avr ii  (New England Biolabs)


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    AvrII
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    AvrII 500 units
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    r0174l
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    290
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    500 units
    Category:
    Restriction Enzymes
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    New England Biolabs avr ii
    AvrII
    AvrII 500 units
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    Average 99 stars, based on 2543 article reviews
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    Images

    1) Product Images from "Murine Gammaherpesvirus 68 Lacking Thymidine Kinase Shows Severe Attenuation of Lytic Cycle Replication In Vivo but Still Establishes Latency"

    Article Title: Murine Gammaherpesvirus 68 Lacking Thymidine Kinase Shows Severe Attenuation of Lytic Cycle Replication In Vivo but Still Establishes Latency

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.4.2410-2417.2003

    Genomic structure of TK + and TK − viruses. (A) The TK − Kan + virus was generated by inserting a FRT-flanked kanamycin resistance gene at genomic coordinates 33301 to 33450. The kanamycin resistance coding sequence was removed by Flp recombination to leave an inset of 167 bp (TK − Kan − ). This virus was reverted by homologous recombination with an unmutated genomic segment (TK + R). An independent mutant (TK − Del) was generated by RecA-mediated recombination of BAC DNA with a genomic plasmid clone in which genomic coordinates 33293 to 34300 are deleted by digestion with Avr II and Pme I. (B) Viral DNA was digested with Xba I, Eco RI, or Sac I, electrophoresed, blotted, and probed with a 32 P-labeled genomic Eco RI fragment spanning the TK locus.
    Figure Legend Snippet: Genomic structure of TK + and TK − viruses. (A) The TK − Kan + virus was generated by inserting a FRT-flanked kanamycin resistance gene at genomic coordinates 33301 to 33450. The kanamycin resistance coding sequence was removed by Flp recombination to leave an inset of 167 bp (TK − Kan − ). This virus was reverted by homologous recombination with an unmutated genomic segment (TK + R). An independent mutant (TK − Del) was generated by RecA-mediated recombination of BAC DNA with a genomic plasmid clone in which genomic coordinates 33293 to 34300 are deleted by digestion with Avr II and Pme I. (B) Viral DNA was digested with Xba I, Eco RI, or Sac I, electrophoresed, blotted, and probed with a 32 P-labeled genomic Eco RI fragment spanning the TK locus.

    Techniques Used: Generated, Sequencing, Homologous Recombination, Mutagenesis, BAC Assay, Plasmid Preparation, Labeling

    2) Product Images from "Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine"

    Article Title: Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine

    Journal: Tropical Medicine and Infectious Disease

    doi: 10.3390/tropicalmed2030037

    Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.
    Figure Legend Snippet: Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.

    Techniques Used: Recombinant

    3) Product Images from "The Highly Virulent 2006 Norwegian EHEC O103:H25 Outbreak Strain Is Related to the 2011 German O104:H4 Outbreak Strain"

    Article Title: The Highly Virulent 2006 Norwegian EHEC O103:H25 Outbreak Strain Is Related to the 2011 German O104:H4 Outbreak Strain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031413

    PFGE of outbreak associated isolates. PFGE of E. coli O103:H25 NOS (lane 2), NVH-847 (lane 3), NVH-848 (lane 4), and NVH-760 (lane 5). Lambda ladder is used as marker (lane 1). Digestion with Xba I (A) showed indistinguishable PFGE patterns, while digestion with Avr II (B) exposed a difference between the stx 2 -positive and the stx -negative isolates.
    Figure Legend Snippet: PFGE of outbreak associated isolates. PFGE of E. coli O103:H25 NOS (lane 2), NVH-847 (lane 3), NVH-848 (lane 4), and NVH-760 (lane 5). Lambda ladder is used as marker (lane 1). Digestion with Xba I (A) showed indistinguishable PFGE patterns, while digestion with Avr II (B) exposed a difference between the stx 2 -positive and the stx -negative isolates.

    Techniques Used: Marker

    4) Product Images from "Heterologous Gene Expression from Transmissible Gastroenteritis Virus Replicon Particles"

    Article Title: Heterologous Gene Expression from Transmissible Gastroenteritis Virus Replicon Particles

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.3.1422-1434.2002

    TGEV-Rep( Avr II) VRPs. Transfected cultures displayed GFP expression at ∼18 h posttransfection (A) [TGEV-Rep( Avr II) pass 0]. Supernatants were harvested ∼36 h posttransfection and used to infect fresh cultures of ST cells for 1 h at room temperature. At ∼18 h postinfection, GFP expression was observed by fluorescent microscopy in these pass 1 cultures (C and D). (C) TGEV VRP production following coelectroporation with VEE-E RNAs [TGEV-Rep( Avr II) plus E protein transcripts pass 1]; (D) TGEV VRP production following infection with VEE VRPs encoding the E protein [TGEV-Rep( Avr II) plus VEE-TGEV(E) pass 1]. Passage of supernatants from cells transfected with TGEV-Rep( Avr II) transcripts without expression of the E protein in trans did not result in detectable GFP expression (B) [TGEV-Rep( Avr II) without E protein pass 1].
    Figure Legend Snippet: TGEV-Rep( Avr II) VRPs. Transfected cultures displayed GFP expression at ∼18 h posttransfection (A) [TGEV-Rep( Avr II) pass 0]. Supernatants were harvested ∼36 h posttransfection and used to infect fresh cultures of ST cells for 1 h at room temperature. At ∼18 h postinfection, GFP expression was observed by fluorescent microscopy in these pass 1 cultures (C and D). (C) TGEV VRP production following coelectroporation with VEE-E RNAs [TGEV-Rep( Avr II) plus E protein transcripts pass 1]; (D) TGEV VRP production following infection with VEE VRPs encoding the E protein [TGEV-Rep( Avr II) plus VEE-TGEV(E) pass 1]. Passage of supernatants from cells transfected with TGEV-Rep( Avr II) transcripts without expression of the E protein in trans did not result in detectable GFP expression (B) [TGEV-Rep( Avr II) without E protein pass 1].

    Techniques Used: Transfection, Expressing, Microscopy, Infection

    Construction of TGEV replicon cDNAs. (a) TGEV F fragments. Structural genes are contained in the TGEV F fragment. FiGFP2( Pfl MI) (∼5.6 kb) was constructed from the wild-type TGEV F fragment (∼5.1 kb) by the deletion of ORF 3A (nt 24828 to 25073) and the insertion of GFP with a 5′ 20-nt N gene IS. Using this construct, we introduced deletions extending from the unique Pfl MI site at the very 3′ end of GFP to the unique Avr II (nucleotide position 25866) and Eco NI (nucleotide position 26935) sites present within the TGEV E and M genes, respectively. (b) Sequence organization of GFP in FiGFP2( Pfl MI). GFP was inserted just downstream of the ORF 3A IS. The TGEV sequence originating at the 3′ end of the S gene (nt 24693) through the start of the GFP gene is shown, and the important IS and restriction sites are labeled. (c) Strategy for assembling recombinant TGEV and replicon cDNAs. The six cDNA subclones (TGEV A, B1, B2, C, DE1, and F deletion fragments) spanning the genome are flanked by unique interconnecting Bgl I and Bst ). TGEV A contained a unique T7 start site at its 5′ end, and the F deletion fragments (FiGFP2- Avr II and FiGFP2- Eco NI) contain GFP and a 25-nt T tail, allowing for the synthesis of capped T7, polyadenylated transcripts in vitro.
    Figure Legend Snippet: Construction of TGEV replicon cDNAs. (a) TGEV F fragments. Structural genes are contained in the TGEV F fragment. FiGFP2( Pfl MI) (∼5.6 kb) was constructed from the wild-type TGEV F fragment (∼5.1 kb) by the deletion of ORF 3A (nt 24828 to 25073) and the insertion of GFP with a 5′ 20-nt N gene IS. Using this construct, we introduced deletions extending from the unique Pfl MI site at the very 3′ end of GFP to the unique Avr II (nucleotide position 25866) and Eco NI (nucleotide position 26935) sites present within the TGEV E and M genes, respectively. (b) Sequence organization of GFP in FiGFP2( Pfl MI). GFP was inserted just downstream of the ORF 3A IS. The TGEV sequence originating at the 3′ end of the S gene (nt 24693) through the start of the GFP gene is shown, and the important IS and restriction sites are labeled. (c) Strategy for assembling recombinant TGEV and replicon cDNAs. The six cDNA subclones (TGEV A, B1, B2, C, DE1, and F deletion fragments) spanning the genome are flanked by unique interconnecting Bgl I and Bst ). TGEV A contained a unique T7 start site at its 5′ end, and the F deletion fragments (FiGFP2- Avr II and FiGFP2- Eco NI) contain GFP and a 25-nt T tail, allowing for the synthesis of capped T7, polyadenylated transcripts in vitro.

    Techniques Used: Construct, Sequencing, Labeling, Recombinant, In Vitro

    Heterologous gene expression from TGEV replicon RNAs. (a) Cultures of BHK cells were transfected with TGEV replicon RNAs encoding GFP that contained ORF 3B and E (A) [TGEV-Rep( Avr II) transcripts] or ORF 3B, E, and M gene deletions (C). Alternatively, transcripts were treated with RNase prior to transfection into BHK cells (B and D) [TGEV-Rep( Avr II) DNA and TGEV-Rep( Eco NI) DNA only, respectively]. At ∼18 h posttransfection, GFP expression was observed by fluorescent microscopy. (b) Intracellular RNA was isolated from transfected cell cultures and used as a template for RT-PCR. Leader-containing GFP subgenomic transcripts were detected using a 5′ leader primer (TGEV-L) and 3′ primers located just downstream of the Avr II [(−)E5546] or Eco NI [M6400(−)] sites. Appropriately sized amplicons of ∼850 bp were generated, corresponding to transcripts encoding GFP. Cells transfected with TGEV-Rep( Avr II) (A) or TGEV-Rep( Eco NI) (B) RNA are shown. A 1-kb ladder is shown in both panels (lanes 1). Arrows indicate leader-containing GFP amplicons (lanes 2).
    Figure Legend Snippet: Heterologous gene expression from TGEV replicon RNAs. (a) Cultures of BHK cells were transfected with TGEV replicon RNAs encoding GFP that contained ORF 3B and E (A) [TGEV-Rep( Avr II) transcripts] or ORF 3B, E, and M gene deletions (C). Alternatively, transcripts were treated with RNase prior to transfection into BHK cells (B and D) [TGEV-Rep( Avr II) DNA and TGEV-Rep( Eco NI) DNA only, respectively]. At ∼18 h posttransfection, GFP expression was observed by fluorescent microscopy. (b) Intracellular RNA was isolated from transfected cell cultures and used as a template for RT-PCR. Leader-containing GFP subgenomic transcripts were detected using a 5′ leader primer (TGEV-L) and 3′ primers located just downstream of the Avr II [(−)E5546] or Eco NI [M6400(−)] sites. Appropriately sized amplicons of ∼850 bp were generated, corresponding to transcripts encoding GFP. Cells transfected with TGEV-Rep( Avr II) (A) or TGEV-Rep( Eco NI) (B) RNA are shown. A 1-kb ladder is shown in both panels (lanes 1). Arrows indicate leader-containing GFP amplicons (lanes 2).

    Techniques Used: Expressing, Transfection, Microscopy, Isolation, Reverse Transcription Polymerase Chain Reaction, Generated

    Strategy to assemble TGEV-Rep( Avr II) VRPs. (a) In the full-length TGEV-Rep( Avr II) cDNA construct, ORF 3A has been replaced with GFP, and ORF 3B and the 5′ end of the E gene have been deleted. To produce packaged replicon particles, replicon RNA-transfected cells were infected with VEE VRPs expressing the TGEV E protein [VEE-TGEV(E)]. Alternatively, TGEV-Rep( Avr II) replicon RNAs can be coelectroporated with pVR21-E1-derived transcripts. TGEV VRPs should be released from cells that can be used as single-hit expression vectors. (b) Cultures of ST cells were infected with either wild-type (wt) TGEV alone or with TGEV and VEE VRPs expressing a G 1 Norwalk-like virus capsid (wt + VEE) at an MOI of 5 for 1 h at room temperature. The inocula were removed, and the cultures were incubated in complete medium at 37°C. Samples were harvested at the indicated times and assayed by plaque assay in ST cells.
    Figure Legend Snippet: Strategy to assemble TGEV-Rep( Avr II) VRPs. (a) In the full-length TGEV-Rep( Avr II) cDNA construct, ORF 3A has been replaced with GFP, and ORF 3B and the 5′ end of the E gene have been deleted. To produce packaged replicon particles, replicon RNA-transfected cells were infected with VEE VRPs expressing the TGEV E protein [VEE-TGEV(E)]. Alternatively, TGEV-Rep( Avr II) replicon RNAs can be coelectroporated with pVR21-E1-derived transcripts. TGEV VRPs should be released from cells that can be used as single-hit expression vectors. (b) Cultures of ST cells were infected with either wild-type (wt) TGEV alone or with TGEV and VEE VRPs expressing a G 1 Norwalk-like virus capsid (wt + VEE) at an MOI of 5 for 1 h at room temperature. The inocula were removed, and the cultures were incubated in complete medium at 37°C. Samples were harvested at the indicated times and assayed by plaque assay in ST cells.

    Techniques Used: Construct, Transfection, Infection, Expressing, Derivative Assay, Incubation, Plaque Assay

    Sequence analysis of leader-containing amplicons encoding GFP. TGEV-Rep( Avr II) and TGEV-Rep( Eco NI) leader-containing amplicons were isolated from agarose gels and subcloned into Topo II TA cloning vectors. Inserts were sequenced using universal primers and an automated sequencer. (A) Sequence of the 5′ end of GFP amplicons generated from TGEV-Rep( Eco NI)-transfected cells, indicating that the leader-containing transcripts initiated from the TGEV ORF 3A IS. (B) Leader-containing GFP transcripts with the Pfl MI- Avr II deletion derived from TGEV-Rep( Avr II) VRP-infected cells. (C) Leader-containing GFP transcripts with the Pfl MI- Eco NI deletion derived from TGEV-Rep( Eco NI)-transfected cells. Underlined bases correspond to the GFP stop codon, and the shaded bases correspond to the deletion and blunt-end ligation sites.
    Figure Legend Snippet: Sequence analysis of leader-containing amplicons encoding GFP. TGEV-Rep( Avr II) and TGEV-Rep( Eco NI) leader-containing amplicons were isolated from agarose gels and subcloned into Topo II TA cloning vectors. Inserts were sequenced using universal primers and an automated sequencer. (A) Sequence of the 5′ end of GFP amplicons generated from TGEV-Rep( Eco NI)-transfected cells, indicating that the leader-containing transcripts initiated from the TGEV ORF 3A IS. (B) Leader-containing GFP transcripts with the Pfl MI- Avr II deletion derived from TGEV-Rep( Avr II) VRP-infected cells. (C) Leader-containing GFP transcripts with the Pfl MI- Eco NI deletion derived from TGEV-Rep( Eco NI)-transfected cells. Underlined bases correspond to the GFP stop codon, and the shaded bases correspond to the deletion and blunt-end ligation sites.

    Techniques Used: Sequencing, Isolation, TA Cloning, Generated, Transfection, Derivative Assay, Infection, Ligation

    5) Product Images from "CTAG-Containing Cleavage Site Profiling to Delineate Salmonella into Natural Clusters"

    Article Title: CTAG-Containing Cleavage Site Profiling to Delineate Salmonella into Natural Clusters

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0103388

    Physical map comparison between S. gallinarum strains 287/91 and SARB21. The map of SARB21 was reported previously [21] ; here letter designations for the cleavage fragments of SARB21 have been changed according to the homologues in strain 287/91 for the convenience of comparison. Note that all XbaI, I-CeuI and AvrII (maps from top to bottom) cleavage sites are conserved in the two strains except the AvrII site between fragments F and J in 287/91 (open arrow), which is missing from SARB21. Lines with solid arrowheads at both ends indicate the ranges of genomic inversions via rrn -mediated recombination between the two strains and filled arrows indicate recombination sites that have resulted in the translocation of I-CeuI fragment D.
    Figure Legend Snippet: Physical map comparison between S. gallinarum strains 287/91 and SARB21. The map of SARB21 was reported previously [21] ; here letter designations for the cleavage fragments of SARB21 have been changed according to the homologues in strain 287/91 for the convenience of comparison. Note that all XbaI, I-CeuI and AvrII (maps from top to bottom) cleavage sites are conserved in the two strains except the AvrII site between fragments F and J in 287/91 (open arrow), which is missing from SARB21. Lines with solid arrowheads at both ends indicate the ranges of genomic inversions via rrn -mediated recombination between the two strains and filled arrows indicate recombination sites that have resulted in the translocation of I-CeuI fragment D.

    Techniques Used: Translocation Assay

    XbaI and AvrII cleavage patterns of S. gallinarum strains 287/91 and SARB21 after PFGE separation. (A) XbaI cleavage. Lanes: 1, SARB21; 2, 287/91; 3, λDNA as molecular size marker. (B) AvrII cleavage. Lanes: 1, λDNA as molecular size marker; 2, SARB21; 3, 287/91. Letter designations are for strain 287/91; the same letters are used for homologous fragments in strain SARB21. In the designation of fragments in SARB21, C′ means a fragment homologous to C in 287/91 but truncated on the right-hand part by genomic rearrangement, and ‘C means truncation on the left-hand part of the fragment.
    Figure Legend Snippet: XbaI and AvrII cleavage patterns of S. gallinarum strains 287/91 and SARB21 after PFGE separation. (A) XbaI cleavage. Lanes: 1, SARB21; 2, 287/91; 3, λDNA as molecular size marker. (B) AvrII cleavage. Lanes: 1, λDNA as molecular size marker; 2, SARB21; 3, 287/91. Letter designations are for strain 287/91; the same letters are used for homologous fragments in strain SARB21. In the designation of fragments in SARB21, C′ means a fragment homologous to C in 287/91 but truncated on the right-hand part by genomic rearrangement, and ‘C means truncation on the left-hand part of the fragment.

    Techniques Used: Marker

    6) Product Images from "A novel approach to the generation of seamless constructs for plant transformation"

    Article Title: A novel approach to the generation of seamless constructs for plant transformation

    Journal: Plant Methods

    doi: 10.1186/1746-4811-10-10

    Vector linearization and RE analysis of cloned gfp . A . pAUrumII in its circular (supercoiled) and LguI treated, linear state with the LguI -spacer removed next to pAUrumIII in its circular (supercoiled) and BaeI treated, linear state. The BaeI treated pAUrumIII also appears in a relaxed, circular state. The short BaeI -spacer is not visible. B . Restriction analysis for pAUrumII- gfp with NotI on plasmid DNA from 15 colonies. The correct construct is separated in two fragments of 2.6 kb and 1.5 kb, respectively. C . Restriction analysis for pAUrumIII- gfp with AvrII . The correct construct is separated in two fragments of 2.6 kb and 1.5 kb, respectively. The construct separated in a 2.6 kb and a 0.86 kb fragment is pAUrumIII. For all gels, L indicates the O’GeneRuler 1 kb DNA Ladder (Thermo Scientific).
    Figure Legend Snippet: Vector linearization and RE analysis of cloned gfp . A . pAUrumII in its circular (supercoiled) and LguI treated, linear state with the LguI -spacer removed next to pAUrumIII in its circular (supercoiled) and BaeI treated, linear state. The BaeI treated pAUrumIII also appears in a relaxed, circular state. The short BaeI -spacer is not visible. B . Restriction analysis for pAUrumII- gfp with NotI on plasmid DNA from 15 colonies. The correct construct is separated in two fragments of 2.6 kb and 1.5 kb, respectively. C . Restriction analysis for pAUrumIII- gfp with AvrII . The correct construct is separated in two fragments of 2.6 kb and 1.5 kb, respectively. The construct separated in a 2.6 kb and a 0.86 kb fragment is pAUrumIII. For all gels, L indicates the O’GeneRuler 1 kb DNA Ladder (Thermo Scientific).

    Techniques Used: Plasmid Preparation, Clone Assay, Construct

    7) Product Images from "Evidence of Metabolic Switching and Implications for Food Safety from the Phenome(s) of Salmonella enterica Serovar Typhimurium DT104 Cultured at Selected Points across the Pork Production Food Chain"

    Article Title: Evidence of Metabolic Switching and Implications for Food Safety from the Phenome(s) of Salmonella enterica Serovar Typhimurium DT104 Cultured at Selected Points across the Pork Production Food Chain

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01041-13

    (A) PFGE analysis of S . Typhimurium DT104 using XbaI and AvrII. Isolate groups were color identified for simplicity as follows, and the color codes were maintained in all of the assays: reference strain (white); group A, environmental (green); group B,
    Figure Legend Snippet: (A) PFGE analysis of S . Typhimurium DT104 using XbaI and AvrII. Isolate groups were color identified for simplicity as follows, and the color codes were maintained in all of the assays: reference strain (white); group A, environmental (green); group B,

    Techniques Used:

    8) Product Images from "Protection conferred by a recombinant Marek's disease virus that expresses the spike protein from infectious bronchitis virus in specific pathogen-free chicken"

    Article Title: Protection conferred by a recombinant Marek's disease virus that expresses the spike protein from infectious bronchitis virus in specific pathogen-free chicken

    Journal: Virology Journal

    doi: 10.1186/1743-422X-9-85

    Identification of the transfer vector by restriction enzyme digestion analysis. M: DNA Marker; Lane 1: pUP-S1-DOWN digested with Avr II and Not I; Lane 2: pUP-LTR-EGFP-S1-DOWN digested with Not I; Lane 3: pUP-LTR-EGFP-S1-DOWN digested with Hin d III.
    Figure Legend Snippet: Identification of the transfer vector by restriction enzyme digestion analysis. M: DNA Marker; Lane 1: pUP-S1-DOWN digested with Avr II and Not I; Lane 2: pUP-LTR-EGFP-S1-DOWN digested with Not I; Lane 3: pUP-LTR-EGFP-S1-DOWN digested with Hin d III.

    Techniques Used: Plasmid Preparation, Marker

    9) Product Images from "Genomic Diversification among Archival Strains of Salmonella enterica Serovar Typhimurium LT7"

    Article Title: Genomic Diversification among Archival Strains of Salmonella enterica Serovar Typhimurium LT7

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.185.7.2131-2142.2003

    Point mutations as inferred from the creation or loss of endonuclease cleavage sites. (A) Xba I-cleaved genomic DNA of LT7 (lane 1) and 9059A (lane 2); (B) Avr II-cleaved genomic DNA of LT7 (lane 1) and 8608D (lane 2); (C) I- Ceu I-cleaved genomic DNA of LT7 (lane 1) and 8111F231213 (lane 2). For these three strains, genomic translocations, duplications, and inversions had been ruled out.
    Figure Legend Snippet: Point mutations as inferred from the creation or loss of endonuclease cleavage sites. (A) Xba I-cleaved genomic DNA of LT7 (lane 1) and 9059A (lane 2); (B) Avr II-cleaved genomic DNA of LT7 (lane 1) and 8608D (lane 2); (C) I- Ceu I-cleaved genomic DNA of LT7 (lane 1) and 8111F231213 (lane 2). For these three strains, genomic translocations, duplications, and inversions had been ruled out.

    Techniques Used:

    Genomic inversion exemplified by strain 8111D323. (A) PFGE gels of LT7 and 8111D323. Lanes: 1, LT7 DNA cleaved by Xba I; 2, 8111D323 DNA cleaved by Xba I; 3, LT7 DNA cleaved by Avr II; and 4, 8111D323 DNA cleaved by Avr II. (B) Local comparison of LT7 and 8111D323 showing the inversion of I- Ceu I A, which resulted in two hybrid rrn operons, the disappearance of Xba I B and Avr II A, and appearance of two new fragments each from Xba I (411 and 322 kb) and Avr II (777 and 757 kb) digestions.
    Figure Legend Snippet: Genomic inversion exemplified by strain 8111D323. (A) PFGE gels of LT7 and 8111D323. Lanes: 1, LT7 DNA cleaved by Xba I; 2, 8111D323 DNA cleaved by Xba I; 3, LT7 DNA cleaved by Avr II; and 4, 8111D323 DNA cleaved by Avr II. (B) Local comparison of LT7 and 8111D323 showing the inversion of I- Ceu I A, which resulted in two hybrid rrn operons, the disappearance of Xba I B and Avr II A, and appearance of two new fragments each from Xba I (411 and 322 kb) and Avr II (777 and 757 kb) digestions.

    Techniques Used:

    PFGE patterns of genomic DNA of the wild serovar Typhimurium LT7 strain stored at −70°C cleaved with the endonucleases Xba I, Avr II, I- Ceu I, and Spe I. Lanes: 1, Xba I cleavage of LT7 (3 small fragments—W [6.5 kb], X [6.4 kb], and Y [1 kb]—had run out of the gel); 2, I- Ceu I cleavage of LT7; 3 and 4, Avr II cleavages of LT2 and LT7, respectively, for a comparison; two small fragments of LT7 had run out of the gel, including L (4 kb) and M (2 kb); 5, Spe I cleavage of LT7 (12 small fragments, X through II, ranging from 49 to 7 kb, had run out of the gel); 6, Spe I cleavage of LT7 serB ::Tn 10 , showing the size shift of S112 with the Tn 10 insertion (it is now 112 + 9, i.e., 121 kb).
    Figure Legend Snippet: PFGE patterns of genomic DNA of the wild serovar Typhimurium LT7 strain stored at −70°C cleaved with the endonucleases Xba I, Avr II, I- Ceu I, and Spe I. Lanes: 1, Xba I cleavage of LT7 (3 small fragments—W [6.5 kb], X [6.4 kb], and Y [1 kb]—had run out of the gel); 2, I- Ceu I cleavage of LT7; 3 and 4, Avr II cleavages of LT2 and LT7, respectively, for a comparison; two small fragments of LT7 had run out of the gel, including L (4 kb) and M (2 kb); 5, Spe I cleavage of LT7 (12 small fragments, X through II, ranging from 49 to 7 kb, had run out of the gel); 6, Spe I cleavage of LT7 serB ::Tn 10 , showing the size shift of S112 with the Tn 10 insertion (it is now 112 + 9, i.e., 121 kb).

    Techniques Used:

    10) Product Images from "Optimization of Pulse-Field Gel Electrophoresis for Subtyping of Klebsiella pneumoniae"

    Article Title: Optimization of Pulse-Field Gel Electrophoresis for Subtyping of Klebsiella pneumoniae

    Journal: International Journal of Environmental Research and Public Health

    doi: 10.3390/ijerph10072720

    Clustering results of patterns obtained with Xba I- and Avr II-PFGE respectively. Charts are shown for 69 K. pneumoniae strains.
    Figure Legend Snippet: Clustering results of patterns obtained with Xba I- and Avr II-PFGE respectively. Charts are shown for 69 K. pneumoniae strains.

    Techniques Used:

    PFGE images of four K. pneumoniae isolates restricted with Avr II (lanes 2 to 5), Pme I (lanes 7 to 10), Spe I (lanes 12 to 15), Xba I (lanes 17 to 20) and Swa I (lanes 22 to 25). The size standard (H9812) was loaded in lanes 1, 6, 11, 16, and 21.
    Figure Legend Snippet: PFGE images of four K. pneumoniae isolates restricted with Avr II (lanes 2 to 5), Pme I (lanes 7 to 10), Spe I (lanes 12 to 15), Xba I (lanes 17 to 20) and Swa I (lanes 22 to 25). The size standard (H9812) was loaded in lanes 1, 6, 11, 16, and 21.

    Techniques Used:

    Clustering results of patterns obtained with Xba I- and Avr II-PFGE of 11 K. pneumoniae strains from one outbreak.
    Figure Legend Snippet: Clustering results of patterns obtained with Xba I- and Avr II-PFGE of 11 K. pneumoniae strains from one outbreak.

    Techniques Used:

    11) Product Images from "A Method for Rapid Genetic Integration into Plasmodium falciparum Utilizing Mycobacteriophage Bxb1 Integrase"

    Article Title: A Method for Rapid Genetic Integration into Plasmodium falciparum Utilizing Mycobacteriophage Bxb1 Integrase

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-60761-652-8_6

    Dual-plasmid approach for integrase-mediated attB × attP recombination. ( a ) Map of the attP -carrying plasmid pLN-ENR-GFP that can be used as a template for engineering the stable site-specific integration of a gene of interest. Digestion with Avr II and Afl II can be performed to replace the enr-gfp fusion by the gene of interest. Alternatively, digestion with Avr II and Bsi WI can place the gene of interest in frame with gfp . The plasmid carries the blasticidin S-deaminase ( bsd ) selectable marker. ( b ) Map of the integrase-expressing plasmid pINT, which harbors a neomycin selectable marker. The plasmid is usually maintained under G418 selection pressure only for the first 6 days following transfection. ( c ) Schematic representation of the attB × attP recombination allowing integration of a gene of interest ( yfg ) at the cg6 - attB locus. Dd2 attB or 3D7 attB parasites are co-transfected with pINT and an attP -plasmid carrying the gene of interest, and selected with 2.5 µg/mL BSD, 2.5 nM WR99210 (to select for the human dihydrofolate reductase (hdhfr) marker in the cg6-attB locus) and 125 or 250 µg/mL G418 (for the Dd2 attB and 3D7 attB parasite lines respectively). Integrase expressed from the pINT plasmid catalyzes insertion of the attP -plasmid harboring the gene of interest into the attB site. PCR reactions using p1 + p2 and p3 + p4 primers as well as Southern blots analyzing digested-DNA hybridization to cg6 or yfg probes ( designated as open boxes ), can be performed to confirm integration of the gene of interest at the cg6-attB locus. Sequences of the primers for PCR screening are as follows: p1: 5′-GAAAATATTATTACAAAGGGTGAGG, p2: 5′-TTAGCTAATTCGCTTGTAAG, p3: 5′-CTCTTCTACTCTTTCGAATTC, p4 is designed based on the sequence of the gene of interest. pBS refers to the pBluescript backbone. UTR : untranslated region. Panel ( c with the permission of the Nature Publishing Group.
    Figure Legend Snippet: Dual-plasmid approach for integrase-mediated attB × attP recombination. ( a ) Map of the attP -carrying plasmid pLN-ENR-GFP that can be used as a template for engineering the stable site-specific integration of a gene of interest. Digestion with Avr II and Afl II can be performed to replace the enr-gfp fusion by the gene of interest. Alternatively, digestion with Avr II and Bsi WI can place the gene of interest in frame with gfp . The plasmid carries the blasticidin S-deaminase ( bsd ) selectable marker. ( b ) Map of the integrase-expressing plasmid pINT, which harbors a neomycin selectable marker. The plasmid is usually maintained under G418 selection pressure only for the first 6 days following transfection. ( c ) Schematic representation of the attB × attP recombination allowing integration of a gene of interest ( yfg ) at the cg6 - attB locus. Dd2 attB or 3D7 attB parasites are co-transfected with pINT and an attP -plasmid carrying the gene of interest, and selected with 2.5 µg/mL BSD, 2.5 nM WR99210 (to select for the human dihydrofolate reductase (hdhfr) marker in the cg6-attB locus) and 125 or 250 µg/mL G418 (for the Dd2 attB and 3D7 attB parasite lines respectively). Integrase expressed from the pINT plasmid catalyzes insertion of the attP -plasmid harboring the gene of interest into the attB site. PCR reactions using p1 + p2 and p3 + p4 primers as well as Southern blots analyzing digested-DNA hybridization to cg6 or yfg probes ( designated as open boxes ), can be performed to confirm integration of the gene of interest at the cg6-attB locus. Sequences of the primers for PCR screening are as follows: p1: 5′-GAAAATATTATTACAAAGGGTGAGG, p2: 5′-TTAGCTAATTCGCTTGTAAG, p3: 5′-CTCTTCTACTCTTTCGAATTC, p4 is designed based on the sequence of the gene of interest. pBS refers to the pBluescript backbone. UTR : untranslated region. Panel ( c with the permission of the Nature Publishing Group.

    Techniques Used: Plasmid Preparation, Marker, Expressing, Selection, Transfection, Polymerase Chain Reaction, DNA Hybridization, Sequencing

    Related Articles

    Isolation:

    Article Title: The Highly Virulent 2006 Norwegian EHEC O103:H25 Outbreak Strain Is Related to the 2011 German O104:H4 Outbreak Strain
    Article Snippet: .. Pulsed-Field Gel Electrophoresis (PFGE) and plasmid isolation The E. coli isolates associated with the outbreak were analyzed by PFGE as described previously using the restriction enzymes Xba I and Avr II (New England BioLabs). .. DNA for detection of colicin E2 encoding plasmids were isolated by Qiagen plasmid purification kit (Qiagen, Hilden, Germany), and separated by electrophoresis in 2% agarose gels.

    Construct:

    Article Title: A novel approach to the generation of seamless constructs for plant transformation
    Article Snippet: .. Plasmid mini-preparations of overnight LB cultures (100 mg/L ampicillin) of randomly selected single white colonies were analyzed with REs NotI for pAUrumII-gfp , AvrII for pAUrumIII-gfp and AseI + NcoI for both constructs (all New England Biolabs). .. A plasmid midi-preparation of a selected positive clone of each of pAUrumII-gfp and pAUrumIII-gfp was made and correct gene insertion was verified by sequencing at Eurofins MWG Operon, Germany.

    Generated:

    Article Title: Heterologous Gene Expression from Transmissible Gastroenteritis Virus Replicon Particles
    Article Snippet: .. TGEV pFiGFP2( Pfl MI) was digested with Pfl MI and Avr II or Eco NI, treated with T4 DNA polymerase under conditions in which the 5′→3′ exonuclease activity generated blunt ends (according to the manufacturer’s directions) (New England BioLabs), and religated using T4 DNA ligase. ..

    Activity Assay:

    Article Title: Heterologous Gene Expression from Transmissible Gastroenteritis Virus Replicon Particles
    Article Snippet: .. TGEV pFiGFP2( Pfl MI) was digested with Pfl MI and Avr II or Eco NI, treated with T4 DNA polymerase under conditions in which the 5′→3′ exonuclease activity generated blunt ends (according to the manufacturer’s directions) (New England BioLabs), and religated using T4 DNA ligase. ..

    Pulsed-Field Gel:

    Article Title: The Highly Virulent 2006 Norwegian EHEC O103:H25 Outbreak Strain Is Related to the 2011 German O104:H4 Outbreak Strain
    Article Snippet: .. Pulsed-Field Gel Electrophoresis (PFGE) and plasmid isolation The E. coli isolates associated with the outbreak were analyzed by PFGE as described previously using the restriction enzymes Xba I and Avr II (New England BioLabs). .. DNA for detection of colicin E2 encoding plasmids were isolated by Qiagen plasmid purification kit (Qiagen, Hilden, Germany), and separated by electrophoresis in 2% agarose gels.

    Article Title: Evidence of Metabolic Switching and Implications for Food Safety from the Phenome(s) of Salmonella enterica Serovar Typhimurium DT104 Cultured at Selected Points across the Pork Production Food Chain
    Article Snippet: .. Pulsed-field gel electrophoresis (PFGE) was performed using the restriction endonucleases XbaI and AvrII (New England BioLabs) by following the CDC PulseNet protocol ( ). .. Salmonella enterica serovar Braenderup H9812 (ATCC BAA-664) was used as the reference strain for the molecular size standard.

    Plasmid Preparation:

    Article Title: The Highly Virulent 2006 Norwegian EHEC O103:H25 Outbreak Strain Is Related to the 2011 German O104:H4 Outbreak Strain
    Article Snippet: .. Pulsed-Field Gel Electrophoresis (PFGE) and plasmid isolation The E. coli isolates associated with the outbreak were analyzed by PFGE as described previously using the restriction enzymes Xba I and Avr II (New England BioLabs). .. DNA for detection of colicin E2 encoding plasmids were isolated by Qiagen plasmid purification kit (Qiagen, Hilden, Germany), and separated by electrophoresis in 2% agarose gels.

    Article Title: A novel approach to the generation of seamless constructs for plant transformation
    Article Snippet: .. Plasmid mini-preparations of overnight LB cultures (100 mg/L ampicillin) of randomly selected single white colonies were analyzed with REs NotI for pAUrumII-gfp , AvrII for pAUrumIII-gfp and AseI + NcoI for both constructs (all New England Biolabs). .. A plasmid midi-preparation of a selected positive clone of each of pAUrumII-gfp and pAUrumIII-gfp was made and correct gene insertion was verified by sequencing at Eurofins MWG Operon, Germany.

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    Genomic structure of TK + and TK − viruses. (A) The TK − Kan + virus was generated by inserting a FRT-flanked kanamycin resistance gene at genomic coordinates 33301 to 33450. The kanamycin resistance coding sequence was removed by Flp recombination to leave an inset of 167 bp (TK − Kan − ). This virus was reverted by homologous recombination with an unmutated genomic segment (TK + R). An independent mutant (TK − Del) was generated by RecA-mediated recombination of BAC DNA with a genomic plasmid clone in which genomic coordinates 33293 to 34300 are deleted by digestion with <t>Avr</t> II and <t>Pme</t> I. (B) Viral DNA was digested with Xba I, Eco RI, or Sac I, electrophoresed, blotted, and probed with a 32 P-labeled genomic Eco RI fragment spanning the TK locus.
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    Genomic structure of TK + and TK − viruses. (A) The TK − Kan + virus was generated by inserting a FRT-flanked kanamycin resistance gene at genomic coordinates 33301 to 33450. The kanamycin resistance coding sequence was removed by Flp recombination to leave an inset of 167 bp (TK − Kan − ). This virus was reverted by homologous recombination with an unmutated genomic segment (TK + R). An independent mutant (TK − Del) was generated by RecA-mediated recombination of BAC DNA with a genomic plasmid clone in which genomic coordinates 33293 to 34300 are deleted by digestion with Avr II and Pme I. (B) Viral DNA was digested with Xba I, Eco RI, or Sac I, electrophoresed, blotted, and probed with a 32 P-labeled genomic Eco RI fragment spanning the TK locus.

    Journal: Journal of Virology

    Article Title: Murine Gammaherpesvirus 68 Lacking Thymidine Kinase Shows Severe Attenuation of Lytic Cycle Replication In Vivo but Still Establishes Latency

    doi: 10.1128/JVI.77.4.2410-2417.2003

    Figure Lengend Snippet: Genomic structure of TK + and TK − viruses. (A) The TK − Kan + virus was generated by inserting a FRT-flanked kanamycin resistance gene at genomic coordinates 33301 to 33450. The kanamycin resistance coding sequence was removed by Flp recombination to leave an inset of 167 bp (TK − Kan − ). This virus was reverted by homologous recombination with an unmutated genomic segment (TK + R). An independent mutant (TK − Del) was generated by RecA-mediated recombination of BAC DNA with a genomic plasmid clone in which genomic coordinates 33293 to 34300 are deleted by digestion with Avr II and Pme I. (B) Viral DNA was digested with Xba I, Eco RI, or Sac I, electrophoresed, blotted, and probed with a 32 P-labeled genomic Eco RI fragment spanning the TK locus.

    Article Snippet: The central portion (33293 to 34300) of the TK open reading frame (ORF) (32879 to 34813) was excised by digestion with Avr II and Pme I, blunting with T4 DNA polymerase (New England Biolabs, Hitchin, United Kingdom), and religation with T4 DNA ligase (New England Biolabs).

    Techniques: Generated, Sequencing, Homologous Recombination, Mutagenesis, BAC Assay, Plasmid Preparation, Labeling

    Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.

    Journal: Tropical Medicine and Infectious Disease

    Article Title: Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine

    doi: 10.3390/tropicalmed2030037

    Figure Lengend Snippet: Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.

    Article Snippet: To replace both the M and G genes of SPBN with those of the LBVAFR1999, the KpnI site was introduced upstream of the pSPBN-LBVG M gene start signal, through digestion of another pSPBN (that contains the KpnI site) with AvrII and KpnI (New England Biolabs, Ipswich, MA, USA) ( ).

    Techniques: Recombinant

    PFGE of outbreak associated isolates. PFGE of E. coli O103:H25 NOS (lane 2), NVH-847 (lane 3), NVH-848 (lane 4), and NVH-760 (lane 5). Lambda ladder is used as marker (lane 1). Digestion with Xba I (A) showed indistinguishable PFGE patterns, while digestion with Avr II (B) exposed a difference between the stx 2 -positive and the stx -negative isolates.

    Journal: PLoS ONE

    Article Title: The Highly Virulent 2006 Norwegian EHEC O103:H25 Outbreak Strain Is Related to the 2011 German O104:H4 Outbreak Strain

    doi: 10.1371/journal.pone.0031413

    Figure Lengend Snippet: PFGE of outbreak associated isolates. PFGE of E. coli O103:H25 NOS (lane 2), NVH-847 (lane 3), NVH-848 (lane 4), and NVH-760 (lane 5). Lambda ladder is used as marker (lane 1). Digestion with Xba I (A) showed indistinguishable PFGE patterns, while digestion with Avr II (B) exposed a difference between the stx 2 -positive and the stx -negative isolates.

    Article Snippet: Pulsed-Field Gel Electrophoresis (PFGE) and plasmid isolation The E. coli isolates associated with the outbreak were analyzed by PFGE as described previously using the restriction enzymes Xba I and Avr II (New England BioLabs).

    Techniques: Marker

    TGEV-Rep( Avr II) VRPs. Transfected cultures displayed GFP expression at ∼18 h posttransfection (A) [TGEV-Rep( Avr II) pass 0]. Supernatants were harvested ∼36 h posttransfection and used to infect fresh cultures of ST cells for 1 h at room temperature. At ∼18 h postinfection, GFP expression was observed by fluorescent microscopy in these pass 1 cultures (C and D). (C) TGEV VRP production following coelectroporation with VEE-E RNAs [TGEV-Rep( Avr II) plus E protein transcripts pass 1]; (D) TGEV VRP production following infection with VEE VRPs encoding the E protein [TGEV-Rep( Avr II) plus VEE-TGEV(E) pass 1]. Passage of supernatants from cells transfected with TGEV-Rep( Avr II) transcripts without expression of the E protein in trans did not result in detectable GFP expression (B) [TGEV-Rep( Avr II) without E protein pass 1].

    Journal: Journal of Virology

    Article Title: Heterologous Gene Expression from Transmissible Gastroenteritis Virus Replicon Particles

    doi: 10.1128/JVI.76.3.1422-1434.2002

    Figure Lengend Snippet: TGEV-Rep( Avr II) VRPs. Transfected cultures displayed GFP expression at ∼18 h posttransfection (A) [TGEV-Rep( Avr II) pass 0]. Supernatants were harvested ∼36 h posttransfection and used to infect fresh cultures of ST cells for 1 h at room temperature. At ∼18 h postinfection, GFP expression was observed by fluorescent microscopy in these pass 1 cultures (C and D). (C) TGEV VRP production following coelectroporation with VEE-E RNAs [TGEV-Rep( Avr II) plus E protein transcripts pass 1]; (D) TGEV VRP production following infection with VEE VRPs encoding the E protein [TGEV-Rep( Avr II) plus VEE-TGEV(E) pass 1]. Passage of supernatants from cells transfected with TGEV-Rep( Avr II) transcripts without expression of the E protein in trans did not result in detectable GFP expression (B) [TGEV-Rep( Avr II) without E protein pass 1].

    Article Snippet: TGEV pFiGFP2( Pfl MI) was digested with Pfl MI and Avr II or Eco NI, treated with T4 DNA polymerase under conditions in which the 5′→3′ exonuclease activity generated blunt ends (according to the manufacturer’s directions) (New England BioLabs), and religated using T4 DNA ligase.

    Techniques: Transfection, Expressing, Microscopy, Infection

    Construction of TGEV replicon cDNAs. (a) TGEV F fragments. Structural genes are contained in the TGEV F fragment. FiGFP2( Pfl MI) (∼5.6 kb) was constructed from the wild-type TGEV F fragment (∼5.1 kb) by the deletion of ORF 3A (nt 24828 to 25073) and the insertion of GFP with a 5′ 20-nt N gene IS. Using this construct, we introduced deletions extending from the unique Pfl MI site at the very 3′ end of GFP to the unique Avr II (nucleotide position 25866) and Eco NI (nucleotide position 26935) sites present within the TGEV E and M genes, respectively. (b) Sequence organization of GFP in FiGFP2( Pfl MI). GFP was inserted just downstream of the ORF 3A IS. The TGEV sequence originating at the 3′ end of the S gene (nt 24693) through the start of the GFP gene is shown, and the important IS and restriction sites are labeled. (c) Strategy for assembling recombinant TGEV and replicon cDNAs. The six cDNA subclones (TGEV A, B1, B2, C, DE1, and F deletion fragments) spanning the genome are flanked by unique interconnecting Bgl I and Bst ). TGEV A contained a unique T7 start site at its 5′ end, and the F deletion fragments (FiGFP2- Avr II and FiGFP2- Eco NI) contain GFP and a 25-nt T tail, allowing for the synthesis of capped T7, polyadenylated transcripts in vitro.

    Journal: Journal of Virology

    Article Title: Heterologous Gene Expression from Transmissible Gastroenteritis Virus Replicon Particles

    doi: 10.1128/JVI.76.3.1422-1434.2002

    Figure Lengend Snippet: Construction of TGEV replicon cDNAs. (a) TGEV F fragments. Structural genes are contained in the TGEV F fragment. FiGFP2( Pfl MI) (∼5.6 kb) was constructed from the wild-type TGEV F fragment (∼5.1 kb) by the deletion of ORF 3A (nt 24828 to 25073) and the insertion of GFP with a 5′ 20-nt N gene IS. Using this construct, we introduced deletions extending from the unique Pfl MI site at the very 3′ end of GFP to the unique Avr II (nucleotide position 25866) and Eco NI (nucleotide position 26935) sites present within the TGEV E and M genes, respectively. (b) Sequence organization of GFP in FiGFP2( Pfl MI). GFP was inserted just downstream of the ORF 3A IS. The TGEV sequence originating at the 3′ end of the S gene (nt 24693) through the start of the GFP gene is shown, and the important IS and restriction sites are labeled. (c) Strategy for assembling recombinant TGEV and replicon cDNAs. The six cDNA subclones (TGEV A, B1, B2, C, DE1, and F deletion fragments) spanning the genome are flanked by unique interconnecting Bgl I and Bst ). TGEV A contained a unique T7 start site at its 5′ end, and the F deletion fragments (FiGFP2- Avr II and FiGFP2- Eco NI) contain GFP and a 25-nt T tail, allowing for the synthesis of capped T7, polyadenylated transcripts in vitro.

    Article Snippet: TGEV pFiGFP2( Pfl MI) was digested with Pfl MI and Avr II or Eco NI, treated with T4 DNA polymerase under conditions in which the 5′→3′ exonuclease activity generated blunt ends (according to the manufacturer’s directions) (New England BioLabs), and religated using T4 DNA ligase.

    Techniques: Construct, Sequencing, Labeling, Recombinant, In Vitro

    Heterologous gene expression from TGEV replicon RNAs. (a) Cultures of BHK cells were transfected with TGEV replicon RNAs encoding GFP that contained ORF 3B and E (A) [TGEV-Rep( Avr II) transcripts] or ORF 3B, E, and M gene deletions (C). Alternatively, transcripts were treated with RNase prior to transfection into BHK cells (B and D) [TGEV-Rep( Avr II) DNA and TGEV-Rep( Eco NI) DNA only, respectively]. At ∼18 h posttransfection, GFP expression was observed by fluorescent microscopy. (b) Intracellular RNA was isolated from transfected cell cultures and used as a template for RT-PCR. Leader-containing GFP subgenomic transcripts were detected using a 5′ leader primer (TGEV-L) and 3′ primers located just downstream of the Avr II [(−)E5546] or Eco NI [M6400(−)] sites. Appropriately sized amplicons of ∼850 bp were generated, corresponding to transcripts encoding GFP. Cells transfected with TGEV-Rep( Avr II) (A) or TGEV-Rep( Eco NI) (B) RNA are shown. A 1-kb ladder is shown in both panels (lanes 1). Arrows indicate leader-containing GFP amplicons (lanes 2).

    Journal: Journal of Virology

    Article Title: Heterologous Gene Expression from Transmissible Gastroenteritis Virus Replicon Particles

    doi: 10.1128/JVI.76.3.1422-1434.2002

    Figure Lengend Snippet: Heterologous gene expression from TGEV replicon RNAs. (a) Cultures of BHK cells were transfected with TGEV replicon RNAs encoding GFP that contained ORF 3B and E (A) [TGEV-Rep( Avr II) transcripts] or ORF 3B, E, and M gene deletions (C). Alternatively, transcripts were treated with RNase prior to transfection into BHK cells (B and D) [TGEV-Rep( Avr II) DNA and TGEV-Rep( Eco NI) DNA only, respectively]. At ∼18 h posttransfection, GFP expression was observed by fluorescent microscopy. (b) Intracellular RNA was isolated from transfected cell cultures and used as a template for RT-PCR. Leader-containing GFP subgenomic transcripts were detected using a 5′ leader primer (TGEV-L) and 3′ primers located just downstream of the Avr II [(−)E5546] or Eco NI [M6400(−)] sites. Appropriately sized amplicons of ∼850 bp were generated, corresponding to transcripts encoding GFP. Cells transfected with TGEV-Rep( Avr II) (A) or TGEV-Rep( Eco NI) (B) RNA are shown. A 1-kb ladder is shown in both panels (lanes 1). Arrows indicate leader-containing GFP amplicons (lanes 2).

    Article Snippet: TGEV pFiGFP2( Pfl MI) was digested with Pfl MI and Avr II or Eco NI, treated with T4 DNA polymerase under conditions in which the 5′→3′ exonuclease activity generated blunt ends (according to the manufacturer’s directions) (New England BioLabs), and religated using T4 DNA ligase.

    Techniques: Expressing, Transfection, Microscopy, Isolation, Reverse Transcription Polymerase Chain Reaction, Generated

    Strategy to assemble TGEV-Rep( Avr II) VRPs. (a) In the full-length TGEV-Rep( Avr II) cDNA construct, ORF 3A has been replaced with GFP, and ORF 3B and the 5′ end of the E gene have been deleted. To produce packaged replicon particles, replicon RNA-transfected cells were infected with VEE VRPs expressing the TGEV E protein [VEE-TGEV(E)]. Alternatively, TGEV-Rep( Avr II) replicon RNAs can be coelectroporated with pVR21-E1-derived transcripts. TGEV VRPs should be released from cells that can be used as single-hit expression vectors. (b) Cultures of ST cells were infected with either wild-type (wt) TGEV alone or with TGEV and VEE VRPs expressing a G 1 Norwalk-like virus capsid (wt + VEE) at an MOI of 5 for 1 h at room temperature. The inocula were removed, and the cultures were incubated in complete medium at 37°C. Samples were harvested at the indicated times and assayed by plaque assay in ST cells.

    Journal: Journal of Virology

    Article Title: Heterologous Gene Expression from Transmissible Gastroenteritis Virus Replicon Particles

    doi: 10.1128/JVI.76.3.1422-1434.2002

    Figure Lengend Snippet: Strategy to assemble TGEV-Rep( Avr II) VRPs. (a) In the full-length TGEV-Rep( Avr II) cDNA construct, ORF 3A has been replaced with GFP, and ORF 3B and the 5′ end of the E gene have been deleted. To produce packaged replicon particles, replicon RNA-transfected cells were infected with VEE VRPs expressing the TGEV E protein [VEE-TGEV(E)]. Alternatively, TGEV-Rep( Avr II) replicon RNAs can be coelectroporated with pVR21-E1-derived transcripts. TGEV VRPs should be released from cells that can be used as single-hit expression vectors. (b) Cultures of ST cells were infected with either wild-type (wt) TGEV alone or with TGEV and VEE VRPs expressing a G 1 Norwalk-like virus capsid (wt + VEE) at an MOI of 5 for 1 h at room temperature. The inocula were removed, and the cultures were incubated in complete medium at 37°C. Samples were harvested at the indicated times and assayed by plaque assay in ST cells.

    Article Snippet: TGEV pFiGFP2( Pfl MI) was digested with Pfl MI and Avr II or Eco NI, treated with T4 DNA polymerase under conditions in which the 5′→3′ exonuclease activity generated blunt ends (according to the manufacturer’s directions) (New England BioLabs), and religated using T4 DNA ligase.

    Techniques: Construct, Transfection, Infection, Expressing, Derivative Assay, Incubation, Plaque Assay

    Sequence analysis of leader-containing amplicons encoding GFP. TGEV-Rep( Avr II) and TGEV-Rep( Eco NI) leader-containing amplicons were isolated from agarose gels and subcloned into Topo II TA cloning vectors. Inserts were sequenced using universal primers and an automated sequencer. (A) Sequence of the 5′ end of GFP amplicons generated from TGEV-Rep( Eco NI)-transfected cells, indicating that the leader-containing transcripts initiated from the TGEV ORF 3A IS. (B) Leader-containing GFP transcripts with the Pfl MI- Avr II deletion derived from TGEV-Rep( Avr II) VRP-infected cells. (C) Leader-containing GFP transcripts with the Pfl MI- Eco NI deletion derived from TGEV-Rep( Eco NI)-transfected cells. Underlined bases correspond to the GFP stop codon, and the shaded bases correspond to the deletion and blunt-end ligation sites.

    Journal: Journal of Virology

    Article Title: Heterologous Gene Expression from Transmissible Gastroenteritis Virus Replicon Particles

    doi: 10.1128/JVI.76.3.1422-1434.2002

    Figure Lengend Snippet: Sequence analysis of leader-containing amplicons encoding GFP. TGEV-Rep( Avr II) and TGEV-Rep( Eco NI) leader-containing amplicons were isolated from agarose gels and subcloned into Topo II TA cloning vectors. Inserts were sequenced using universal primers and an automated sequencer. (A) Sequence of the 5′ end of GFP amplicons generated from TGEV-Rep( Eco NI)-transfected cells, indicating that the leader-containing transcripts initiated from the TGEV ORF 3A IS. (B) Leader-containing GFP transcripts with the Pfl MI- Avr II deletion derived from TGEV-Rep( Avr II) VRP-infected cells. (C) Leader-containing GFP transcripts with the Pfl MI- Eco NI deletion derived from TGEV-Rep( Eco NI)-transfected cells. Underlined bases correspond to the GFP stop codon, and the shaded bases correspond to the deletion and blunt-end ligation sites.

    Article Snippet: TGEV pFiGFP2( Pfl MI) was digested with Pfl MI and Avr II or Eco NI, treated with T4 DNA polymerase under conditions in which the 5′→3′ exonuclease activity generated blunt ends (according to the manufacturer’s directions) (New England BioLabs), and religated using T4 DNA ligase.

    Techniques: Sequencing, Isolation, TA Cloning, Generated, Transfection, Derivative Assay, Infection, Ligation