mlv  (Promega)

 
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    Name:
    M MLV Reverse Transcriptase
    Description:
    RNA dependent DNA polymerase for cDNA synthesis
    Catalog Number:
    m1701
    Price:
    None
    Category:
    Nucleic Acid Extraction Analysis PCR RT PCR
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    Structured Review

    Promega mlv
    Mapping the IN binding interface in the BRD2 ET domain. (A to C) Coimmunoprecipitation of <t>FLAG-tagged</t> <t>MLV</t> IN with GFP-tagged WT BRD2 or its point mutants. Proteins recovered after coimmunoprecipitation with a GFP affinity matrix were analyzed by Western
    RNA dependent DNA polymerase for cDNA synthesis
    https://www.bioz.com/result/mlv/product/Promega
    Average 99 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    mlv - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Bromo- and Extraterminal Domain Chromatin Regulators Serve as Cofactors for Murine Leukemia Virus Integration"

    Article Title: Bromo- and Extraterminal Domain Chromatin Regulators Serve as Cofactors for Murine Leukemia Virus Integration

    Journal: Journal of Virology

    doi: 10.1128/JVI.01942-13

    Mapping the IN binding interface in the BRD2 ET domain. (A to C) Coimmunoprecipitation of FLAG-tagged MLV IN with GFP-tagged WT BRD2 or its point mutants. Proteins recovered after coimmunoprecipitation with a GFP affinity matrix were analyzed by Western
    Figure Legend Snippet: Mapping the IN binding interface in the BRD2 ET domain. (A to C) Coimmunoprecipitation of FLAG-tagged MLV IN with GFP-tagged WT BRD2 or its point mutants. Proteins recovered after coimmunoprecipitation with a GFP affinity matrix were analyzed by Western

    Techniques Used: Binding Assay, Western Blot

    BET bromodomain inhibitors, JQ1 and I-BET, reduce integration of MLV, but not of HIV-1, in HEK293T cells. (A to D) HEK293T cells were transduced with MLV-based (A and C) and HIV-1-based (B and D) vectors carrying EGFP as a reporter gene at an MOI of 0.1
    Figure Legend Snippet: BET bromodomain inhibitors, JQ1 and I-BET, reduce integration of MLV, but not of HIV-1, in HEK293T cells. (A to D) HEK293T cells were transduced with MLV-based (A and C) and HIV-1-based (B and D) vectors carrying EGFP as a reporter gene at an MOI of 0.1

    Techniques Used: Transduction

    Overexpression of IN binding domain of BRD2 (residues 640 to 801) increases MLV integration. (A and B) HEK293T-based cell lines stably expressing BRD2(640–801) were challenged with MLV-based (A) and HIV-1-based (B) retroviral vectors expressing
    Figure Legend Snippet: Overexpression of IN binding domain of BRD2 (residues 640 to 801) increases MLV integration. (A and B) HEK293T-based cell lines stably expressing BRD2(640–801) were challenged with MLV-based (A) and HIV-1-based (B) retroviral vectors expressing

    Techniques Used: Over Expression, Binding Assay, Stable Transfection, Expressing

    2) Product Images from "Nucleic Acid, Antibody, and Virus Culture Methods to Detect Xenotropic MLV-Related Virus in Human Blood Samples"

    Article Title: Nucleic Acid, Antibody, and Virus Culture Methods to Detect Xenotropic MLV-Related Virus in Human Blood Samples

    Journal: Advances in Virology

    doi: 10.1155/2011/272193

    XMRV single-copy assay (X-SCA) . X-SCA primers anneal to conserved regions in XMRV/MLV gag leader region while the probe spans a 24 nt deletion in XMRV compared to MLV (a) allowing for differential amplification profiles for XMRV and MLV (b). X-SCA amplification products run on a 2% agarose gel distinguish between the products being amplified since the XMRV product is 24 nt smaller than the MLV product. Lane 1 is the X-SCA product from the XMRV standard curve, Lane 2 is the MLV product from the genomic DNA extracted from TA3.Cyc-T1 mouse cells, and Lane 3 is the “no template” negative control (c).
    Figure Legend Snippet: XMRV single-copy assay (X-SCA) . X-SCA primers anneal to conserved regions in XMRV/MLV gag leader region while the probe spans a 24 nt deletion in XMRV compared to MLV (a) allowing for differential amplification profiles for XMRV and MLV (b). X-SCA amplification products run on a 2% agarose gel distinguish between the products being amplified since the XMRV product is 24 nt smaller than the MLV product. Lane 1 is the X-SCA product from the XMRV standard curve, Lane 2 is the MLV product from the genomic DNA extracted from TA3.Cyc-T1 mouse cells, and Lane 3 is the “no template” negative control (c).

    Techniques Used: Amplification, Agarose Gel Electrophoresis, Negative Control

    3) Product Images from "The Breadth of Antiviral Activity of Apobec3DE in Chimpanzees Has Been Driven by Positive Selection ▿"

    Article Title: The Breadth of Antiviral Activity of Apobec3DE in Chimpanzees Has Been Driven by Positive Selection ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.05046-11

    Restriction of retroviruses by human and chimpanzee Apobec3DE. Human Apobec3DE (hA3DE) and chimpanzee Apobec3DE (cA3DE) were expressed in single-round infectivity assays to determine antiviral activity. Infections in the presence of human Apobec3G (hA3G) were used as a positive control. Infectivity is normalized to 100% in the absence of Apobec3 (none). (A) Infectivity of MLV. (B) Infectivity of HIV-2 without Vif (HIV-2Δ vif ) and of HIV-2 containing Vif (HIV-2 WT). (C) Infectivity of HIV-1 without Vif (HIVΔ vif ) and HIV-1 containing Vif (HIV WT). (D) Infectivity of SIVagmTAN without Vif (SIVagmTANΔ vif ) and SIVagmTAN containing Vif (SIVagmTAN WT). Experiments were performed at least 2 times, and results from one representative experiment are shown. Error bars represent the standard deviation of triplicate infections within one experiment. P values were calculated using paired two-tailed Student's t test.
    Figure Legend Snippet: Restriction of retroviruses by human and chimpanzee Apobec3DE. Human Apobec3DE (hA3DE) and chimpanzee Apobec3DE (cA3DE) were expressed in single-round infectivity assays to determine antiviral activity. Infections in the presence of human Apobec3G (hA3G) were used as a positive control. Infectivity is normalized to 100% in the absence of Apobec3 (none). (A) Infectivity of MLV. (B) Infectivity of HIV-2 without Vif (HIV-2Δ vif ) and of HIV-2 containing Vif (HIV-2 WT). (C) Infectivity of HIV-1 without Vif (HIVΔ vif ) and HIV-1 containing Vif (HIV WT). (D) Infectivity of SIVagmTAN without Vif (SIVagmTANΔ vif ) and SIVagmTAN containing Vif (SIVagmTAN WT). Experiments were performed at least 2 times, and results from one representative experiment are shown. Error bars represent the standard deviation of triplicate infections within one experiment. P values were calculated using paired two-tailed Student's t test.

    Techniques Used: Infection, Activity Assay, Positive Control, Standard Deviation, Two Tailed Test

    4) Product Images from "Construction of Retroviral Vectors with Improved Safety, Gene Expression, and Versatility"

    Article Title: Construction of Retroviral Vectors with Improved Safety, Gene Expression, and Versatility

    Journal: Journal of Virology

    doi:

    Schematic representation of the retroviral vector MFG. In MFG, the gene of interest (dotted box) is cloned into the Nco I site, containing the start codon in it, and expressed as a spliced mRNA. MFG contains the 420- and 99-bp coding sequences for gag and env , respectively. U3 of Moloney MLV is 448 bp long. ATG, start codon of gag .
    Figure Legend Snippet: Schematic representation of the retroviral vector MFG. In MFG, the gene of interest (dotted box) is cloned into the Nco I site, containing the start codon in it, and expressed as a spliced mRNA. MFG contains the 420- and 99-bp coding sequences for gag and env , respectively. U3 of Moloney MLV is 448 bp long. ATG, start codon of gag .

    Techniques Used: Plasmid Preparation, Clone Assay

    5) Product Images from "Bromo- and Extraterminal Domain Chromatin Regulators Serve as Cofactors for Murine Leukemia Virus Integration"

    Article Title: Bromo- and Extraterminal Domain Chromatin Regulators Serve as Cofactors for Murine Leukemia Virus Integration

    Journal: Journal of Virology

    doi: 10.1128/JVI.01942-13

    Mapping the IN binding interface in the BRD2 ET domain. (A to C) Coimmunoprecipitation of FLAG-tagged MLV IN with GFP-tagged WT BRD2 or its point mutants. Proteins recovered after coimmunoprecipitation with a GFP affinity matrix were analyzed by Western
    Figure Legend Snippet: Mapping the IN binding interface in the BRD2 ET domain. (A to C) Coimmunoprecipitation of FLAG-tagged MLV IN with GFP-tagged WT BRD2 or its point mutants. Proteins recovered after coimmunoprecipitation with a GFP affinity matrix were analyzed by Western

    Techniques Used: Binding Assay, Western Blot

    BET bromodomain inhibitors, JQ1 and I-BET, reduce integration of MLV, but not of HIV-1, in HEK293T cells. (A to D) HEK293T cells were transduced with MLV-based (A and C) and HIV-1-based (B and D) vectors carrying EGFP as a reporter gene at an MOI of 0.1
    Figure Legend Snippet: BET bromodomain inhibitors, JQ1 and I-BET, reduce integration of MLV, but not of HIV-1, in HEK293T cells. (A to D) HEK293T cells were transduced with MLV-based (A and C) and HIV-1-based (B and D) vectors carrying EGFP as a reporter gene at an MOI of 0.1

    Techniques Used: Transduction

    Overexpression of IN binding domain of BRD2 (residues 640 to 801) increases MLV integration. (A and B) HEK293T-based cell lines stably expressing BRD2(640–801) were challenged with MLV-based (A) and HIV-1-based (B) retroviral vectors expressing
    Figure Legend Snippet: Overexpression of IN binding domain of BRD2 (residues 640 to 801) increases MLV integration. (A and B) HEK293T-based cell lines stably expressing BRD2(640–801) were challenged with MLV-based (A) and HIV-1-based (B) retroviral vectors expressing

    Techniques Used: Over Expression, Binding Assay, Stable Transfection, Expressing

    6) Product Images from "Transcriptional inaccuracy threshold attenuates differences in RNA-dependent DNA synthesis fidelity between retroviral reverse transcriptases"

    Article Title: Transcriptional inaccuracy threshold attenuates differences in RNA-dependent DNA synthesis fidelity between retroviral reverse transcriptases

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-18974-8

    Comparison of RNA- and DNA-dependent DNA synthesis fidelities of retroviral RTs. Estimates of fidelity of RNA-dependent DNA synthesis are based on mutant frequencies obtained with the adapted M13mp2 lacZα forward mutation assay, reported in this paper (orange bars). Blue bars represent previously reported values, obtained using M13mp2-based assays measuring the accuracy of DNA-dependent DNA synthesis of HIV-1 BH10 29 , HIV-1 ESP49 36 , AMV 14 , and MLV, O_K65R/V75I and O_K65R RTs 18 .
    Figure Legend Snippet: Comparison of RNA- and DNA-dependent DNA synthesis fidelities of retroviral RTs. Estimates of fidelity of RNA-dependent DNA synthesis are based on mutant frequencies obtained with the adapted M13mp2 lacZα forward mutation assay, reported in this paper (orange bars). Blue bars represent previously reported values, obtained using M13mp2-based assays measuring the accuracy of DNA-dependent DNA synthesis of HIV-1 BH10 29 , HIV-1 ESP49 36 , AMV 14 , and MLV, O_K65R/V75I and O_K65R RTs 18 .

    Techniques Used: DNA Synthesis, Mutagenesis

    Comparison of mutational spectra induced by retroviral RTs during RNA- and DNA-dependent DNA synthesis. (Up) Combined mutational spectra derived from RNA-dependent DNA synthesis reactions including all RTs analysed in this study (Supplementary Figs S2 to S7). RTs included are those of WT HIV-1 BH10 (pink), HIV-1 ESP49 (purple), AMV (red), and MLV (green), and mutants O_K65R/V75I (blue) and O_K65R (brown). (Bottom) Combined mutational spectra induced during DNA-dependent DNA synthesis reactions, and taken from previously published reports 15 , 18 , 36 . RT colour codes are the same as above. For all RTs and mutational spectra, single-nucleotide substitutions are indicated by the letter corresponding to the new base above the template sequence of the lac Zα target. Open upright triangles represent insertions and inverted triangles indicate deletions. The triangles are positioned at the 3′ end of the frameshift, with the number of inserted/deleted nucleotides indicated between parentheses. If not specified, one-nucleotide insertions correspond to the duplication of the base where the triangle is positioned.
    Figure Legend Snippet: Comparison of mutational spectra induced by retroviral RTs during RNA- and DNA-dependent DNA synthesis. (Up) Combined mutational spectra derived from RNA-dependent DNA synthesis reactions including all RTs analysed in this study (Supplementary Figs S2 to S7). RTs included are those of WT HIV-1 BH10 (pink), HIV-1 ESP49 (purple), AMV (red), and MLV (green), and mutants O_K65R/V75I (blue) and O_K65R (brown). (Bottom) Combined mutational spectra induced during DNA-dependent DNA synthesis reactions, and taken from previously published reports 15 , 18 , 36 . RT colour codes are the same as above. For all RTs and mutational spectra, single-nucleotide substitutions are indicated by the letter corresponding to the new base above the template sequence of the lac Zα target. Open upright triangles represent insertions and inverted triangles indicate deletions. The triangles are positioned at the 3′ end of the frameshift, with the number of inserted/deleted nucleotides indicated between parentheses. If not specified, one-nucleotide insertions correspond to the duplication of the base where the triangle is positioned.

    Techniques Used: DNA Synthesis, Derivative Assay, Sequencing

    Related Articles

    RT Lamp Assay:

    Article Title: Rapid detection of wheat yellow mosaic virus by reverse transcription loop-mediated isothermal amplification
    Article Snippet: .. For each primer set, AMV and M-MLV RT-LAMP was performed and similar results were obtained, therefore, M-MLV reverse transcriptase was chosen for the subsequent RT-LAMP assay because of its relatively cheap price (Figure ). ..

    Purification:

    Article Title: APOBEC3A deaminates transiently exposed single-strand DNA during LINE-1 retrotransposition
    Article Snippet: .. RT-PCR was carried out on 0.5 μl of purified RNA, using MMLV reverse transcriptase (Promega, Madison, WI) and 0.8 μM LEAP adapter (5np1) at 42°C for 30 min. ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: OsTIR1 and OsAFB2 Downregulation via OsmiR393 Overexpression Leads to More Tillers, Early Flowering and Less Tolerance to Salt and Drought in Rice
    Article Snippet: .. The total RNA reverse transcription reaction was performed with a two-step RT-PCR kit (Promega, Cat.# M 1701) on 2 µg of total RNA according to the product manual. .. Expression detection by real-time quantitative PCR Gene expression was analyzed by quantitative real time RT-PCR and semi-quantitative RT-PCR using the primers listed in .

    Article Title: APOBEC3A deaminates transiently exposed single-strand DNA during LINE-1 retrotransposition
    Article Snippet: .. RT-PCR was carried out on 0.5 μl of purified RNA, using MMLV reverse transcriptase (Promega, Madison, WI) and 0.8 μM LEAP adapter (5np1) at 42°C for 30 min. ..

    Incubation:

    Article Title: A cypovirus VP5 displays the RNA chaperone-like activity that destabilizes RNA helices and accelerates strand annealing
    Article Snippet: .. After incubation, 4 μl of the RT buffer, 1 μl of 0.1 M DTT, 5 U RNasin and 0.5 μl M-MLV reverse transcriptase (Promega) were supplemented and reacted at 25°C for 30 min. ..

    other:

    Article Title: Rapid detection of wheat yellow mosaic virus by reverse transcription loop-mediated isothermal amplification
    Article Snippet: Discussion WYMV could be detected by the RT-LAMP method using the designed four sets of primers (I-IV) and M-MLV reverse transcriptase instead of AMV reverse transcriptase under isothermal condition at 65°C for 80 min.

    Polymerase Chain Reaction:

    Article Title: Calcium-dependent regulation of the cell cycle via a novel MAPK-NF-?B pathway in Swiss 3T3 cells
    Article Snippet: .. 2 μg of RNA was used to perform reverse transcriptase PCR (using 200 U M-MLV reverse transcriptase; Promega). .. For PCR amplification, 5 μl of RT products were incubated with 20 pmol of specific primers and 1 U of Taq DNA polymerase (Promega).

    Clear Native PAGE:

    Article Title: Characterization of HelD, an interacting partner of RNA polymerase from Bacillus subtilis
    Article Snippet: .. Native PAGE assays Five picomoles of RNAP and 25 pmol of HelD, T4 DNA ligase (TaKaRa) or MLV reverse transcriptase (Promega), respectively, were used. .. Proteins tested for mutual interactions were incubated for 15 min at 30°C in 10 μl in the storage buffer.

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  • 85
    Promega mlv ltr luc supernatants
    Effects of mutations in the PCE on <t>MLV</t> transcription. (A) Nucleotide sequences of the wild-type (WT) and mutant PCEs. (B) A gel shift assay was performed with 293 cell nuclear extracts with different DNA probes including wild-type PCE (WT), PCE 1mt (1mt), or PCE 2mt (2mt). C1 and C2, PCE-protein complexes. (C) 293 cells were transiently transfected with the <t>WZL-Luc</t> vector containing wild-type or mutant PCEs. Luciferase activity was measured 48 h after transfection. Results were reproducible, and the assay was repeated more than three times in triplicate.
    Mlv Ltr Luc Supernatants, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 1 article reviews
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    90
    Promega mlv r env
    Endocytosis in Env-expressing cells is required for the cell-cell fusion. (A) The E-MLV R-Env (left panel) or HIV-1 Env (right panel) was transfected to <t>HEK293T</t> cells together with the nls-LacZ expression plasmid. The transfected HEK293T cells were co-culture with TE671mCAT1 or NP2/CD4/X4 cells pretreated with indicated inhibitor (black bars). The transfected cells were treated with indicated inhibitors, and were co-cultured with TE671mCAT1 or NP2/CD4/X4 cells (gray bar). This experiment was repeated three times. Fusion indexes in the control cells were always set to 1, and relative fusion indexes ± SD are shown. Asterisks indicate statistically significant differences between treatments of the Env-expressing or target cells. (B) HEK293T, TE671mCAT1, SC-1, and HeLamCAT1 cells were treated with indicated inhibitor. Ratios (%) of live cells per total cells are indicated. This experiment was repeated three times. (C) HEK293T cells were transfected with the MLV R-Env (left panel) or HIV-1 Env (right panel) expression plsmid together with the nls-LacZ expression plasmid. The cells were also simultaneously transfected with the dynamin dominant negative mutant expression plasmid or pcDNA3.1. This experiment was repeated three times. Fusion indexes in the control cells were always set to 1, and relative fusion indexes ± SD are shown. Asterisks indicate statistically significant differences between transfection with pcDNA3.1 and dynamin mutant.
    Mlv R Env, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega mlv ltr luc
    Effects of mutations in the PCE on <t>MLV</t> transcription. (A) Nucleotide sequences of the wild-type (WT) and mutant PCEs. (B) A gel shift assay was performed with 293 cell nuclear extracts with different DNA probes including wild-type PCE (WT), PCE 1mt (1mt), or PCE 2mt (2mt). C1 and C2, PCE-protein complexes. (C) 293 cells were transiently transfected with the <t>WZL-Luc</t> vector containing wild-type or mutant PCEs. Luciferase activity was measured 48 h after transfection. Results were reproducible, and the assay was repeated more than three times in triplicate.
    Mlv Ltr Luc, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mlv ltr luc/product/Promega
    Average 85 stars, based on 1 article reviews
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    85
    Promega mlv nc
    Binding of the Gypsy NC-like peptide to Gypsy 5′ and 3′ RNAs. Gypsy NC-like peptide and 32 P-labelled 5′ and 3′ RNAs were synthesized as described in Materials and Methods. Binding of Gypsy NC (G-NC) was monitored by gel retardation (data not shown) and nucleoprotein complex formation. Complexes were recovered by centrifugation as reported in methods. The same experiments were carried out with the TYA1-D peptide and with <t>MLV</t> and HIV-NC peptides (see Materials and Methods for peptide sequences). Optimal complex formation with the G-NC and TYA1-D peptides was found to take place at a protein to nt molar ratio of 1 to 6, as previously observed for retroviral NC proteins, such as HIV-1 NCp7 and MuLV NCp10 ( 20 , 45 ). Note that the basic HIV-NC and MLV-NC peptides did not form large amounts of ribonucleoprotein complexes (HIV-NC and MLV-NC on both panels); although they bind <t>RNA</t> [see Refs ( 20 – 22 )]. CT stands for RNA alone.
    Mlv Nc, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of mutations in the PCE on MLV transcription. (A) Nucleotide sequences of the wild-type (WT) and mutant PCEs. (B) A gel shift assay was performed with 293 cell nuclear extracts with different DNA probes including wild-type PCE (WT), PCE 1mt (1mt), or PCE 2mt (2mt). C1 and C2, PCE-protein complexes. (C) 293 cells were transiently transfected with the WZL-Luc vector containing wild-type or mutant PCEs. Luciferase activity was measured 48 h after transfection. Results were reproducible, and the assay was repeated more than three times in triplicate.

    Journal: Molecular and Cellular Biology

    Article Title: Identification of Homeodomain Proteins, PBX1 and PREP1, Involved in the Transcription of Murine Leukemia Virus

    doi: 10.1128/MCB.23.3.831-841.2003

    Figure Lengend Snippet: Effects of mutations in the PCE on MLV transcription. (A) Nucleotide sequences of the wild-type (WT) and mutant PCEs. (B) A gel shift assay was performed with 293 cell nuclear extracts with different DNA probes including wild-type PCE (WT), PCE 1mt (1mt), or PCE 2mt (2mt). C1 and C2, PCE-protein complexes. (C) 293 cells were transiently transfected with the WZL-Luc vector containing wild-type or mutant PCEs. Luciferase activity was measured 48 h after transfection. Results were reproducible, and the assay was repeated more than three times in triplicate.

    Article Snippet: 3T3 or 293 cells were grown to 50 to 80% confluence in 96-well plates and incubated with individual CDKIs for 30 min. MLV-LTR-Luc supernatants were then added and incubated for 24 h. Luciferase activity was measured with the Bright-Glo assay system (Promega), and the activity was determined with an Acquest Ultra-HTS system (LJL Biosystems, Inc.).

    Techniques: Mutagenesis, Electrophoretic Mobility Shift Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay

    Inhibition of MLV transcription by blocking PBX1 protein synthesis. (A) 293 cells were cotransfected with WZL-Luc and a Pbx1 -targeted antisense oligonucleotide. Each Pbx1 antisense nucleotide was paired with a sense nucleotide as control. Luciferase activity was measured 24 h after transfection. A pcDNA3.1 reverse primer, used for sequencing the insert of pcDNA plasmids (Invitrogen), served as another control. The pcDNA3.1 primer and two sense Pbx1 oligonucleotides had similar effects on viral transcription (data not shown). (B) 293 cells were transiently transfected with an siRNA of Pbx1 for 24 h and then incubated with MLV viral supernatants for another 24 h. An siRNA of Renilla luciferase was used as control. The assay was repeated twice in triplicate with the same results.

    Journal: Molecular and Cellular Biology

    Article Title: Identification of Homeodomain Proteins, PBX1 and PREP1, Involved in the Transcription of Murine Leukemia Virus

    doi: 10.1128/MCB.23.3.831-841.2003

    Figure Lengend Snippet: Inhibition of MLV transcription by blocking PBX1 protein synthesis. (A) 293 cells were cotransfected with WZL-Luc and a Pbx1 -targeted antisense oligonucleotide. Each Pbx1 antisense nucleotide was paired with a sense nucleotide as control. Luciferase activity was measured 24 h after transfection. A pcDNA3.1 reverse primer, used for sequencing the insert of pcDNA plasmids (Invitrogen), served as another control. The pcDNA3.1 primer and two sense Pbx1 oligonucleotides had similar effects on viral transcription (data not shown). (B) 293 cells were transiently transfected with an siRNA of Pbx1 for 24 h and then incubated with MLV viral supernatants for another 24 h. An siRNA of Renilla luciferase was used as control. The assay was repeated twice in triplicate with the same results.

    Article Snippet: 3T3 or 293 cells were grown to 50 to 80% confluence in 96-well plates and incubated with individual CDKIs for 30 min. MLV-LTR-Luc supernatants were then added and incubated for 24 h. Luciferase activity was measured with the Bright-Glo assay system (Promega), and the activity was determined with an Acquest Ultra-HTS system (LJL Biosystems, Inc.).

    Techniques: Inhibition, Blocking Assay, Luciferase, Activity Assay, Transfection, Sequencing, Incubation

    Involvement of PBX1 and PBX1-associated proteins in MLV transcription. (A) 293 cells were transiently transfected with an expression plasmid (i.e., CMV-PBX1a, -PBX1b, -MEIS1, -PREP1, or -NFAT) for 24 h before retroviral supernatants of MLV were added. Luciferase activity was determined 48 h after addition of viral supernatants. (B) 293 cells were transiently transfected with mock or CMV-PBX1a plasmids. MLV supernatants were added 24 h after transfection in the presence of the CDKIs (i.e., 1 μM Purv, 1 μM MeO-Ros, or 0.03 μM Flavo). Luciferase activity was measured 48 h after MLV was included. Results were reproducible, and data from one experiment are presented here.

    Journal: Molecular and Cellular Biology

    Article Title: Identification of Homeodomain Proteins, PBX1 and PREP1, Involved in the Transcription of Murine Leukemia Virus

    doi: 10.1128/MCB.23.3.831-841.2003

    Figure Lengend Snippet: Involvement of PBX1 and PBX1-associated proteins in MLV transcription. (A) 293 cells were transiently transfected with an expression plasmid (i.e., CMV-PBX1a, -PBX1b, -MEIS1, -PREP1, or -NFAT) for 24 h before retroviral supernatants of MLV were added. Luciferase activity was determined 48 h after addition of viral supernatants. (B) 293 cells were transiently transfected with mock or CMV-PBX1a plasmids. MLV supernatants were added 24 h after transfection in the presence of the CDKIs (i.e., 1 μM Purv, 1 μM MeO-Ros, or 0.03 μM Flavo). Luciferase activity was measured 48 h after MLV was included. Results were reproducible, and data from one experiment are presented here.

    Article Snippet: 3T3 or 293 cells were grown to 50 to 80% confluence in 96-well plates and incubated with individual CDKIs for 30 min. MLV-LTR-Luc supernatants were then added and incubated for 24 h. Luciferase activity was measured with the Bright-Glo assay system (Promega), and the activity was determined with an Acquest Ultra-HTS system (LJL Biosystems, Inc.).

    Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay

    Endocytosis in Env-expressing cells is required for the cell-cell fusion. (A) The E-MLV R-Env (left panel) or HIV-1 Env (right panel) was transfected to HEK293T cells together with the nls-LacZ expression plasmid. The transfected HEK293T cells were co-culture with TE671mCAT1 or NP2/CD4/X4 cells pretreated with indicated inhibitor (black bars). The transfected cells were treated with indicated inhibitors, and were co-cultured with TE671mCAT1 or NP2/CD4/X4 cells (gray bar). This experiment was repeated three times. Fusion indexes in the control cells were always set to 1, and relative fusion indexes ± SD are shown. Asterisks indicate statistically significant differences between treatments of the Env-expressing or target cells. (B) HEK293T, TE671mCAT1, SC-1, and HeLamCAT1 cells were treated with indicated inhibitor. Ratios (%) of live cells per total cells are indicated. This experiment was repeated three times. (C) HEK293T cells were transfected with the MLV R-Env (left panel) or HIV-1 Env (right panel) expression plsmid together with the nls-LacZ expression plasmid. The cells were also simultaneously transfected with the dynamin dominant negative mutant expression plasmid or pcDNA3.1. This experiment was repeated three times. Fusion indexes in the control cells were always set to 1, and relative fusion indexes ± SD are shown. Asterisks indicate statistically significant differences between transfection with pcDNA3.1 and dynamin mutant.

    Journal: Frontiers in Microbiology

    Article Title: Fragments of Target Cells are Internalized into Retroviral Envelope Protein-Expressing Cells during Cell-Cell Fusion by Endocytosis

    doi: 10.3389/fmicb.2015.01552

    Figure Lengend Snippet: Endocytosis in Env-expressing cells is required for the cell-cell fusion. (A) The E-MLV R-Env (left panel) or HIV-1 Env (right panel) was transfected to HEK293T cells together with the nls-LacZ expression plasmid. The transfected HEK293T cells were co-culture with TE671mCAT1 or NP2/CD4/X4 cells pretreated with indicated inhibitor (black bars). The transfected cells were treated with indicated inhibitors, and were co-cultured with TE671mCAT1 or NP2/CD4/X4 cells (gray bar). This experiment was repeated three times. Fusion indexes in the control cells were always set to 1, and relative fusion indexes ± SD are shown. Asterisks indicate statistically significant differences between treatments of the Env-expressing or target cells. (B) HEK293T, TE671mCAT1, SC-1, and HeLamCAT1 cells were treated with indicated inhibitor. Ratios (%) of live cells per total cells are indicated. This experiment was repeated three times. (C) HEK293T cells were transfected with the MLV R-Env (left panel) or HIV-1 Env (right panel) expression plsmid together with the nls-LacZ expression plasmid. The cells were also simultaneously transfected with the dynamin dominant negative mutant expression plasmid or pcDNA3.1. This experiment was repeated three times. Fusion indexes in the control cells were always set to 1, and relative fusion indexes ± SD are shown. Asterisks indicate statistically significant differences between transfection with pcDNA3.1 and dynamin mutant.

    Article Snippet: HEK293T cells seeded in 3-cm plates were transfected with pcDNA3.1, MLV R-Env, or HIV-1 Env (1 μg) together with nls-LacZ (1 μg) by the Fugene HD transfection reagent (Promega), and incubated for 24–48 h at 37°C.

    Techniques: Expressing, Transfection, Plasmid Preparation, Co-Culture Assay, Cell Culture, Dominant Negative Mutation, Mutagenesis

    Target cells are internalized into E-MLV R-Env-expressing cells. (A) Expression plasmids of the E-MLV R-Env (upper panel) or HIV-1 Env (lower panel) were transfected to HEK293T cells together with the GFP expression plasmid. TE671mCAT1 or NP2/CD4/X4 cells were stained with the cell tracker orange, and co-cultured with the transfected cells. These cells were observed under a fluorescent microscopy. Interfaces of syncytia are indicated by blue lines, and nucleic assemblies are surrounded by red lines. Single cells are surrounded by green line for scale. (B) HEK293T cells were transfected with the R+Env and GFP expression plasmids, and co-cultured with cell tracker orange-stained TE671mCAT1 cells (upper panel). HEK293T cells were transfected with the R-Env and GFP expression plasmids, and co-cultured with cell tracker orange-stained TE671 plus unstained TE671mCAT1 cells (middle and lower panels).

    Journal: Frontiers in Microbiology

    Article Title: Fragments of Target Cells are Internalized into Retroviral Envelope Protein-Expressing Cells during Cell-Cell Fusion by Endocytosis

    doi: 10.3389/fmicb.2015.01552

    Figure Lengend Snippet: Target cells are internalized into E-MLV R-Env-expressing cells. (A) Expression plasmids of the E-MLV R-Env (upper panel) or HIV-1 Env (lower panel) were transfected to HEK293T cells together with the GFP expression plasmid. TE671mCAT1 or NP2/CD4/X4 cells were stained with the cell tracker orange, and co-cultured with the transfected cells. These cells were observed under a fluorescent microscopy. Interfaces of syncytia are indicated by blue lines, and nucleic assemblies are surrounded by red lines. Single cells are surrounded by green line for scale. (B) HEK293T cells were transfected with the R+Env and GFP expression plasmids, and co-cultured with cell tracker orange-stained TE671mCAT1 cells (upper panel). HEK293T cells were transfected with the R-Env and GFP expression plasmids, and co-cultured with cell tracker orange-stained TE671 plus unstained TE671mCAT1 cells (middle and lower panels).

    Article Snippet: HEK293T cells seeded in 3-cm plates were transfected with pcDNA3.1, MLV R-Env, or HIV-1 Env (1 μg) together with nls-LacZ (1 μg) by the Fugene HD transfection reagent (Promega), and incubated for 24–48 h at 37°C.

    Techniques: Expressing, Transfection, Plasmid Preparation, Staining, Cell Culture, Microscopy

    Effects of mutations in the PCE on MLV transcription. (A) Nucleotide sequences of the wild-type (WT) and mutant PCEs. (B) A gel shift assay was performed with 293 cell nuclear extracts with different DNA probes including wild-type PCE (WT), PCE 1mt (1mt), or PCE 2mt (2mt). C1 and C2, PCE-protein complexes. (C) 293 cells were transiently transfected with the WZL-Luc vector containing wild-type or mutant PCEs. Luciferase activity was measured 48 h after transfection. Results were reproducible, and the assay was repeated more than three times in triplicate.

    Journal: Molecular and Cellular Biology

    Article Title: Identification of Homeodomain Proteins, PBX1 and PREP1, Involved in the Transcription of Murine Leukemia Virus

    doi: 10.1128/MCB.23.3.831-841.2003

    Figure Lengend Snippet: Effects of mutations in the PCE on MLV transcription. (A) Nucleotide sequences of the wild-type (WT) and mutant PCEs. (B) A gel shift assay was performed with 293 cell nuclear extracts with different DNA probes including wild-type PCE (WT), PCE 1mt (1mt), or PCE 2mt (2mt). C1 and C2, PCE-protein complexes. (C) 293 cells were transiently transfected with the WZL-Luc vector containing wild-type or mutant PCEs. Luciferase activity was measured 48 h after transfection. Results were reproducible, and the assay was repeated more than three times in triplicate.

    Article Snippet: The first, MLV-LTR-Luc, used exclusively in retroviral infection assays, was derived from pBabe-neo , wherein (i) the simian virus 40 promoter was replaced with the internal ribosome entry site (IRES) of hepatitis C virus, (ii) the neomycin resistance gene was changed to the luciferase gene from the pGL2 control plasmid (Promega), and (iii) the U3 region of the 5′ LTR was replaced with the strong cytomegalovirus (CMV) promoter for higher-titer virus production.

    Techniques: Mutagenesis, Electrophoretic Mobility Shift Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay

    Inhibition of MLV transcription by blocking PBX1 protein synthesis. (A) 293 cells were cotransfected with WZL-Luc and a Pbx1 -targeted antisense oligonucleotide. Each Pbx1 antisense nucleotide was paired with a sense nucleotide as control. Luciferase activity was measured 24 h after transfection. A pcDNA3.1 reverse primer, used for sequencing the insert of pcDNA plasmids (Invitrogen), served as another control. The pcDNA3.1 primer and two sense Pbx1 oligonucleotides had similar effects on viral transcription (data not shown). (B) 293 cells were transiently transfected with an siRNA of Pbx1 for 24 h and then incubated with MLV viral supernatants for another 24 h. An siRNA of Renilla luciferase was used as control. The assay was repeated twice in triplicate with the same results.

    Journal: Molecular and Cellular Biology

    Article Title: Identification of Homeodomain Proteins, PBX1 and PREP1, Involved in the Transcription of Murine Leukemia Virus

    doi: 10.1128/MCB.23.3.831-841.2003

    Figure Lengend Snippet: Inhibition of MLV transcription by blocking PBX1 protein synthesis. (A) 293 cells were cotransfected with WZL-Luc and a Pbx1 -targeted antisense oligonucleotide. Each Pbx1 antisense nucleotide was paired with a sense nucleotide as control. Luciferase activity was measured 24 h after transfection. A pcDNA3.1 reverse primer, used for sequencing the insert of pcDNA plasmids (Invitrogen), served as another control. The pcDNA3.1 primer and two sense Pbx1 oligonucleotides had similar effects on viral transcription (data not shown). (B) 293 cells were transiently transfected with an siRNA of Pbx1 for 24 h and then incubated with MLV viral supernatants for another 24 h. An siRNA of Renilla luciferase was used as control. The assay was repeated twice in triplicate with the same results.

    Article Snippet: The first, MLV-LTR-Luc, used exclusively in retroviral infection assays, was derived from pBabe-neo , wherein (i) the simian virus 40 promoter was replaced with the internal ribosome entry site (IRES) of hepatitis C virus, (ii) the neomycin resistance gene was changed to the luciferase gene from the pGL2 control plasmid (Promega), and (iii) the U3 region of the 5′ LTR was replaced with the strong cytomegalovirus (CMV) promoter for higher-titer virus production.

    Techniques: Inhibition, Blocking Assay, Luciferase, Activity Assay, Transfection, Sequencing, Incubation

    Involvement of PBX1 and PBX1-associated proteins in MLV transcription. (A) 293 cells were transiently transfected with an expression plasmid (i.e., CMV-PBX1a, -PBX1b, -MEIS1, -PREP1, or -NFAT) for 24 h before retroviral supernatants of MLV were added. Luciferase activity was determined 48 h after addition of viral supernatants. (B) 293 cells were transiently transfected with mock or CMV-PBX1a plasmids. MLV supernatants were added 24 h after transfection in the presence of the CDKIs (i.e., 1 μM Purv, 1 μM MeO-Ros, or 0.03 μM Flavo). Luciferase activity was measured 48 h after MLV was included. Results were reproducible, and data from one experiment are presented here.

    Journal: Molecular and Cellular Biology

    Article Title: Identification of Homeodomain Proteins, PBX1 and PREP1, Involved in the Transcription of Murine Leukemia Virus

    doi: 10.1128/MCB.23.3.831-841.2003

    Figure Lengend Snippet: Involvement of PBX1 and PBX1-associated proteins in MLV transcription. (A) 293 cells were transiently transfected with an expression plasmid (i.e., CMV-PBX1a, -PBX1b, -MEIS1, -PREP1, or -NFAT) for 24 h before retroviral supernatants of MLV were added. Luciferase activity was determined 48 h after addition of viral supernatants. (B) 293 cells were transiently transfected with mock or CMV-PBX1a plasmids. MLV supernatants were added 24 h after transfection in the presence of the CDKIs (i.e., 1 μM Purv, 1 μM MeO-Ros, or 0.03 μM Flavo). Luciferase activity was measured 48 h after MLV was included. Results were reproducible, and data from one experiment are presented here.

    Article Snippet: The first, MLV-LTR-Luc, used exclusively in retroviral infection assays, was derived from pBabe-neo , wherein (i) the simian virus 40 promoter was replaced with the internal ribosome entry site (IRES) of hepatitis C virus, (ii) the neomycin resistance gene was changed to the luciferase gene from the pGL2 control plasmid (Promega), and (iii) the U3 region of the 5′ LTR was replaced with the strong cytomegalovirus (CMV) promoter for higher-titer virus production.

    Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay

    Binding of the Gypsy NC-like peptide to Gypsy 5′ and 3′ RNAs. Gypsy NC-like peptide and 32 P-labelled 5′ and 3′ RNAs were synthesized as described in Materials and Methods. Binding of Gypsy NC (G-NC) was monitored by gel retardation (data not shown) and nucleoprotein complex formation. Complexes were recovered by centrifugation as reported in methods. The same experiments were carried out with the TYA1-D peptide and with MLV and HIV-NC peptides (see Materials and Methods for peptide sequences). Optimal complex formation with the G-NC and TYA1-D peptides was found to take place at a protein to nt molar ratio of 1 to 6, as previously observed for retroviral NC proteins, such as HIV-1 NCp7 and MuLV NCp10 ( 20 , 45 ). Note that the basic HIV-NC and MLV-NC peptides did not form large amounts of ribonucleoprotein complexes (HIV-NC and MLV-NC on both panels); although they bind RNA [see Refs ( 20 – 22 )]. CT stands for RNA alone.

    Journal: Nucleic Acids Research

    Article Title: Characterization of a nucleocapsid-like region and of two distinct primer tRNALys,2 binding sites in the endogenous retrovirus Gypsy

    doi: 10.1093/nar/gkl722

    Figure Lengend Snippet: Binding of the Gypsy NC-like peptide to Gypsy 5′ and 3′ RNAs. Gypsy NC-like peptide and 32 P-labelled 5′ and 3′ RNAs were synthesized as described in Materials and Methods. Binding of Gypsy NC (G-NC) was monitored by gel retardation (data not shown) and nucleoprotein complex formation. Complexes were recovered by centrifugation as reported in methods. The same experiments were carried out with the TYA1-D peptide and with MLV and HIV-NC peptides (see Materials and Methods for peptide sequences). Optimal complex formation with the G-NC and TYA1-D peptides was found to take place at a protein to nt molar ratio of 1 to 6, as previously observed for retroviral NC proteins, such as HIV-1 NCp7 and MuLV NCp10 ( 20 , 45 ). Note that the basic HIV-NC and MLV-NC peptides did not form large amounts of ribonucleoprotein complexes (HIV-NC and MLV-NC on both panels); although they bind RNA [see Refs ( 20 – 22 )]. CT stands for RNA alone.

    Article Snippet: Binding of the Gypsy NC peptide to RNA 32 P-labelled Gypsy 5′ or 3′ RNA (5 × 10−8 M) was incubated with Gypsy NC, TYA1-D, MLV-NC or HIV-1 NC at protein to nucleotide molar ratios as indicated in the figure legend, at 30°C for 5 min in 10 μl assays containing 20 mM Tris–HCl (pH 7.5), 30 mM NaCl, 0.1 mM MgCl2 , 5 mM DTT, 0.01 mM ZnCl2 and 8 U RNasin (Promega).

    Techniques: Binding Assay, Synthesized, Electrophoretic Mobility Shift Assay, Centrifugation

    Role of the Gypsy NC peptide in cDNA synthesis in vitro . ( A ) Schematic representation of the initiation of reverse transcription on Gypsy 5′ RNA. Reverse transcription of Gypsy 5′ U5 and R RNA sequences leads to the synthesis of the so-called minus strand strong stop cDNA, ss-cDNA(−), by RT extension of primer tRNA Lys,2 . ( B ) Gypsy 5′ RNA, 3′ RNA or recombinant 5′–3′ RNA and 32 P-tRNA Lys,2 were incubated with or without TYA1-D or Gypsy NC peptide. MLV RT was added together with dNTPs to allow reverse transcription. Assays were processed as described in Materials and Methods and ss-cDNA(−) was denatured and analysed by 10% PAGE in 7 M urea. Controls without protein are shown in lanes 1, 8, 11, 18 and 21. Protein to RNA nucleotide molar ratios were 1:48 (lanes 2, 5, 12, 15, 22 and 25), 1:24 (lanes 3, 6, 13, 16, 23 and 26) and 1:12 (lanes 4, 7, 9, 10, 14, 17, 19, 20, 24 and 27), corresponding to 1.25 × 10 −7 , 2.5 × 10 −7 and 5 × 10 −7 M for 5′ RNA and 2.5 × 10 −7 , 5 × 10 −7 and 10 −6 M for 5′–3′ RNA. Note that hybridization of tRNA Lys,2 to the 3′ PBS caused an inhibition of the initiation of Gypsy reverse transcription which was severe with the TYA1-D peptide (compare lanes 2–4 with 12–14) and moderate with the homologous peptide (compare lanes 5–7 with 15–17).

    Journal: Nucleic Acids Research

    Article Title: Characterization of a nucleocapsid-like region and of two distinct primer tRNALys,2 binding sites in the endogenous retrovirus Gypsy

    doi: 10.1093/nar/gkl722

    Figure Lengend Snippet: Role of the Gypsy NC peptide in cDNA synthesis in vitro . ( A ) Schematic representation of the initiation of reverse transcription on Gypsy 5′ RNA. Reverse transcription of Gypsy 5′ U5 and R RNA sequences leads to the synthesis of the so-called minus strand strong stop cDNA, ss-cDNA(−), by RT extension of primer tRNA Lys,2 . ( B ) Gypsy 5′ RNA, 3′ RNA or recombinant 5′–3′ RNA and 32 P-tRNA Lys,2 were incubated with or without TYA1-D or Gypsy NC peptide. MLV RT was added together with dNTPs to allow reverse transcription. Assays were processed as described in Materials and Methods and ss-cDNA(−) was denatured and analysed by 10% PAGE in 7 M urea. Controls without protein are shown in lanes 1, 8, 11, 18 and 21. Protein to RNA nucleotide molar ratios were 1:48 (lanes 2, 5, 12, 15, 22 and 25), 1:24 (lanes 3, 6, 13, 16, 23 and 26) and 1:12 (lanes 4, 7, 9, 10, 14, 17, 19, 20, 24 and 27), corresponding to 1.25 × 10 −7 , 2.5 × 10 −7 and 5 × 10 −7 M for 5′ RNA and 2.5 × 10 −7 , 5 × 10 −7 and 10 −6 M for 5′–3′ RNA. Note that hybridization of tRNA Lys,2 to the 3′ PBS caused an inhibition of the initiation of Gypsy reverse transcription which was severe with the TYA1-D peptide (compare lanes 2–4 with 12–14) and moderate with the homologous peptide (compare lanes 5–7 with 15–17).

    Article Snippet: Binding of the Gypsy NC peptide to RNA 32 P-labelled Gypsy 5′ or 3′ RNA (5 × 10−8 M) was incubated with Gypsy NC, TYA1-D, MLV-NC or HIV-1 NC at protein to nucleotide molar ratios as indicated in the figure legend, at 30°C for 5 min in 10 μl assays containing 20 mM Tris–HCl (pH 7.5), 30 mM NaCl, 0.1 mM MgCl2 , 5 mM DTT, 0.01 mM ZnCl2 and 8 U RNasin (Promega).

    Techniques: In Vitro, Recombinant, Incubation, Polyacrylamide Gel Electrophoresis, Hybridization, Inhibition

    Hybridization of primer tRNA Lys,2 to the Gypsy 5′ and 3′ PBSs. Gypsy RNA and 32 P-labelled primer tRNA Lys,2 were incubated at 30°C for 5 min. RNA complexes were purified by SDS-PK treatment and phenol extraction (see Materials and Methods). The nature of the RNA, 5′ wt, Δ5′ PBS, 3′ wt, Δ3′ PBS, recombinant 5′–3′ (either wt, Δ5′ PBS, Δ3′ PBS or Δ5′–Δ3′ PBS) is indicated at the top of each panel. Panel ( A ) stands for 5′ RNA, ( B ) for 3′ RNA and ( C ) for 5′–3′ RNA. Control was with Gypsy RNA and 32 P-labelled tRNA Lys,2 but without NC protein as shown in lanes 1 and 12 (A and B), and 1 and 19 (C). (A and B): TYA1-D and Gypsy NC (G-NC) were added to the assays at protein to nucleotide molar ratios of 1:30, 1:15 and 1:7 (lanes 2–4 and 5–7, respectively). MLV and HIV-NC peptides were at peptide to nt molar ratios of 1:15 and 1:7 (lanes 8–9 and 10–11, respectively). When the Δ5′ PBS RNA or the Δ3′ PBS RNA was used, the peptide to nt ratio was 1:7 (lanes 13–14). Note that primer tRNA Lys,2 annealed at a low level to the 5′ RNA without the chaperone [(A) lane 1] and annealing was optimal at protein to nt molar ratios of 1:15 to 1:7 [lanes 3–4 and 6–7 in (A)]. Quantifications made by laser scanning indicated that total percentages of tRNA annealed to the 5′ PBS increased from 15–20 to 55–65% upon addition of TYA1-D and G-NC, but did not change after addition of the MLV and HIV peptides (average values of three independent assays). Primer tRNA Lys,2 did not anneal to the 3′ RNA without chaperone [(B), lane 1] and annealing was optimal at protein to nt molar ratio 1:7 (lanes 4 and 7). Quantifications made by laser scanning indicated that total percentages of tRNA annealed to the 3′ PBS increased from 0 to 40–50% upon addition of TYA1-D and G-NC, but did not change after addition of the MLV and HIV peptides (average values of three independent assays). (C): TYA1-D and Gypsy NC (G-NC) were added to the assays at protein to nucleotide molar ratios of 1:15 and 1:7 (lanes 2–3, 7–8 and 12–13, and 4–5, 9–10, and 14–15, respectively). For Δ 5′–Δ3′ PBS RNA, ratio was 1:7 (lanes 17–18). Additional experiments with TYA1-D and G-NC were at molar NC to nt ratios of 1:30, 1:15 and 1:7 (lanes 20–22 and 23–25, respectively) and with MLV and HIV peptides ratios were 1:15 and 1:7 (lanes 26–27 and 28–29, respectively). Quantifications made by laser scanning indicated that total percentages of tRNA annealed to the 5′ and 3′ PBSs increased from 8–12 to 40–85% upon addition of TYA1-D and G-NC, but did not change after addition of the MLV and HIV peptides (average values of three independent assays; see also Figure 7 ). Sizes of tRNA and Gypsy RNAs (in nt) are indicated on the right for 5′ RNA (433 nt), 3′ RNA (525 nt), 5′–3′ RNA (959 nt) and tRNA Lys,2 (76 nt).

    Journal: Nucleic Acids Research

    Article Title: Characterization of a nucleocapsid-like region and of two distinct primer tRNALys,2 binding sites in the endogenous retrovirus Gypsy

    doi: 10.1093/nar/gkl722

    Figure Lengend Snippet: Hybridization of primer tRNA Lys,2 to the Gypsy 5′ and 3′ PBSs. Gypsy RNA and 32 P-labelled primer tRNA Lys,2 were incubated at 30°C for 5 min. RNA complexes were purified by SDS-PK treatment and phenol extraction (see Materials and Methods). The nature of the RNA, 5′ wt, Δ5′ PBS, 3′ wt, Δ3′ PBS, recombinant 5′–3′ (either wt, Δ5′ PBS, Δ3′ PBS or Δ5′–Δ3′ PBS) is indicated at the top of each panel. Panel ( A ) stands for 5′ RNA, ( B ) for 3′ RNA and ( C ) for 5′–3′ RNA. Control was with Gypsy RNA and 32 P-labelled tRNA Lys,2 but without NC protein as shown in lanes 1 and 12 (A and B), and 1 and 19 (C). (A and B): TYA1-D and Gypsy NC (G-NC) were added to the assays at protein to nucleotide molar ratios of 1:30, 1:15 and 1:7 (lanes 2–4 and 5–7, respectively). MLV and HIV-NC peptides were at peptide to nt molar ratios of 1:15 and 1:7 (lanes 8–9 and 10–11, respectively). When the Δ5′ PBS RNA or the Δ3′ PBS RNA was used, the peptide to nt ratio was 1:7 (lanes 13–14). Note that primer tRNA Lys,2 annealed at a low level to the 5′ RNA without the chaperone [(A) lane 1] and annealing was optimal at protein to nt molar ratios of 1:15 to 1:7 [lanes 3–4 and 6–7 in (A)]. Quantifications made by laser scanning indicated that total percentages of tRNA annealed to the 5′ PBS increased from 15–20 to 55–65% upon addition of TYA1-D and G-NC, but did not change after addition of the MLV and HIV peptides (average values of three independent assays). Primer tRNA Lys,2 did not anneal to the 3′ RNA without chaperone [(B), lane 1] and annealing was optimal at protein to nt molar ratio 1:7 (lanes 4 and 7). Quantifications made by laser scanning indicated that total percentages of tRNA annealed to the 3′ PBS increased from 0 to 40–50% upon addition of TYA1-D and G-NC, but did not change after addition of the MLV and HIV peptides (average values of three independent assays). (C): TYA1-D and Gypsy NC (G-NC) were added to the assays at protein to nucleotide molar ratios of 1:15 and 1:7 (lanes 2–3, 7–8 and 12–13, and 4–5, 9–10, and 14–15, respectively). For Δ 5′–Δ3′ PBS RNA, ratio was 1:7 (lanes 17–18). Additional experiments with TYA1-D and G-NC were at molar NC to nt ratios of 1:30, 1:15 and 1:7 (lanes 20–22 and 23–25, respectively) and with MLV and HIV peptides ratios were 1:15 and 1:7 (lanes 26–27 and 28–29, respectively). Quantifications made by laser scanning indicated that total percentages of tRNA annealed to the 5′ and 3′ PBSs increased from 8–12 to 40–85% upon addition of TYA1-D and G-NC, but did not change after addition of the MLV and HIV peptides (average values of three independent assays; see also Figure 7 ). Sizes of tRNA and Gypsy RNAs (in nt) are indicated on the right for 5′ RNA (433 nt), 3′ RNA (525 nt), 5′–3′ RNA (959 nt) and tRNA Lys,2 (76 nt).

    Article Snippet: Binding of the Gypsy NC peptide to RNA 32 P-labelled Gypsy 5′ or 3′ RNA (5 × 10−8 M) was incubated with Gypsy NC, TYA1-D, MLV-NC or HIV-1 NC at protein to nucleotide molar ratios as indicated in the figure legend, at 30°C for 5 min in 10 μl assays containing 20 mM Tris–HCl (pH 7.5), 30 mM NaCl, 0.1 mM MgCl2 , 5 mM DTT, 0.01 mM ZnCl2 and 8 U RNasin (Promega).

    Techniques: Hybridization, Incubation, Purification, Recombinant