Structured Review

GE Healthcare puc19
Analysis of the I- Uma I target site specificity. CF from induced pAP2 cells was incubated with various substrate plasmids under standard conditions for analysis of cleavage efficiencies (see Materials and Methods). Letters denote the individual substrate plasmids ( e.g. D for <t>pUC19-D).</t> Reaction products were additionally cleaved with Ssp I to yield 2.1 and 0.63 kb fragments in case of cleavage (schematic on the right: the I- Uma I target site fragment is indicated as black box. Xba I cleaves at the right border and in combination with Ssp I produces fragments similar in size to those produced by I- Uma I/ Ssp I). Marker lanes: lin. S, pUC19-D cleaved with Ssp I; lin. X/S, pUC19-D cleaved with Xba I/ Ssp I; s.c./circ., uncleaved pUC19-D showing the supercoiled and circular forms. The + and - symbols refer to the cleavage efficiencies. +++, complete cleavage; ++/+++, > 50% cleavage; ++, ∼50% cleavage; +,
Puc19, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility"

Article Title: The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility

Journal: PLoS ONE

doi: 10.1371/journal.pone.0049551

Analysis of the I- Uma I target site specificity. CF from induced pAP2 cells was incubated with various substrate plasmids under standard conditions for analysis of cleavage efficiencies (see Materials and Methods). Letters denote the individual substrate plasmids ( e.g. D for pUC19-D). Reaction products were additionally cleaved with Ssp I to yield 2.1 and 0.63 kb fragments in case of cleavage (schematic on the right: the I- Uma I target site fragment is indicated as black box. Xba I cleaves at the right border and in combination with Ssp I produces fragments similar in size to those produced by I- Uma I/ Ssp I). Marker lanes: lin. S, pUC19-D cleaved with Ssp I; lin. X/S, pUC19-D cleaved with Xba I/ Ssp I; s.c./circ., uncleaved pUC19-D showing the supercoiled and circular forms. The + and - symbols refer to the cleavage efficiencies. +++, complete cleavage; ++/+++, > 50% cleavage; ++, ∼50% cleavage; +,
Figure Legend Snippet: Analysis of the I- Uma I target site specificity. CF from induced pAP2 cells was incubated with various substrate plasmids under standard conditions for analysis of cleavage efficiencies (see Materials and Methods). Letters denote the individual substrate plasmids ( e.g. D for pUC19-D). Reaction products were additionally cleaved with Ssp I to yield 2.1 and 0.63 kb fragments in case of cleavage (schematic on the right: the I- Uma I target site fragment is indicated as black box. Xba I cleaves at the right border and in combination with Ssp I produces fragments similar in size to those produced by I- Uma I/ Ssp I). Marker lanes: lin. S, pUC19-D cleaved with Ssp I; lin. X/S, pUC19-D cleaved with Xba I/ Ssp I; s.c./circ., uncleaved pUC19-D showing the supercoiled and circular forms. The + and - symbols refer to the cleavage efficiencies. +++, complete cleavage; ++/+++, > 50% cleavage; ++, ∼50% cleavage; +,

Techniques Used: Incubation, Produced, Marker

2) Product Images from "Complementary intrastrand base pairing during initiation of Herpes simplex virus type 1 DNA replication"

Article Title: Complementary intrastrand base pairing during initiation of Herpes simplex virus type 1 DNA replication

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

doi: 10.1073/pnas.121177198

A role for base pairing between box I and box III in DNA replication. The autoradiograph shows a Southern blot analysis of a DNA replication experiment. The plasmids pUC19, pORI(wt), pORI(mut8), pORI(mut9), pORI(mut10), and pORI(mut11) were transfected into BHK cells. The cells were superinfected with HSV-1. Total DNA was isolated and cleaved with Hin dIII and Dpn I. Replicated DNA consists of Dpn I-resistant Hin dIII fragments.
Figure Legend Snippet: A role for base pairing between box I and box III in DNA replication. The autoradiograph shows a Southern blot analysis of a DNA replication experiment. The plasmids pUC19, pORI(wt), pORI(mut8), pORI(mut9), pORI(mut10), and pORI(mut11) were transfected into BHK cells. The cells were superinfected with HSV-1. Total DNA was isolated and cleaved with Hin dIII and Dpn I. Replicated DNA consists of Dpn I-resistant Hin dIII fragments.

Techniques Used: Autoradiography, Southern Blot, Transfection, Isolation

3) Product Images from "The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility"

Article Title: The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility

Journal: PLoS ONE

doi: 10.1371/journal.pone.0049551

Analysis of the I- Uma I target site specificity. CF from induced pAP2 cells was incubated with various substrate plasmids under standard conditions for analysis of cleavage efficiencies (see Materials and Methods). Letters denote the individual substrate plasmids ( e.g. D for pUC19-D). Reaction products were additionally cleaved with Ssp I to yield 2.1 and 0.63 kb fragments in case of cleavage (schematic on the right: the I- Uma I target site fragment is indicated as black box. Xba I cleaves at the right border and in combination with Ssp I produces fragments similar in size to those produced by I- Uma I/ Ssp I). Marker lanes: lin. S, pUC19-D cleaved with Ssp I; lin. X/S, pUC19-D cleaved with Xba I/ Ssp I; s.c./circ., uncleaved pUC19-D showing the supercoiled and circular forms. The + and - symbols refer to the cleavage efficiencies. +++, complete cleavage; ++/+++, > 50% cleavage; ++, ∼50% cleavage; +,
Figure Legend Snippet: Analysis of the I- Uma I target site specificity. CF from induced pAP2 cells was incubated with various substrate plasmids under standard conditions for analysis of cleavage efficiencies (see Materials and Methods). Letters denote the individual substrate plasmids ( e.g. D for pUC19-D). Reaction products were additionally cleaved with Ssp I to yield 2.1 and 0.63 kb fragments in case of cleavage (schematic on the right: the I- Uma I target site fragment is indicated as black box. Xba I cleaves at the right border and in combination with Ssp I produces fragments similar in size to those produced by I- Uma I/ Ssp I). Marker lanes: lin. S, pUC19-D cleaved with Ssp I; lin. X/S, pUC19-D cleaved with Xba I/ Ssp I; s.c./circ., uncleaved pUC19-D showing the supercoiled and circular forms. The + and - symbols refer to the cleavage efficiencies. +++, complete cleavage; ++/+++, > 50% cleavage; ++, ∼50% cleavage; +,

Techniques Used: Incubation, Produced, Marker

4) Product Images from "A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil"

Article Title: A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil

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

doi:

Long-range primer extension reactions on normal and single-uracil templates. ( a ) Denaturing polyacrylamide gel showing products of reactions in which a 31-mer primer was extended against a 119-nucleotide template (see Methods ) containing either a deoxyuridine (U) or a deoxythymidine (T) 23 nucleotides from the 5′ end. Reactions were performed with DNA polymerases from Pyrococcus furiosus ( Pfu ), Pyrococcus woesei ( Pwo ), and Thermus aquaticus ( Taq ). All three polymerases produce full-length products on T templates, but the archaeal enzymes produce smaller major products on U templates. ( b ) Polyacrylamide gel showing the position of the major premature termination product from the primer extension reaction against the U template with Pyrococcus furiosus DNA polymerase ( Pfu exo + ), relative to a sequencing ladder of the segment of pUC19 used in the long-range primer extension reaction (see Methods ). The sequence for the template strand is given, reading 3′→5′ from the bottom (shorter primer extension products) to the top (longer primer extension products). The position of the deoxyuridine (U) is indicated. The major Pfu products correspond to termination of the polymerase reaction 4–6 bases upstream of the template deoxyuridine. The position of the full-length product obtained from primer-extension against the T template (not shown) was consistent with termination at the end of the 119-base template.
Figure Legend Snippet: Long-range primer extension reactions on normal and single-uracil templates. ( a ) Denaturing polyacrylamide gel showing products of reactions in which a 31-mer primer was extended against a 119-nucleotide template (see Methods ) containing either a deoxyuridine (U) or a deoxythymidine (T) 23 nucleotides from the 5′ end. Reactions were performed with DNA polymerases from Pyrococcus furiosus ( Pfu ), Pyrococcus woesei ( Pwo ), and Thermus aquaticus ( Taq ). All three polymerases produce full-length products on T templates, but the archaeal enzymes produce smaller major products on U templates. ( b ) Polyacrylamide gel showing the position of the major premature termination product from the primer extension reaction against the U template with Pyrococcus furiosus DNA polymerase ( Pfu exo + ), relative to a sequencing ladder of the segment of pUC19 used in the long-range primer extension reaction (see Methods ). The sequence for the template strand is given, reading 3′→5′ from the bottom (shorter primer extension products) to the top (longer primer extension products). The position of the deoxyuridine (U) is indicated. The major Pfu products correspond to termination of the polymerase reaction 4–6 bases upstream of the template deoxyuridine. The position of the full-length product obtained from primer-extension against the T template (not shown) was consistent with termination at the end of the 119-base template.

Techniques Used: Sequencing

5) Product Images from "One recognition sequence, seven restriction enzymes, five reaction mechanisms"

Article Title: One recognition sequence, seven restriction enzymes, five reaction mechanisms

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkh685

Mly113I on one- and two-site DNA at varied salt. Reactions at 37°C contained 5 U/ml Mly113II and 5 nM SC DNA in either buffer A ( A and B ) or buffer A + 150 mM NaCl ( C and D ). The DNA was either pUC19, with one Mly113I site (A
Figure Legend Snippet: Mly113I on one- and two-site DNA at varied salt. Reactions at 37°C contained 5 U/ml Mly113II and 5 nM SC DNA in either buffer A ( A and B ) or buffer A + 150 mM NaCl ( C and D ). The DNA was either pUC19, with one Mly113I site (A

Techniques Used:

NarI on one- and two-site plasmids. Reactions contained 40 U/ml NarI and 5 nM supercoiled (SC) DNA in buffer A at 37°C. The DNA was in ( A ), pUC19, with one NarI site; in ( B ), pDG5, with two NarI sites. In both cases, the upper panel shows
Figure Legend Snippet: NarI on one- and two-site plasmids. Reactions contained 40 U/ml NarI and 5 nM supercoiled (SC) DNA in buffer A at 37°C. The DNA was in ( A ), pUC19, with one NarI site; in ( B ), pDG5, with two NarI sites. In both cases, the upper panel shows

Techniques Used:

KasI on one- and two-site DNA. Reactions contained 40 U/ml KasI and 5 nM SC DNA in buffer B at 37°C. The DNA was in ( A ), pUC19, with one KasI site; in ( B ), pDG5, with two KasI sites; in ( C ), Cat (Figure B), with one
Figure Legend Snippet: KasI on one- and two-site DNA. Reactions contained 40 U/ml KasI and 5 nM SC DNA in buffer B at 37°C. The DNA was in ( A ), pUC19, with one KasI site; in ( B ), pDG5, with two KasI sites; in ( C ), Cat (Figure B), with one

Techniques Used:

Enzymes and substrates. ( A ) Shown, within the grey box, are the sequence GGCGCC and the sequences flanking this site in pUC19. The enzymes KasI, NarI, Mly113I, SfoI, EgeI, EheI and BbeI cleave GGCGCC at the positions shown. ( B ) The plasmids pUC19 (2686
Figure Legend Snippet: Enzymes and substrates. ( A ) Shown, within the grey box, are the sequence GGCGCC and the sequences flanking this site in pUC19. The enzymes KasI, NarI, Mly113I, SfoI, EgeI, EheI and BbeI cleave GGCGCC at the positions shown. ( B ) The plasmids pUC19 (2686

Techniques Used: Sequencing

SfoI on one- and two-site DNA. Reactions contained 12 U/ml SfoI and 5 nM SC DNA in buffer B at 21°C. The DNA was: in ( A ), pUC19, with one SfoI site; in ( B ), pDG5, with two SfoI sites. Samples were taken from the reactions at various times
Figure Legend Snippet: SfoI on one- and two-site DNA. Reactions contained 12 U/ml SfoI and 5 nM SC DNA in buffer B at 21°C. The DNA was: in ( A ), pUC19, with one SfoI site; in ( B ), pDG5, with two SfoI sites. Samples were taken from the reactions at various times

Techniques Used:

6) Product Images from "Short-Tailed Stx Phages Exploit the Conserved YaeT Protein To Disseminate Shiga Toxin Genes among Enterobacteria ▿"

Article Title: Short-Tailed Stx Phages Exploit the Conserved YaeT Protein To Disseminate Shiga Toxin Genes among Enterobacteria ▿

Journal: Journal of Bacteriology

doi: 10.1128/JB.00824-07

Subcloning of the open reading frame associated with adsorption of φ24 B to MRL1. Construct pUCR1 was one of the four original clones that complemented the φ24 B phage adsorption defect. All four clones shared the two open reading frames present in pUCR1; the solid box represents yaeT , and the striped box represents skp . When fragments from pUCR1 were subcloned into pUC19 or pUC18 (indicated by the vector promoter direction: leftward, pUC19; rightward, pUC18), only the complete yaeT gene found in pUCR1 and pUCR1D complemented the MRL1 adsorption defect. Restriction sites are as follows: X, XhoI; H, HincII; K, KpnI; P, PstI.
Figure Legend Snippet: Subcloning of the open reading frame associated with adsorption of φ24 B to MRL1. Construct pUCR1 was one of the four original clones that complemented the φ24 B phage adsorption defect. All four clones shared the two open reading frames present in pUCR1; the solid box represents yaeT , and the striped box represents skp . When fragments from pUCR1 were subcloned into pUC19 or pUC18 (indicated by the vector promoter direction: leftward, pUC19; rightward, pUC18), only the complete yaeT gene found in pUCR1 and pUCR1D complemented the MRL1 adsorption defect. Restriction sites are as follows: X, XhoI; H, HincII; K, KpnI; P, PstI.

Techniques Used: Subcloning, Adsorption, Construct, Clone Assay, Plasmid Preparation

Related Articles

Clone Assay:

Article Title: Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis
Article Snippet: .. The amplicons were joined using mixed PCR ( ) or the In-fusion HD Cloning kit (Takara Bio, Otsu, Japan), and subcloned into pUC19 (GE Healthcare Japan, Hino, Japan). .. Ligated fragments were introduced into P. gingivalis ATCC 33277 by electroporation.

Ligation:

Article Title: Short-Tailed Stx Phages Exploit the Conserved YaeT Protein To Disseminate Shiga Toxin Genes among Enterobacteria ▿
Article Snippet: .. A genetic library from E. coli MC1061 was created following the ligation of partially Sau3AI-digested chromosomal DNA (2- to 5-kb fragments) into BamHI-restricted, dephosphorylated expression vectors pUC18 and pUC19 (Amersham-Pharmacia). .. This library was transformed into MC1061-MRL1.

Isolation:

Article Title: The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility
Article Snippet: .. Blunt-ended plasmids were religated either in the absence or presence of the 692 bp Dra I fragment isolated from pUC19. .. The cleavage sites of three independent clones (two linearized by AP1 and AP2, respectively, and ligated to the Dra I-fragment, and one linearized by AP2 and directly religated) were analyzed by sequencing.

Subcloning:

Article Title: Evidence for a Ustilago maydis Steroid 5?-Reductase by Functional Expression in Arabidopsis det2-1 Mutants 1
Article Snippet: .. For subcloning and sequencing, plasmids pUC18, pUC19 (Amersham-Pharmacia Biotech), and pCR2.1-TOPO (Invitrogen) were used. ..

Cell Culture:

Article Title: One recognition sequence, seven restriction enzymes, five reaction mechanisms
Article Snippet: .. Transformants of Escherichia coli HB101 ( ) with pUC19 or pDG5 were cultured in M9 minimal media containing 37 MBq/l [ methyl -3 H]thymidine (Amersham Biosciences) and the plasmids purified by CsCl density-gradient centrifugations ( ). .. The preparations generally contained 85–95% supercoiled monomeric plasmid, with 5–15% as open-circle (OC) and dimeric forms.

Purification:

Article Title: One recognition sequence, seven restriction enzymes, five reaction mechanisms
Article Snippet: .. Transformants of Escherichia coli HB101 ( ) with pUC19 or pDG5 were cultured in M9 minimal media containing 37 MBq/l [ methyl -3 H]thymidine (Amersham Biosciences) and the plasmids purified by CsCl density-gradient centrifugations ( ). .. The preparations generally contained 85–95% supercoiled monomeric plasmid, with 5–15% as open-circle (OC) and dimeric forms.

Polymerase Chain Reaction:

Article Title: Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis
Article Snippet: .. The amplicons were joined using mixed PCR ( ) or the In-fusion HD Cloning kit (Takara Bio, Otsu, Japan), and subcloned into pUC19 (GE Healthcare Japan, Hino, Japan). .. Ligated fragments were introduced into P. gingivalis ATCC 33277 by electroporation.

Generated:

Article Title: A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil
Article Snippet: .. A sequencing ladder for pUC19 was generated by using a T7 Sequenase v2.0, DNA chain-termination sequencing kit (Amersham Life Sciences) in accordance with the manufacturers instructions, using primer C (see above). .. The sequencing reaction products, and the products of the long-range primer extension reaction with Pfu exo+ were run alongside each other on a denaturing gel (Stratagene CastAway, 42 × 18 cm), and visualized by autoradiography.

Expressing:

Article Title: Short-Tailed Stx Phages Exploit the Conserved YaeT Protein To Disseminate Shiga Toxin Genes among Enterobacteria ▿
Article Snippet: .. A genetic library from E. coli MC1061 was created following the ligation of partially Sau3AI-digested chromosomal DNA (2- to 5-kb fragments) into BamHI-restricted, dephosphorylated expression vectors pUC18 and pUC19 (Amersham-Pharmacia). .. This library was transformed into MC1061-MRL1.

Sequencing:

Article Title: A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil
Article Snippet: .. A sequencing ladder for pUC19 was generated by using a T7 Sequenase v2.0, DNA chain-termination sequencing kit (Amersham Life Sciences) in accordance with the manufacturers instructions, using primer C (see above). .. The sequencing reaction products, and the products of the long-range primer extension reaction with Pfu exo+ were run alongside each other on a denaturing gel (Stratagene CastAway, 42 × 18 cm), and visualized by autoradiography.

Article Title: Evidence for a Ustilago maydis Steroid 5?-Reductase by Functional Expression in Arabidopsis det2-1 Mutants 1
Article Snippet: .. For subcloning and sequencing, plasmids pUC18, pUC19 (Amersham-Pharmacia Biotech), and pCR2.1-TOPO (Invitrogen) were used. ..

Plasmid Preparation:

Article Title: Complementary intrastrand base pairing during initiation of Herpes simplex virus type 1 DNA replication
Article Snippet: .. The plasmid pORI(wt) has been described ( ). pUC19 was purchased from Amersham Pharmacia. .. Plasmids pORI(mut8), pORI(mut9), pORI(mut10), and pORImut(11) were constructed as described below.

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    GE Healthcare puc19 dna probe
    Replication of HPV16 <t>DNA</t> independently of E1 and E2 proteins . (A) Maps of PA99GFP and its mutant derivatives. The top bar represents a linear map of the HPV16 genome with responsive eight ORFs shown in arrows and the LCR indicated by a gray bar. The lines below show HPV16 segments, designated wild type (WT) and respective deletion mutants. PA99GFP contains an entire HPV16 DNA, an enhanced green fluorescent protein (EGFP) under a CMV promoter in a <t>pUC19</t> backbone. The mutants ΔE1-E2-#13, ΔE1-E2#15, and ΔE1-E2#18 harboring HPV16 DNA sequenced denoted by solid bars were created from pPA99GFP as described in Materials and Methods. Autoradiograms of representative Southern blots show extrachromosomal DNA (HindIII-digested products) and replicated DNA (DpnI-resistant) from transient replication assays with WT HPV16 and mutants in C33A (B) or 293 (C). Cells were transfected with WT or mutant HPV16 DNA containing plasmids as indicated. At 4 days post-transfection, extrachromosamal DNA were isolated from 1 × 10 7 cells. For Southern analysis, 10% of each sample was linearized with single cutting enzyme, HindIII and 90% was digested with HindIII and DpnI. Blots were hybridized with random-primed radiolabeled pUC19 DNA. The blots with HindIII digested DNA were exposed for 2 h whereas the ones with double digestion (HindIII + DpnI) were exposed for 4 days. The pUC19 backbone plasmid was used as the
    Puc19 Dna Probe, supplied by GE Healthcare, 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/puc19 dna probe/product/GE Healthcare
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    puc19 dna probe - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    92
    GE Healthcare puc19
    Analysis of the I- Uma I target site specificity. CF from induced pAP2 cells was incubated with various substrate plasmids under standard conditions for analysis of cleavage efficiencies (see Materials and Methods). Letters denote the individual substrate plasmids ( e.g. D for <t>pUC19-D).</t> Reaction products were additionally cleaved with Ssp I to yield 2.1 and 0.63 kb fragments in case of cleavage (schematic on the right: the I- Uma I target site fragment is indicated as black box. Xba I cleaves at the right border and in combination with Ssp I produces fragments similar in size to those produced by I- Uma I/ Ssp I). Marker lanes: lin. S, pUC19-D cleaved with Ssp I; lin. X/S, pUC19-D cleaved with Xba I/ Ssp I; s.c./circ., uncleaved pUC19-D showing the supercoiled and circular forms. The + and - symbols refer to the cleavage efficiencies. +++, complete cleavage; ++/+++, > 50% cleavage; ++, ∼50% cleavage; +,
    Puc19, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/puc19/product/GE Healthcare
    Average 92 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    puc19 - by Bioz Stars, 2020-05
    92/100 stars
      Buy from Supplier

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    Replication of HPV16 DNA independently of E1 and E2 proteins . (A) Maps of PA99GFP and its mutant derivatives. The top bar represents a linear map of the HPV16 genome with responsive eight ORFs shown in arrows and the LCR indicated by a gray bar. The lines below show HPV16 segments, designated wild type (WT) and respective deletion mutants. PA99GFP contains an entire HPV16 DNA, an enhanced green fluorescent protein (EGFP) under a CMV promoter in a pUC19 backbone. The mutants ΔE1-E2-#13, ΔE1-E2#15, and ΔE1-E2#18 harboring HPV16 DNA sequenced denoted by solid bars were created from pPA99GFP as described in Materials and Methods. Autoradiograms of representative Southern blots show extrachromosomal DNA (HindIII-digested products) and replicated DNA (DpnI-resistant) from transient replication assays with WT HPV16 and mutants in C33A (B) or 293 (C). Cells were transfected with WT or mutant HPV16 DNA containing plasmids as indicated. At 4 days post-transfection, extrachromosamal DNA were isolated from 1 × 10 7 cells. For Southern analysis, 10% of each sample was linearized with single cutting enzyme, HindIII and 90% was digested with HindIII and DpnI. Blots were hybridized with random-primed radiolabeled pUC19 DNA. The blots with HindIII digested DNA were exposed for 2 h whereas the ones with double digestion (HindIII + DpnI) were exposed for 4 days. The pUC19 backbone plasmid was used as the

    Journal: Virology Journal

    Article Title: Viral trans-factor independent replication of human papillomavirus genomes

    doi: 10.1186/1743-422X-7-123

    Figure Lengend Snippet: Replication of HPV16 DNA independently of E1 and E2 proteins . (A) Maps of PA99GFP and its mutant derivatives. The top bar represents a linear map of the HPV16 genome with responsive eight ORFs shown in arrows and the LCR indicated by a gray bar. The lines below show HPV16 segments, designated wild type (WT) and respective deletion mutants. PA99GFP contains an entire HPV16 DNA, an enhanced green fluorescent protein (EGFP) under a CMV promoter in a pUC19 backbone. The mutants ΔE1-E2-#13, ΔE1-E2#15, and ΔE1-E2#18 harboring HPV16 DNA sequenced denoted by solid bars were created from pPA99GFP as described in Materials and Methods. Autoradiograms of representative Southern blots show extrachromosomal DNA (HindIII-digested products) and replicated DNA (DpnI-resistant) from transient replication assays with WT HPV16 and mutants in C33A (B) or 293 (C). Cells were transfected with WT or mutant HPV16 DNA containing plasmids as indicated. At 4 days post-transfection, extrachromosamal DNA were isolated from 1 × 10 7 cells. For Southern analysis, 10% of each sample was linearized with single cutting enzyme, HindIII and 90% was digested with HindIII and DpnI. Blots were hybridized with random-primed radiolabeled pUC19 DNA. The blots with HindIII digested DNA were exposed for 2 h whereas the ones with double digestion (HindIII + DpnI) were exposed for 4 days. The pUC19 backbone plasmid was used as the "spiked plasmid" to monitor the efficiency of plasmid recovery and completeness of DpnI digestion. The control DNA shown on the left, contains 1 ng,

    Article Snippet: Blots were hybridized at 42°C overnight with a pUC19 DNA probe radiolabeled by random priming kit (GE Healthcare).

    Techniques: Mutagenesis, Transfection, Isolation, Random Primed, Plasmid Preparation

    Transient replication of HPV16 DNA in different cell types . Five μg of plasmid containing HPV16 genomic DNA were transfected into various cell lines; baby hamster kidney cell (BHK), American green monkey kidney cell (Vero), human osteoblastoma cell (U2OS), human cervical carcinoma cell (C-33A) and human embryonic kidney cell (293). After 4 days of transfection, low molecular weight DNA was extracted from 1 × 10 7 cells by Hirt method. During extraction, 10 ng of pUC19 DNA was added as a

    Journal: Virology Journal

    Article Title: Viral trans-factor independent replication of human papillomavirus genomes

    doi: 10.1186/1743-422X-7-123

    Figure Lengend Snippet: Transient replication of HPV16 DNA in different cell types . Five μg of plasmid containing HPV16 genomic DNA were transfected into various cell lines; baby hamster kidney cell (BHK), American green monkey kidney cell (Vero), human osteoblastoma cell (U2OS), human cervical carcinoma cell (C-33A) and human embryonic kidney cell (293). After 4 days of transfection, low molecular weight DNA was extracted from 1 × 10 7 cells by Hirt method. During extraction, 10 ng of pUC19 DNA was added as a "spiked plasmid" to monitor the recovery of the plasmid DNA from transfected cells and to test the completeness of DpnI digestion. For the Southern analysis, 10% of Hirt-DNA was digested with HindIII to linearize the plasmid and 90% was digested with both HindIII and DpnI. The blot was probed with random-primed 32 -P labeled pUC19 DNA. As a control, linearized plasmid containing HPV16 at concentrations of 100 and 10 pg was loaded on the left-most lanes of the blot.

    Article Snippet: Blots were hybridized at 42°C overnight with a pUC19 DNA probe radiolabeled by random priming kit (GE Healthcare).

    Techniques: Plasmid Preparation, Transfection, Molecular Weight, Random Primed, Labeling

    Transient replication of HPV16 mutants with LCR and E1-E2 deletions . (A) Schematic representation of PUCGFP comprising an EGFP in a pUC19 backbone. An entire sequence and partial fragments of HPV16 DNA shown in solid lines were inserted into PUCGFP at BamHI to generate WT and mutants designated ΔLCR, ΔE1-E2, ΔLCR-E2, and L1 as described in Materials and Methods. Replication efficiencies of the HPV16 WT and mutants are evaluated by a transient transfection assay. Autoradiograms of representative Southern blots show replicated DNAs from transient transfection assays in C33A (B) and 293 (B). The assay was performed as described in Figure 2 legend. Ten pg to 1 ng of linear plasmid containing the entire HPV16 DNA, was loaded on the left-most lanes, as copy number controls.

    Journal: Virology Journal

    Article Title: Viral trans-factor independent replication of human papillomavirus genomes

    doi: 10.1186/1743-422X-7-123

    Figure Lengend Snippet: Transient replication of HPV16 mutants with LCR and E1-E2 deletions . (A) Schematic representation of PUCGFP comprising an EGFP in a pUC19 backbone. An entire sequence and partial fragments of HPV16 DNA shown in solid lines were inserted into PUCGFP at BamHI to generate WT and mutants designated ΔLCR, ΔE1-E2, ΔLCR-E2, and L1 as described in Materials and Methods. Replication efficiencies of the HPV16 WT and mutants are evaluated by a transient transfection assay. Autoradiograms of representative Southern blots show replicated DNAs from transient transfection assays in C33A (B) and 293 (B). The assay was performed as described in Figure 2 legend. Ten pg to 1 ng of linear plasmid containing the entire HPV16 DNA, was loaded on the left-most lanes, as copy number controls.

    Article Snippet: Blots were hybridized at 42°C overnight with a pUC19 DNA probe radiolabeled by random priming kit (GE Healthcare).

    Techniques: Sequencing, Transient Transfection Assay, Transfection, Plasmid Preparation

    Analysis of the I- Uma I target site specificity. CF from induced pAP2 cells was incubated with various substrate plasmids under standard conditions for analysis of cleavage efficiencies (see Materials and Methods). Letters denote the individual substrate plasmids ( e.g. D for pUC19-D). Reaction products were additionally cleaved with Ssp I to yield 2.1 and 0.63 kb fragments in case of cleavage (schematic on the right: the I- Uma I target site fragment is indicated as black box. Xba I cleaves at the right border and in combination with Ssp I produces fragments similar in size to those produced by I- Uma I/ Ssp I). Marker lanes: lin. S, pUC19-D cleaved with Ssp I; lin. X/S, pUC19-D cleaved with Xba I/ Ssp I; s.c./circ., uncleaved pUC19-D showing the supercoiled and circular forms. The + and - symbols refer to the cleavage efficiencies. +++, complete cleavage; ++/+++, > 50% cleavage; ++, ∼50% cleavage; +,

    Journal: PLoS ONE

    Article Title: The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility

    doi: 10.1371/journal.pone.0049551

    Figure Lengend Snippet: Analysis of the I- Uma I target site specificity. CF from induced pAP2 cells was incubated with various substrate plasmids under standard conditions for analysis of cleavage efficiencies (see Materials and Methods). Letters denote the individual substrate plasmids ( e.g. D for pUC19-D). Reaction products were additionally cleaved with Ssp I to yield 2.1 and 0.63 kb fragments in case of cleavage (schematic on the right: the I- Uma I target site fragment is indicated as black box. Xba I cleaves at the right border and in combination with Ssp I produces fragments similar in size to those produced by I- Uma I/ Ssp I). Marker lanes: lin. S, pUC19-D cleaved with Ssp I; lin. X/S, pUC19-D cleaved with Xba I/ Ssp I; s.c./circ., uncleaved pUC19-D showing the supercoiled and circular forms. The + and - symbols refer to the cleavage efficiencies. +++, complete cleavage; ++/+++, > 50% cleavage; ++, ∼50% cleavage; +,

    Article Snippet: Blunt-ended plasmids were religated either in the absence or presence of the 692 bp Dra I fragment isolated from pUC19.

    Techniques: Incubation, Produced, Marker

    SPR analysis of the DB293 interaction with wild-type or various mutated Pit-1 transcription factor binding sites. ( A ) SPR sensorgrams for binding of increasing concentrations of DB293 (1 nM to 0.4 µM) to the wild-type (WT) Pit-1 consensus-binding sites (red) or mutated sequences Pit-1-M1 (blue) or Pit-1-M2 (purple) sequences within biotinylated hairpin oligonucleotides in MES buffer, 25°C. The minimal consensus-binding sites are visualized in red. ( B ) Binding plots derived from SPR sensorgrams used to calculate the affinity constants for DB293 bound to the various sequences. Written in red are the specific consensus-binding sites with mutation points in blue (M1, removing the ATGA) or purple (M2, removing the AT-rich sequence 3′ to the ATGA). ( C ) Equilibrium constants for DB293 binding to Pit-1-WT, Pit-1-M1 and Pit-1-M2 sequences. The deduced cooperativity factor identifies the potential cooperativity of DB293 molecules for DNA binding. A weak nonspecific binding (factor of 10- to 50-fold less than the strong consensus binding) was also obtained but could not be accurately determined.

    Journal: Nucleic Acids Research

    Article Title: Direct inhibition of the DNA-binding activity of POU transcription factors Pit-1 and Brn-3 by selective binding of a phenyl-furan-benzimidazole dication

    doi: 10.1093/nar/gkn208

    Figure Lengend Snippet: SPR analysis of the DB293 interaction with wild-type or various mutated Pit-1 transcription factor binding sites. ( A ) SPR sensorgrams for binding of increasing concentrations of DB293 (1 nM to 0.4 µM) to the wild-type (WT) Pit-1 consensus-binding sites (red) or mutated sequences Pit-1-M1 (blue) or Pit-1-M2 (purple) sequences within biotinylated hairpin oligonucleotides in MES buffer, 25°C. The minimal consensus-binding sites are visualized in red. ( B ) Binding plots derived from SPR sensorgrams used to calculate the affinity constants for DB293 bound to the various sequences. Written in red are the specific consensus-binding sites with mutation points in blue (M1, removing the ATGA) or purple (M2, removing the AT-rich sequence 3′ to the ATGA). ( C ) Equilibrium constants for DB293 binding to Pit-1-WT, Pit-1-M1 and Pit-1-M2 sequences. The deduced cooperativity factor identifies the potential cooperativity of DB293 molecules for DNA binding. A weak nonspecific binding (factor of 10- to 50-fold less than the strong consensus binding) was also obtained but could not be accurately determined.

    Article Snippet: DNase I footprinting The pUC19-Brn-3, pUC19-IRF-1 and pUC19-Pit-1 plasmids containing the sequences established from Panomics, as well as the pUC19-Pit-1-WT, pUC19-Pit-1-M1, pUC19-Pit-1-M2, pUC19-Pit-1-M3 and pUC19-Pit-1-M4 were digested using EcoRI and PstI restriction enzymes, 3′-32 P-end labeled using [α-32 P]dATP (GE Healthcare, Vélizy, France, 3000 Ci/mmol) and experiments were conducted as recently described ( ).

    Techniques: SPR Assay, Binding Assay, Derivative Assay, Mutagenesis, Sequencing