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  • 99
    New England Biolabs bamhi bamhi
    Effect of partial APRT1 knockout on cell sensitivity to 6mp. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector <t>pΔAPRT1-NEO5,</t> with paromomycin resistance cassette ( NEO5 ) (middle), and after homologous recombination (lower). ( B ) Southern blot analysis of XhoI- and <t>BamHI-digested</t> genomic DNA from wild-type cells and partial APRT1 knockout cells. Molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h. ( D ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after measurement of cell growth rates in 6mp. ( E ) Southern blot analysis of XhoI-and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after the induction of phenotypic assortment with 5 mg/mL paromomycin for 2 months. Molecular weight of signals against the probe corresponds to that predicted in the schematic in ( A ).
    Bamhi Bamhi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher bamhi
    Joining of incompatible <t>DNA</t> ends by MRN and DNA ligase IIIα/XRCC1: microhomology-mediated joining of DNA ends with incompatible 5′ overhangs. A , circular plasmid DNA was digested with either KpnI ( left ) and PstI ( right ) to generate noncomplementary 3′ overhangs or EcoRI ( left ) and <t>BamHI</t> ( right ), to generate noncomplementary 5′ overhangs. Joining of the incompatible DNA ends was detected by the PCR using the indicated primers. B , DNA substrate (0.8 pmol) with noncomplementary 3′ overhangs was incubated with 0.01 pmol of DNA ligase IIIα/XRCC1 alone or in combination with equimolar amounts of hMre11 and MRN as indicated. C , DNA substrate (0.8 pmol) with noncomplementary 5′ overhangs was incubated with 0.01 pmol of DNA ligase IIIα/XRCC1 alone or in combination with equimolar amounts of hMre11 and MRN as indicated. D , DNA substrate (0.8 pmol) with noncomplementary 5′ overhangs was incubated with 0.01 pmol of either DNA ligase IIIα/XRCC1 or DNA ligase IV/XRCC4 in the absence or presence of an equimolar amount of MRN. E , sequences of the junctions generated by the joining of incompatible 5′ ends by DNA ligase IIIα/XRCC1 in combination with either hMre11 or MRN are shown. Bolded residues indicate microhomologies.
    Bamhi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13718 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher bamhi fragment
    Golden Gate-based multisite mutagenesis. A) The FlpO ORF contains two internal BsaI sites. These are mutated by amplifying fragments of the FlpO ORF via PCR. The primers contain flanking BsaI sites that lead to compatible overhangs after restriction digest. The site-directed mutagenesis vector (pGG-sdm) contains BsaI sites for the mutagenesis assembly. Additionally, <t>BamHI</t> and <t>KpnI</t> sites allow the transfer of the mutated DNA assembly to Golden Gate entry vectors. From there, mRNA can be generated using SP6 RNA polymerase. Note that the pGG-sdm vector represents a standard Gateway TM middle entry vector. B) A Golden Gate reaction is used to prepare a dual FRT-based recombination construct in the Gateway TM middle entry vector. An LR reaction was prepared to generate a final expression construct. ISceI sites flank the expression cassette. The dual FRT element is driven by the zebrafish beta actin 2 promoter and a human globin intron with SV40 polyA is used in the 3’ entry vector. C) Zebrafish embryos were injected with the expression construct alone or in combination with FlpO mRNA. Without FlpO mRNA 64% (n=98) of the fish were positively injected and showed eGFP expression and the absence of mCherry. In combination with FlpO mRNA 60% (n=79) of the fish were positively injected and all of them showed the complete absence of green fluorescence and the appearance of red fluorescence, which is indicative for proper FlpO activity.
    Bamhi Fragment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 262 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bamhi
    Plasmids used for TLS assays. ( A ) Construction of an AP site-containing plasmid. Plasmid pVP15 is linearized by BbrPI digestion. The <t>PstI-BamHI</t> fragment of <t>pVP9</t> is removed by digestion and purification on a microcon YM-30 column. Both linearized plasmids
    Bamhi, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6617 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher fastdigest bamhi
    Plasmids used for TLS assays. ( A ) Construction of an AP site-containing plasmid. Plasmid pVP15 is linearized by BbrPI digestion. The <t>PstI-BamHI</t> fragment of <t>pVP9</t> is removed by digestion and purification on a microcon YM-30 column. Both linearized plasmids
    Fastdigest Bamhi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 263 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore restriction enzymes bamhi
    Plasmids used for TLS assays. ( A ) Construction of an AP site-containing plasmid. Plasmid pVP15 is linearized by BbrPI digestion. The <t>PstI-BamHI</t> fragment of <t>pVP9</t> is removed by digestion and purification on a microcon YM-30 column. Both linearized plasmids
    Restriction Enzymes Bamhi, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effect of partial APRT1 knockout on cell sensitivity to 6mp. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pΔAPRT1-NEO5, with paromomycin resistance cassette ( NEO5 ) (middle), and after homologous recombination (lower). ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and partial APRT1 knockout cells. Molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h. ( D ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after measurement of cell growth rates in 6mp. ( E ) Southern blot analysis of XhoI-and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after the induction of phenotypic assortment with 5 mg/mL paromomycin for 2 months. Molecular weight of signals against the probe corresponds to that predicted in the schematic in ( A ).

    Journal: Genes

    Article Title: Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena

    doi: 10.3390/genes9040179

    Figure Lengend Snippet: Effect of partial APRT1 knockout on cell sensitivity to 6mp. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pΔAPRT1-NEO5, with paromomycin resistance cassette ( NEO5 ) (middle), and after homologous recombination (lower). ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and partial APRT1 knockout cells. Molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h. ( D ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after measurement of cell growth rates in 6mp. ( E ) Southern blot analysis of XhoI-and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after the induction of phenotypic assortment with 5 mg/mL paromomycin for 2 months. Molecular weight of signals against the probe corresponds to that predicted in the schematic in ( A ).

    Article Snippet: Amplified forward and reverse target fragments were cloned into the BamHI–BamHI and PstI–PstI I sites, respectively, of pAkRNAi-NEO5 with the NEBuilder HF DNA Assembly Kit (New England BioLabs) to create the hairpin cassette.

    Techniques: Knock-Out, Plasmid Preparation, Homologous Recombination, Southern Blot, Molecular Weight

    Distinct Influences of the Ku80 C-terminus on DNA-PK Activation with Linearized Plasmid DNA. a) DNA-PK kinase stimulation with plasmid DNA linearized with EcoRV generating blunt-ended termini and with KpnI generating 4 base 3’ single stranded overhangs. b) DNA-PK kinase stimulation with plasmid DNA linearized with XhoI and BamHI generating 4 base 5’ single stranded overhangs. DNA substrates are depicted pictorially. DNA termini generated by digestion are depicted below each graph indicating locations of pyrimidines (Py) and purines (Pu). Kinase activity is reported as the mean and SD of pmol of phosphate transferred. Asterisks indicate statistically significant differences compared to wild type (p

    Journal: PLoS ONE

    Article Title: Recognition of DNA Termini by the C-Terminal Region of the Ku80 and the DNA-Dependent Protein Kinase Catalytic Subunit

    doi: 10.1371/journal.pone.0127321

    Figure Lengend Snippet: Distinct Influences of the Ku80 C-terminus on DNA-PK Activation with Linearized Plasmid DNA. a) DNA-PK kinase stimulation with plasmid DNA linearized with EcoRV generating blunt-ended termini and with KpnI generating 4 base 3’ single stranded overhangs. b) DNA-PK kinase stimulation with plasmid DNA linearized with XhoI and BamHI generating 4 base 5’ single stranded overhangs. DNA substrates are depicted pictorially. DNA termini generated by digestion are depicted below each graph indicating locations of pyrimidines (Py) and purines (Pu). Kinase activity is reported as the mean and SD of pmol of phosphate transferred. Asterisks indicate statistically significant differences compared to wild type (p

    Article Snippet: Kinase assays using plasmid DNA substrates were performed with pcDNA3.1 digested with either XhoI, BamHI, EcoRV, and KpnI or pCAG-GFP digested with XbaI or EcoRI (New England Biolabs).

    Techniques: Activation Assay, Plasmid Preparation, Generated, Activity Assay

    Joining of incompatible DNA ends by MRN and DNA ligase IIIα/XRCC1: microhomology-mediated joining of DNA ends with incompatible 5′ overhangs. A , circular plasmid DNA was digested with either KpnI ( left ) and PstI ( right ) to generate noncomplementary 3′ overhangs or EcoRI ( left ) and BamHI ( right ), to generate noncomplementary 5′ overhangs. Joining of the incompatible DNA ends was detected by the PCR using the indicated primers. B , DNA substrate (0.8 pmol) with noncomplementary 3′ overhangs was incubated with 0.01 pmol of DNA ligase IIIα/XRCC1 alone or in combination with equimolar amounts of hMre11 and MRN as indicated. C , DNA substrate (0.8 pmol) with noncomplementary 5′ overhangs was incubated with 0.01 pmol of DNA ligase IIIα/XRCC1 alone or in combination with equimolar amounts of hMre11 and MRN as indicated. D , DNA substrate (0.8 pmol) with noncomplementary 5′ overhangs was incubated with 0.01 pmol of either DNA ligase IIIα/XRCC1 or DNA ligase IV/XRCC4 in the absence or presence of an equimolar amount of MRN. E , sequences of the junctions generated by the joining of incompatible 5′ ends by DNA ligase IIIα/XRCC1 in combination with either hMre11 or MRN are shown. Bolded residues indicate microhomologies.

    Journal: The Journal of Biological Chemistry

    Article Title: Human Mre11/Human Rad50/Nbs1 and DNA Ligase III?/XRCC1 Protein Complexes Act Together in an Alternative Nonhomologous End Joining Pathway *

    doi: 10.1074/jbc.M111.274159

    Figure Lengend Snippet: Joining of incompatible DNA ends by MRN and DNA ligase IIIα/XRCC1: microhomology-mediated joining of DNA ends with incompatible 5′ overhangs. A , circular plasmid DNA was digested with either KpnI ( left ) and PstI ( right ) to generate noncomplementary 3′ overhangs or EcoRI ( left ) and BamHI ( right ), to generate noncomplementary 5′ overhangs. Joining of the incompatible DNA ends was detected by the PCR using the indicated primers. B , DNA substrate (0.8 pmol) with noncomplementary 3′ overhangs was incubated with 0.01 pmol of DNA ligase IIIα/XRCC1 alone or in combination with equimolar amounts of hMre11 and MRN as indicated. C , DNA substrate (0.8 pmol) with noncomplementary 5′ overhangs was incubated with 0.01 pmol of DNA ligase IIIα/XRCC1 alone or in combination with equimolar amounts of hMre11 and MRN as indicated. D , DNA substrate (0.8 pmol) with noncomplementary 5′ overhangs was incubated with 0.01 pmol of either DNA ligase IIIα/XRCC1 or DNA ligase IV/XRCC4 in the absence or presence of an equimolar amount of MRN. E , sequences of the junctions generated by the joining of incompatible 5′ ends by DNA ligase IIIα/XRCC1 in combination with either hMre11 or MRN are shown. Bolded residues indicate microhomologies.

    Article Snippet: Linear DNA molecules were generated by digestion of pGADT7 (Invitrogen) with BamHI and EcoRI for the four nucleotide 5′ overhangs and of pcDNA4HisMax C (Invitrogen) with KpnI and PstI for the four nucleotide 3′ overhangs.

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Incubation, Generated

    Golden Gate-based multisite mutagenesis. A) The FlpO ORF contains two internal BsaI sites. These are mutated by amplifying fragments of the FlpO ORF via PCR. The primers contain flanking BsaI sites that lead to compatible overhangs after restriction digest. The site-directed mutagenesis vector (pGG-sdm) contains BsaI sites for the mutagenesis assembly. Additionally, BamHI and KpnI sites allow the transfer of the mutated DNA assembly to Golden Gate entry vectors. From there, mRNA can be generated using SP6 RNA polymerase. Note that the pGG-sdm vector represents a standard Gateway TM middle entry vector. B) A Golden Gate reaction is used to prepare a dual FRT-based recombination construct in the Gateway TM middle entry vector. An LR reaction was prepared to generate a final expression construct. ISceI sites flank the expression cassette. The dual FRT element is driven by the zebrafish beta actin 2 promoter and a human globin intron with SV40 polyA is used in the 3’ entry vector. C) Zebrafish embryos were injected with the expression construct alone or in combination with FlpO mRNA. Without FlpO mRNA 64% (n=98) of the fish were positively injected and showed eGFP expression and the absence of mCherry. In combination with FlpO mRNA 60% (n=79) of the fish were positively injected and all of them showed the complete absence of green fluorescence and the appearance of red fluorescence, which is indicative for proper FlpO activity.

    Journal: PLoS ONE

    Article Title: Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs

    doi: 10.1371/journal.pone.0076117

    Figure Lengend Snippet: Golden Gate-based multisite mutagenesis. A) The FlpO ORF contains two internal BsaI sites. These are mutated by amplifying fragments of the FlpO ORF via PCR. The primers contain flanking BsaI sites that lead to compatible overhangs after restriction digest. The site-directed mutagenesis vector (pGG-sdm) contains BsaI sites for the mutagenesis assembly. Additionally, BamHI and KpnI sites allow the transfer of the mutated DNA assembly to Golden Gate entry vectors. From there, mRNA can be generated using SP6 RNA polymerase. Note that the pGG-sdm vector represents a standard Gateway TM middle entry vector. B) A Golden Gate reaction is used to prepare a dual FRT-based recombination construct in the Gateway TM middle entry vector. An LR reaction was prepared to generate a final expression construct. ISceI sites flank the expression cassette. The dual FRT element is driven by the zebrafish beta actin 2 promoter and a human globin intron with SV40 polyA is used in the 3’ entry vector. C) Zebrafish embryos were injected with the expression construct alone or in combination with FlpO mRNA. Without FlpO mRNA 64% (n=98) of the fish were positively injected and showed eGFP expression and the absence of mCherry. In combination with FlpO mRNA 60% (n=79) of the fish were positively injected and all of them showed the complete absence of green fluorescence and the appearance of red fluorescence, which is indicative for proper FlpO activity.

    Article Snippet: For that, we used standard BamHI, KpnI enzymes from Thermo, Fisher together with the Fastdigest buffer (Thermo, Fisher).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Plasmid Preparation, Generated, Construct, Expressing, Injection, Fluorescence In Situ Hybridization, Fluorescence, Activity Assay

    Golden Gate entry vector design and cloning. Cloning cassette used to fill Golden Gate entry vectors. Inserts can be introduced in two ways. XcmI restriction digest generates overhangs that can be used for TA cloning. Second, BamHI and KpnI sites can be used to clone inserts with different length either via standard ligation or an oligo annealing and cloning procedure. This cassette is flanked with BsaI sites used for the Golden Gate assembly. Each entry vector contains an SP6 promoter and a globin intron and SV40 polyA site flanking the insert for the generation of mRNA.

    Journal: PLoS ONE

    Article Title: Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs

    doi: 10.1371/journal.pone.0076117

    Figure Lengend Snippet: Golden Gate entry vector design and cloning. Cloning cassette used to fill Golden Gate entry vectors. Inserts can be introduced in two ways. XcmI restriction digest generates overhangs that can be used for TA cloning. Second, BamHI and KpnI sites can be used to clone inserts with different length either via standard ligation or an oligo annealing and cloning procedure. This cassette is flanked with BsaI sites used for the Golden Gate assembly. Each entry vector contains an SP6 promoter and a globin intron and SV40 polyA site flanking the insert for the generation of mRNA.

    Article Snippet: For that, we used standard BamHI, KpnI enzymes from Thermo, Fisher together with the Fastdigest buffer (Thermo, Fisher).

    Techniques: Plasmid Preparation, Clone Assay, TA Cloning, Ligation

    Genotyping of EBVaGC strains . A: PCR -RFLP of the BamHI-F region showing the f variant (presence of two bands of 128 bp and 71 bp) for the control C666-1 cell line (lane 1) and one band of 199 bp for the F variant for cases GC2 to GC6 (lane 2 to 6). B: PCR-RFLP of the BamHI-W1/I1 region. DNA fragments were digested with BamHI giving two bands of 139 and 67 bp (type D) for GC2 to GC6 (lane 2 to 6) or one band of 245 bp (type C) for the control C666-1 cell line (lane 1). C: EBNA3C region. EBV strains of types A and B correspond to DNA fragment of 153 and 246 bp respectively. Case GC6 shows dual infection with both types A and B viruses. B95.8 is a type A virus that was used as a control (lane 1). D: XhoI polymorphism in exon 1 of the BNLF1 gene. PCR product was digested by XhoI to yield two bands of 343 bp and 154 bp in XhoI + variant for GC2 to GC6 (lane 2 to 6). C666-1 cell line is a positive of loss- XhoI variant (lane 1).

    Journal: Virology Journal

    Article Title: Characteristics of epstein barr virus variants associated with gastric carcinoma in Southern Tunisia

    doi: 10.1186/1743-422X-8-500

    Figure Lengend Snippet: Genotyping of EBVaGC strains . A: PCR -RFLP of the BamHI-F region showing the f variant (presence of two bands of 128 bp and 71 bp) for the control C666-1 cell line (lane 1) and one band of 199 bp for the F variant for cases GC2 to GC6 (lane 2 to 6). B: PCR-RFLP of the BamHI-W1/I1 region. DNA fragments were digested with BamHI giving two bands of 139 and 67 bp (type D) for GC2 to GC6 (lane 2 to 6) or one band of 245 bp (type C) for the control C666-1 cell line (lane 1). C: EBNA3C region. EBV strains of types A and B correspond to DNA fragment of 153 and 246 bp respectively. Case GC6 shows dual infection with both types A and B viruses. B95.8 is a type A virus that was used as a control (lane 1). D: XhoI polymorphism in exon 1 of the BNLF1 gene. PCR product was digested by XhoI to yield two bands of 343 bp and 154 bp in XhoI + variant for GC2 to GC6 (lane 2 to 6). C666-1 cell line is a positive of loss- XhoI variant (lane 1).

    Article Snippet: The enzymatic reactions were carried out in a final volume of 20 μl containing 10 μl of PCR product, 1X digestion buffer and 10 units of BamHI enzyme (Fermentas).

    Techniques: Polymerase Chain Reaction, Variant Assay, Infection

    Electrophoresis of digested pBSC-ptr using enzyme/ Lane 1: Digested pBSC-ptr by KpnI and BamHI/Lane 2: 100-bp DNA ladder marker

    Journal: Iranian Journal of Parasitology

    Article Title: Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct

    doi:

    Figure Lengend Snippet: Electrophoresis of digested pBSC-ptr using enzyme/ Lane 1: Digested pBSC-ptr by KpnI and BamHI/Lane 2: 100-bp DNA ladder marker

    Article Snippet: Construction of the antisense ptr1 gene Recombinant pBluescript containing L. major ptr1 gene (accession no. EF113119) was digested with KpnI and BamHI enzymes and purified using a Fermentas DNA purification kit (Cat. No. k0513) and then subcloned into pcDNA3 digested with KpnI and BamHI.

    Techniques: Electrophoresis, Marker

    Identification of recombinant plasmid pcDNA-rPTR using enzyme digestion. Lane 1:100-bp DNA ladder marker. Lane 2: pcDNA-rPTR digested with KpnI and BamHI

    Journal: Iranian Journal of Parasitology

    Article Title: Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct

    doi:

    Figure Lengend Snippet: Identification of recombinant plasmid pcDNA-rPTR using enzyme digestion. Lane 1:100-bp DNA ladder marker. Lane 2: pcDNA-rPTR digested with KpnI and BamHI

    Article Snippet: Construction of the antisense ptr1 gene Recombinant pBluescript containing L. major ptr1 gene (accession no. EF113119) was digested with KpnI and BamHI enzymes and purified using a Fermentas DNA purification kit (Cat. No. k0513) and then subcloned into pcDNA3 digested with KpnI and BamHI.

    Techniques: Recombinant, Plasmid Preparation, Marker

    Umbravirus 3′ UTRs contain a BTE. (A) Representative predicted (Mfold) secondary structure of the BTE in the 3′ UTR of TBTV genomic RNA, and relative translation activities of selected mutants. 17 nt CS is in bold italics. The shaded bases were deleted in mutants dm1 and dm2 as indicated. In mutants sm1 and sm2, bases in dashed boxes were replaced by bases in solid boxes (dashed arrows). The luciferase activity generated by translation of uncapped mutant RNAs, as a percentage of wild type (100%) are indicated. (B) Genome organization of TBTV RNA and maps of translation reporter constructs containing TBTV UTRs. TUlucTU contains both complete UTRs of TBTV flanking the firefly luciferase coding sequence (fLUC). TUluc has only the TBTV 5′UTR. TUlucTUBF differs from TUlucTU by only a 4 nt insertion (underlined) in the BamH I site. Bases are numbered according to their position in the TBTV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities obtained from the indicated RNAs are normalized to uncapped TUlucTU (defined as 100%) and shown as RLU (relative luciferase units). Error bars indicate standard error. (D) Predicted secondary structure of the GRV BTE. The 17 nt CS (italics) is in the dashed box, with bases that deviate from consensus indicated in bold italics. Mutant sequence (CS) which is identical to the consensus 17 nt CS is indicated at right by dashed arrow. (E) Map of reporter RNA GlucG. GlucG contains the GRV 5′UTR and a chimeric 3′UTR consisting of the TBTV 3′ UTR with the putative GRV BTE in place of the TBTV BTE. Positions of bases from each virus are indicated. (F) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract.

    Journal: Virology

    Article Title: Structural plasticity of Barley yellow dwarf virus-like cap-independent translation elements in four genera of plant viral RNAs

    doi: 10.1016/j.virol.2010.03.025

    Figure Lengend Snippet: Umbravirus 3′ UTRs contain a BTE. (A) Representative predicted (Mfold) secondary structure of the BTE in the 3′ UTR of TBTV genomic RNA, and relative translation activities of selected mutants. 17 nt CS is in bold italics. The shaded bases were deleted in mutants dm1 and dm2 as indicated. In mutants sm1 and sm2, bases in dashed boxes were replaced by bases in solid boxes (dashed arrows). The luciferase activity generated by translation of uncapped mutant RNAs, as a percentage of wild type (100%) are indicated. (B) Genome organization of TBTV RNA and maps of translation reporter constructs containing TBTV UTRs. TUlucTU contains both complete UTRs of TBTV flanking the firefly luciferase coding sequence (fLUC). TUluc has only the TBTV 5′UTR. TUlucTUBF differs from TUlucTU by only a 4 nt insertion (underlined) in the BamH I site. Bases are numbered according to their position in the TBTV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities obtained from the indicated RNAs are normalized to uncapped TUlucTU (defined as 100%) and shown as RLU (relative luciferase units). Error bars indicate standard error. (D) Predicted secondary structure of the GRV BTE. The 17 nt CS (italics) is in the dashed box, with bases that deviate from consensus indicated in bold italics. Mutant sequence (CS) which is identical to the consensus 17 nt CS is indicated at right by dashed arrow. (E) Map of reporter RNA GlucG. GlucG contains the GRV 5′UTR and a chimeric 3′UTR consisting of the TBTV 3′ UTR with the putative GRV BTE in place of the TBTV BTE. Positions of bases from each virus are indicated. (F) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract.

    Article Snippet: It was generated by digesting with BamH I and then filling in the sticky ends with DNA polymerase Pfx (Invitrogen) followed by blunt-end re-ligation. pGlucG was derived by inserting the PCR-amplified 12 nt 5′UTR of GRV into EcoR I/Sca I-digested pTUlucTU, in place of the TBTV 5′ UTR.

    Techniques: Luciferase, Activity Assay, Generated, Mutagenesis, Construct, Sequencing

    ). 17 nt CS is shown in bold italic. (B) Genome organization of RSDaV RNA and maps of translation reporter constructs. RSlucRS has both UTRs of RSDaV flanking the luciferase coding sequence (fLUC). RSluc has only the RSDaV 5′UTR. RSlucRSBF differs from RSlucRS only by a 4 nt GAUC insertion (underlined) in the BamH I site. Bases are numbered according to their position in the RSDaV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities are normalized to uncapped RSlucRS (defined as 100%). Error bars indicate standard error.

    Journal: Virology

    Article Title: Structural plasticity of Barley yellow dwarf virus-like cap-independent translation elements in four genera of plant viral RNAs

    doi: 10.1016/j.virol.2010.03.025

    Figure Lengend Snippet: ). 17 nt CS is shown in bold italic. (B) Genome organization of RSDaV RNA and maps of translation reporter constructs. RSlucRS has both UTRs of RSDaV flanking the luciferase coding sequence (fLUC). RSluc has only the RSDaV 5′UTR. RSlucRSBF differs from RSlucRS only by a 4 nt GAUC insertion (underlined) in the BamH I site. Bases are numbered according to their position in the RSDaV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities are normalized to uncapped RSlucRS (defined as 100%). Error bars indicate standard error.

    Article Snippet: It was generated by digesting with BamH I and then filling in the sticky ends with DNA polymerase Pfx (Invitrogen) followed by blunt-end re-ligation. pGlucG was derived by inserting the PCR-amplified 12 nt 5′UTR of GRV into EcoR I/Sca I-digested pTUlucTU, in place of the TBTV 5′ UTR.

    Techniques: Construct, Luciferase, Sequencing

    Plasmids used for TLS assays. ( A ) Construction of an AP site-containing plasmid. Plasmid pVP15 is linearized by BbrPI digestion. The PstI-BamHI fragment of pVP9 is removed by digestion and purification on a microcon YM-30 column. Both linearized plasmids

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

    Article Title: Mutational specificity and genetic control of replicative bypass of an abasic site in yeast

    doi: 10.1073/pnas.0711227105

    Figure Lengend Snippet: Plasmids used for TLS assays. ( A ) Construction of an AP site-containing plasmid. Plasmid pVP15 is linearized by BbrPI digestion. The PstI-BamHI fragment of pVP9 is removed by digestion and purification on a microcon YM-30 column. Both linearized plasmids

    Article Snippet: Monoadducted heteroduplex plasmids were generated as follows: Plasmids pVP9 and pVP10 were digested with BamHI and PstI ( A ), and the vector portion was separated from the 25-bp insert by centrifugation in a YM-30 microcon filter unit (Millipore).

    Techniques: Plasmid Preparation, Purification