 7SK RNA

    Article Snippet: For 2D‐TLC, 7SK RNA isolated from CDK9‐Flag immunoprecipitates or in vitro transcribed was digested to completion with 0.5–1 U of RNase T2 (Worthington) in 50 mM ammonium acetate, 0.05% SDS, and 1 mM EDTA.

    Techniques:

    Ψ250 in 7SK RNA is critical for 7SK RNP formation

    Journal: EMBO Reports

    Article Title: Pseudouridylation of 7 SK sn RNA promotes 7 SK sn RNP formation to suppress HIV‐1 transcription and escape from latency

    doi: 10.15252/embr.201642682

    Figure Lengend Snippet: Ψ250 in 7SK RNA is critical for 7SK RNP formation

    Article Snippet: For 2D‐TLC, 7SK RNA isolated from CDK9‐Flag immunoprecipitates or in vitro transcribed was digested to completion with 0.5–1 U of RNase T2 (Worthington) in 50 mM ammonium acetate, 0.05% SDS, and 1 mM EDTA.

    Techniques: