Structured Review

Roche t4 dna polymerase
Trf4 and Trf5 exhibit a poly(A) polymerase activity but no DNA polymerase activity. (A) DNA polymerase assays on a 29/75-nt primer/template partial duplex DNA substrate. The 29-nt primer was 32 P labeled at the 5′ end and annealed to a 75-nt template. Wild-type (wt) Trf4 (lane 3), Trf4 DD236,238AA (lane 4), and wild-type Trf5 (lane 5) (10 nM each) were incubated with the DNA substrate (20 nM) in the presence of each of the four dNTPs (100 μM). For positive and negative controls, parallel reactions were also carried out with <t>T4</t> DNA polymerase (lane 2), and no added protein (NP) (lane 1). Reaction products were analyzed on a 15% polyacrylamide gel containing 8 M urea and analyzed by a PhosphorImager. (B) DNA polymerase assays on an oligo(dT)/poly(dA) DNA substrate. A mixture of 12- to 18-nt oligo(dT) primers was 5′ end labeled and annealed to poly(dA) template. DNA polymerase reactions were carried out as described for panel A. (C) Poly(A) polymerase assays on a poly(A) substrate. Trf4 (lane 2), Trf4 DD236,238AA (lane 3), and Trf5 (lane 4) (10 nM each) were incubated with poly(A) RNA and [α- 32 P]ATP (50 μM) in the presence of 5 mM Mg 2+ and 0.5 mM Mn 2+ . A control reaction (lane 1) included no added protein (NP). Reaction products were resolved on a 15% polyacrylamide gel containing 8 M urea followed by PhosphorImager analyses of the incorporation of AMP into RNA tails. The sizes of the reaction products (in nucleotides) are indicated to the right of the gel.
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1) Product Images from "Trf4 and Trf5 Proteins of Saccharomyces cerevisiae Exhibit Poly(A) RNA Polymerase Activity but No DNA Polymerase Activity"

Article Title: Trf4 and Trf5 Proteins of Saccharomyces cerevisiae Exhibit Poly(A) RNA Polymerase Activity but No DNA Polymerase Activity

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.25.22.10183-10189.2005

Trf4 and Trf5 exhibit a poly(A) polymerase activity but no DNA polymerase activity. (A) DNA polymerase assays on a 29/75-nt primer/template partial duplex DNA substrate. The 29-nt primer was 32 P labeled at the 5′ end and annealed to a 75-nt template. Wild-type (wt) Trf4 (lane 3), Trf4 DD236,238AA (lane 4), and wild-type Trf5 (lane 5) (10 nM each) were incubated with the DNA substrate (20 nM) in the presence of each of the four dNTPs (100 μM). For positive and negative controls, parallel reactions were also carried out with T4 DNA polymerase (lane 2), and no added protein (NP) (lane 1). Reaction products were analyzed on a 15% polyacrylamide gel containing 8 M urea and analyzed by a PhosphorImager. (B) DNA polymerase assays on an oligo(dT)/poly(dA) DNA substrate. A mixture of 12- to 18-nt oligo(dT) primers was 5′ end labeled and annealed to poly(dA) template. DNA polymerase reactions were carried out as described for panel A. (C) Poly(A) polymerase assays on a poly(A) substrate. Trf4 (lane 2), Trf4 DD236,238AA (lane 3), and Trf5 (lane 4) (10 nM each) were incubated with poly(A) RNA and [α- 32 P]ATP (50 μM) in the presence of 5 mM Mg 2+ and 0.5 mM Mn 2+ . A control reaction (lane 1) included no added protein (NP). Reaction products were resolved on a 15% polyacrylamide gel containing 8 M urea followed by PhosphorImager analyses of the incorporation of AMP into RNA tails. The sizes of the reaction products (in nucleotides) are indicated to the right of the gel.
Figure Legend Snippet: Trf4 and Trf5 exhibit a poly(A) polymerase activity but no DNA polymerase activity. (A) DNA polymerase assays on a 29/75-nt primer/template partial duplex DNA substrate. The 29-nt primer was 32 P labeled at the 5′ end and annealed to a 75-nt template. Wild-type (wt) Trf4 (lane 3), Trf4 DD236,238AA (lane 4), and wild-type Trf5 (lane 5) (10 nM each) were incubated with the DNA substrate (20 nM) in the presence of each of the four dNTPs (100 μM). For positive and negative controls, parallel reactions were also carried out with T4 DNA polymerase (lane 2), and no added protein (NP) (lane 1). Reaction products were analyzed on a 15% polyacrylamide gel containing 8 M urea and analyzed by a PhosphorImager. (B) DNA polymerase assays on an oligo(dT)/poly(dA) DNA substrate. A mixture of 12- to 18-nt oligo(dT) primers was 5′ end labeled and annealed to poly(dA) template. DNA polymerase reactions were carried out as described for panel A. (C) Poly(A) polymerase assays on a poly(A) substrate. Trf4 (lane 2), Trf4 DD236,238AA (lane 3), and Trf5 (lane 4) (10 nM each) were incubated with poly(A) RNA and [α- 32 P]ATP (50 μM) in the presence of 5 mM Mg 2+ and 0.5 mM Mn 2+ . A control reaction (lane 1) included no added protein (NP). Reaction products were resolved on a 15% polyacrylamide gel containing 8 M urea followed by PhosphorImager analyses of the incorporation of AMP into RNA tails. The sizes of the reaction products (in nucleotides) are indicated to the right of the gel.

Techniques Used: Activity Assay, Labeling, Incubation

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Amplification:

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Synthesized:

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Construct:

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Electrophoresis:

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Incubation:

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Stripping Membranes:

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Activity Assay:

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Expressing:

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Transformation Assay:

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Derivative Assay:

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Hybridization:

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Ligation:

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Infection:

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Generated:

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DNA Sequencing:

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Article Title: Human antibody responses against non-covalently cell wall-bound Staphylococcus aureus proteins
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Ligase Independent Cloning:

Article Title: Human antibody responses against non-covalently cell wall-bound Staphylococcus aureus proteins
Article Snippet: Primers listed in Table were designed to amplify gene sequences without the region coding for the natural secretion signals and with 5′ end extensions for ligase-independent cloning (LIC) . .. Briefly, Swa I-digested pRE-USP plasmid and PCR fragments were treated with T4 DNA polymerase (20 °C, 20 min; 75 °C, 20 min; Roche Diagnostics) before incubation for 5 min at room temperature (3:1 vector:insert).

Article Title: Human antibody responses against non-covalently cell wall-bound Staphylococcus aureus proteins
Article Snippet: Primers listed in Table were designed to amplify gene sequences without the region coding for the natural secretion signals and with 5′ end extensions for ligase-independent cloning (LIC) . .. Briefly, Swa I-digested pRE-USP plasmid and PCR fragments were treated with T4 DNA polymerase (20 °C, 20 min; 75 °C, 20 min; Roche Diagnostics) before incubation for 5 min at room temperature (3:1 vector:insert).

Sequencing:

Article Title: A Robust High Throughput Platform to Generate Functional Recombinant Monoclonal Antibodies Using Rabbit B Cells from Peripheral Blood
Article Snippet: The PCR-products derived from the individual rabbit B-cells were cloned into the respective expression vectors using a “sequence and ligation independent cloning” (SLIC) method , . .. The purified linear vector and the insert were treated with 0.5 U T4 DNA polymerase (Roche Applied Sciences, Mannheim, Germany) per 1 µg DNA for 45 min at 25°C in the absence of dNTPs to generate matching overhangs.

Article Title: Resveratrol: brain effects on SIRT1, GPR50 and photoperiodic signaling
Article Snippet: Cloning of SIRT1-Specific DNA Targets Aliquots of 5 μl of the purified DNA solution obtained with monoclonal anti-SIRT1 antibody were treated with T4 DNA polymerase and dNTPs (Roche Applied Science, Mannheim, Germany) for 15 min at 16°C and subsequently heat inactivated at 75°C for 10 min. .. Purified DNA was amplified by standard PCR, using primer matching the EcoRI adaptor sequence (forward and reverse primer: AATTCCGTTGCTGTCG).

Article Title: Unique secreted–surface protein complex of Lactobacillus rhamnosus, identified by phage display
Article Snippet: Paragraph title: Construction of the phagemid pYW01 carrying PelB signal sequence and colD origin of replication ... This was achieved by cleavage of the psp -pelB -c-myc-gIIIC cassette and origin of replication from pDJ08 using PstI-ScaI followed by removal of 3′ protruding end from PstI by T4 DNA polymerase (Roche, Basel, Switzerland) and ligation of the resulting fragment to pGZ119EH EcoRV-ScaI fragment carrying colD origin of replication.

Article Title: Efficient Subtractive Cloning of Genes Activated by Lipopolysaccharide and Interferon ? in Primary-Cultured Cortical Cells of Newborn Mice
Article Snippet: The bead-fixed 3′-trimmed cDNA was blunt-ended with 0.5 units of T4 DNA polymerase (Roche Diagnostics, Tokyo, Japan) in 20 µL of a mixture containing 50 mM Tris·HCl (pH 8.8), 15 mM (NH4 )2 SO4 , 7 mM MgCl2 , 0.1 mM EDTA, 10 mM 2-mercaptoethanol, 0.02 mg/mL bovine serum albumin, and 0.1 mM each of dATP, dCTP, dGTP, and dTTP at 16°C for 10 min. .. The bead-fixed blunt-ended cDNA was ligated with a linker containing the T7 promoter sequence (Step 3).

Article Title: Tea (Camellia sinensis) infusions ameliorate cancer in 4TI metastatic breast cancer model
Article Snippet: Paragraph title: Preparation of cDNA library for Transcriptome sequencing ... The mixture was spun for 2 s and incubated at 16 °C for 2 h before adding 20μlof T4 DNA polymerase (Roche, Germany).

Article Title: Transcriptome analysis of pigeon milk production – role of cornification and triglyceride synthesis genes
Article Snippet: The sequencing libraries were synthesised from the fragmented poly(A)+ RNA according to the Roche cDNA Rapid Library Preparation Method Manual, Rev. .. Briefly, fragmented RNA was reverse transcribed into cDNA and made double-stranded using a mixture of DNA polymerase I, ligase and RNase H and blunt ended with T4 DNA Polymerase (Roche cDNA synthesis system).

Recombinant:

Article Title: A Robust High Throughput Platform to Generate Functional Recombinant Monoclonal Antibodies Using Rabbit B Cells from Peripheral Blood
Article Snippet: Paragraph title: Cloning and Recombinant Expression of Cognate VH and VL Gene Segments ... The purified linear vector and the insert were treated with 0.5 U T4 DNA polymerase (Roche Applied Sciences, Mannheim, Germany) per 1 µg DNA for 45 min at 25°C in the absence of dNTPs to generate matching overhangs.

Magnetic Beads:

Article Title: Efficient Subtractive Cloning of Genes Activated by Lipopolysaccharide and Interferon ? in Primary-Cultured Cortical Cells of Newborn Mice
Article Snippet: Briefly, poly(A)+ RNA derived from 0.5 µg of total RNA was absorbed onto 50 µg of oligo(dT) magnetic beads Dynabeads Oligo(dT)25 (Dynal, Oslo, Norway), and was subjected to first-strand and second-strand cDNA synthesis ( , Step 1). .. The bead-fixed 3′-trimmed cDNA was blunt-ended with 0.5 units of T4 DNA polymerase (Roche Diagnostics, Tokyo, Japan) in 20 µL of a mixture containing 50 mM Tris·HCl (pH 8.8), 15 mM (NH4 )2 SO4 , 7 mM MgCl2 , 0.1 mM EDTA, 10 mM 2-mercaptoethanol, 0.02 mg/mL bovine serum albumin, and 0.1 mM each of dATP, dCTP, dGTP, and dTTP at 16°C for 10 min.

Mutagenesis:

Article Title: The tep1 gene of Sinorhizobium meliloti coding for a putative transmembrane efflux protein and N-acetyl glucosamine affect nod gene expression and nodulation of alfalfa plants
Article Snippet: Paragraph title: Construction of a S. meliloti tep1 mutant ... Next, the Sac I fragment containing the disrupted ORF was treated with T4 DNA polymerase (Roche Biochemicals) to make blunt ends and then cloned into the Sma I site of the suicide vector pK18mobsac [ ] to give pTrans3.

Article Title: Mutations in the β-Subunit of the RNA Polymerase Impair the Surface-Associated Motility and Virulence of Acinetobacter baumannii
Article Snippet: Paragraph title: Mutant complementation. ... The plasmid pBAV1K-T5-gfp was used to complement the Rifr 3, Rifr 5, and Rifr 8 mutants by blunt-end cloning the wild-type rpoB gene from the A. baumannii ATCC 17978 wild-type genome into the XbaI site of the vector, whose overhangs were filled with T4 DNA polymerase (Roche).

Article Title: Trf4 and Trf5 Proteins of Saccharomyces cerevisiae Exhibit Poly(A) RNA Polymerase Activity but No DNA Polymerase Activity
Article Snippet: The mutant Trf4 DD236,238,AA protein was then purified in parallel with the wild-type Trf4 and Trf5 proteins. .. The T4 DNA polymerase was purchased from Roche (Indianapolis, IN).

Purification:

Article Title: A Robust High Throughput Platform to Generate Functional Recombinant Monoclonal Antibodies Using Rabbit B Cells from Peripheral Blood
Article Snippet: .. The purified linear vector and the insert were treated with 0.5 U T4 DNA polymerase (Roche Applied Sciences, Mannheim, Germany) per 1 µg DNA for 45 min at 25°C in the absence of dNTPs to generate matching overhangs. .. The reaction was stopped by adding 1/10th of the reaction volume of a 10 mM dCTP solution (Invitrogen).

Article Title: Resveratrol: brain effects on SIRT1, GPR50 and photoperiodic signaling
Article Snippet: .. Cloning of SIRT1-Specific DNA Targets Aliquots of 5 μl of the purified DNA solution obtained with monoclonal anti-SIRT1 antibody were treated with T4 DNA polymerase and dNTPs (Roche Applied Science, Mannheim, Germany) for 15 min at 16°C and subsequently heat inactivated at 75°C for 10 min. .. According to the manufacturer’s instructions, EcoRI adaptor oligonucleotides were ligated to the DNA pieces and excess adaptors subsequently removed with Sephacryl S-400 spin columns (Universal Riboclone cDNA Synthesis System, Promega, Madison, WI, USA).

Article Title: The Sas3p and Gcn5p histone acetyltransferases are recruited to similar genes
Article Snippet: For immunoprecipitated DNA samples, the aqueous phase was subsequently purified with Montage PCR columns (Millipore Corp., Billerica, MA, USA). .. The immunoprecipitated DNA was blunted by T4 phage DNA polymerase in a reaction volume of 124 μL (T4 DNA Pol buffer, 40 μg/μl BSA, 80 μM dNTPs, 0.6 U T4 DNA Polymerase from Roche).

Article Title: Dynamic remodeling of histone modifications in response to osmotic stress in Saccharomyces cerevisiae
Article Snippet: The aqueous phase was subsequently purified with Montage PCR columns (Millipore). .. Immunoprecipitated DNA was blunted by T4 phage DNA polymerase in a reaction volume of 124 μL (T4 DNA Pol buffer, 40 μg/μL BSA, 80 μM dNTPs, 0.6 U T4 DNA Polymerase from Roche).

Article Title: Transcriptome analysis of pigeon milk production – role of cornification and triglyceride synthesis genes
Article Snippet: Poly(A)+ RNA was twice purified from total RNA using Dynal beads (Life Technologies), according to the manufacturer’s instructions. .. Briefly, fragmented RNA was reverse transcribed into cDNA and made double-stranded using a mixture of DNA polymerase I, ligase and RNase H and blunt ended with T4 DNA Polymerase (Roche cDNA synthesis system).

Article Title: Trf4 and Trf5 Proteins of Saccharomyces cerevisiae Exhibit Poly(A) RNA Polymerase Activity but No DNA Polymerase Activity
Article Snippet: The mutant Trf4 DD236,238,AA protein was then purified in parallel with the wild-type Trf4 and Trf5 proteins. .. The T4 DNA polymerase was purchased from Roche (Indianapolis, IN).

Polymerase Chain Reaction:

Article Title: A Robust High Throughput Platform to Generate Functional Recombinant Monoclonal Antibodies Using Rabbit B Cells from Peripheral Blood
Article Snippet: The PCR-products derived from the individual rabbit B-cells were cloned into the respective expression vectors using a “sequence and ligation independent cloning” (SLIC) method , . .. The purified linear vector and the insert were treated with 0.5 U T4 DNA polymerase (Roche Applied Sciences, Mannheim, Germany) per 1 µg DNA for 45 min at 25°C in the absence of dNTPs to generate matching overhangs.

Article Title: Resveratrol: brain effects on SIRT1, GPR50 and photoperiodic signaling
Article Snippet: Cloning of SIRT1-Specific DNA Targets Aliquots of 5 μl of the purified DNA solution obtained with monoclonal anti-SIRT1 antibody were treated with T4 DNA polymerase and dNTPs (Roche Applied Science, Mannheim, Germany) for 15 min at 16°C and subsequently heat inactivated at 75°C for 10 min. .. Purified DNA was amplified by standard PCR, using primer matching the EcoRI adaptor sequence (forward and reverse primer: AATTCCGTTGCTGTCG).

Article Title: Unique secreted–surface protein complex of Lactobacillus rhamnosus, identified by phage display
Article Snippet: The PCR product was cleaved and ligated into the EarI-NsiI digested phagemid pDJ01 ( ). .. This was achieved by cleavage of the psp -pelB -c-myc-gIIIC cassette and origin of replication from pDJ08 using PstI-ScaI followed by removal of 3′ protruding end from PstI by T4 DNA polymerase (Roche, Basel, Switzerland) and ligation of the resulting fragment to pGZ119EH EcoRV-ScaI fragment carrying colD origin of replication.

Article Title: Human antibody responses against non-covalently cell wall-bound Staphylococcus aureus proteins
Article Snippet: .. Briefly, Swa I-digested pRE-USP plasmid and PCR fragments were treated with T4 DNA polymerase (20 °C, 20 min; 75 °C, 20 min; Roche Diagnostics) before incubation for 5 min at room temperature (3:1 vector:insert). .. Z-Competent E. coli MC1061 cells (Zymo Research, Orange, CA, USA) were transformed with the plasmid:vector mixtures.

Article Title: The Sas3p and Gcn5p histone acetyltransferases are recruited to similar genes
Article Snippet: For immunoprecipitated DNA samples, the aqueous phase was subsequently purified with Montage PCR columns (Millipore Corp., Billerica, MA, USA). .. The immunoprecipitated DNA was blunted by T4 phage DNA polymerase in a reaction volume of 124 μL (T4 DNA Pol buffer, 40 μg/μl BSA, 80 μM dNTPs, 0.6 U T4 DNA Polymerase from Roche).

Article Title: Context-Dependent Functional Divergence of the Notch Ligands DLL1 and DLL4 In Vivo
Article Snippet: Dll1-attB , Dll4-attB and chimeric Dll1-4-attB The eGFP gene was removed from pNC-attB vector [ ] by AfeI/HindIII digest, blunting ends with T4 DNA-Polymerase (Roche) and religation; Flag-tagged Dll1 and Dll4 ORFs were released from pTracer-CMV and inserted into pNC-attB-deltaGFP using restriction enzymes EcoRI/BamHI. .. To generate an alternative construct with HA-tagged DLL4, the HA-tag was added to the Dll4 ORF by PCR with primer pair Dll4.up (EcoRI) GAA TTC ACC ATG ACG CCT GCG TCC CGG AGC G / Dll4.lowHA (NotI) GCG GCC GCT TAT TAT TAA GCG TAG TCT GGA ACG TCG TAT GGG TAT ACC TCT GTG GCA ATC ACA CAC TCG.

Article Title: Mutations in the β-Subunit of the RNA Polymerase Impair the Surface-Associated Motility and Virulence of Acinetobacter baumannii
Article Snippet: The plasmid pBAV1K-T5-gfp was used to complement the Rifr 3, Rifr 5, and Rifr 8 mutants by blunt-end cloning the wild-type rpoB gene from the A. baumannii ATCC 17978 wild-type genome into the XbaI site of the vector, whose overhangs were filled with T4 DNA polymerase (Roche). .. As this plasmid carries a gene encoding kanamycin resistance, complementation of the mutants constructed using the pCR-BluntII-TOPO vector, which also encodes kanamycin resistance, was carried out using a variant of the pBAV1K-T5-gfp vector (pBAV1TIC-T5-gfp), constructed in this work as follows.

Article Title: Human antibody responses against non-covalently cell wall-bound Staphylococcus aureus proteins
Article Snippet: .. Briefly, Swa I-digested pRE-USP plasmid and PCR fragments were treated with T4 DNA polymerase (20 °C, 20 min; 75 °C, 20 min; Roche Diagnostics) before incubation for 5 min at room temperature (3:1 vector:insert). .. Z-Competent E. coli MC1061 cells (Zymo Research, Orange, CA, USA) were transformed with the plasmid:vector mixtures.

Article Title: Dynamic remodeling of histone modifications in response to osmotic stress in Saccharomyces cerevisiae
Article Snippet: The aqueous phase was subsequently purified with Montage PCR columns (Millipore). .. Immunoprecipitated DNA was blunted by T4 phage DNA polymerase in a reaction volume of 124 μL (T4 DNA Pol buffer, 40 μg/μL BSA, 80 μM dNTPs, 0.6 U T4 DNA Polymerase from Roche).

Article Title: Efficient Subtractive Cloning of Genes Activated by Lipopolysaccharide and Interferon ? in Primary-Cultured Cortical Cells of Newborn Mice
Article Snippet: Paragraph title: cDNA Synthesis and PCR Amplification ... The bead-fixed 3′-trimmed cDNA was blunt-ended with 0.5 units of T4 DNA polymerase (Roche Diagnostics, Tokyo, Japan) in 20 µL of a mixture containing 50 mM Tris·HCl (pH 8.8), 15 mM (NH4 )2 SO4 , 7 mM MgCl2 , 0.1 mM EDTA, 10 mM 2-mercaptoethanol, 0.02 mg/mL bovine serum albumin, and 0.1 mM each of dATP, dCTP, dGTP, and dTTP at 16°C for 10 min.

Article Title: Tea (Camellia sinensis) infusions ameliorate cancer in 4TI metastatic breast cancer model
Article Snippet: The mixture was spun for 2 s and incubated at 16 °C for 2 h before adding 20μlof T4 DNA polymerase (Roche, Germany). .. The beads were allowed to air dry at room temperature for 3 min, washed using 16 μl of 10 mM Tris-HCL, allowed to pellet and the pellets containing the ds-cDNA transferred to a fresh 200 μl PCR tube.

Immunoprecipitation:

Article Title: The Sas3p and Gcn5p histone acetyltransferases are recruited to similar genes
Article Snippet: .. The immunoprecipitated DNA was blunted by T4 phage DNA polymerase in a reaction volume of 124 μL (T4 DNA Pol buffer, 40 μg/μl BSA, 80 μM dNTPs, 0.6 U T4 DNA Polymerase from Roche). ..

Article Title: Dynamic remodeling of histone modifications in response to osmotic stress in Saccharomyces cerevisiae
Article Snippet: .. Immunoprecipitated DNA was blunted by T4 phage DNA polymerase in a reaction volume of 124 μL (T4 DNA Pol buffer, 40 μg/μL BSA, 80 μM dNTPs, 0.6 U T4 DNA Polymerase from Roche). ..

cDNA Library Assay:

Article Title: Tea (Camellia sinensis) infusions ameliorate cancer in 4TI metastatic breast cancer model
Article Snippet: Paragraph title: Preparation of cDNA library for Transcriptome sequencing ... The mixture was spun for 2 s and incubated at 16 °C for 2 h before adding 20μlof T4 DNA polymerase (Roche, Germany).

Article Title: Transcriptome analysis of pigeon milk production – role of cornification and triglyceride synthesis genes
Article Snippet: Paragraph title: cDNA library synthesis ... Briefly, fragmented RNA was reverse transcribed into cDNA and made double-stranded using a mixture of DNA polymerase I, ligase and RNase H and blunt ended with T4 DNA Polymerase (Roche cDNA synthesis system).

Plasmid Preparation:

Article Title: A Robust High Throughput Platform to Generate Functional Recombinant Monoclonal Antibodies Using Rabbit B Cells from Peripheral Blood
Article Snippet: .. The purified linear vector and the insert were treated with 0.5 U T4 DNA polymerase (Roche Applied Sciences, Mannheim, Germany) per 1 µg DNA for 45 min at 25°C in the absence of dNTPs to generate matching overhangs. .. The reaction was stopped by adding 1/10th of the reaction volume of a 10 mM dCTP solution (Invitrogen).

Article Title: The tep1 gene of Sinorhizobium meliloti coding for a putative transmembrane efflux protein and N-acetyl glucosamine affect nod gene expression and nodulation of alfalfa plants
Article Snippet: .. Next, the Sac I fragment containing the disrupted ORF was treated with T4 DNA polymerase (Roche Biochemicals) to make blunt ends and then cloned into the Sma I site of the suicide vector pK18mobsac [ ] to give pTrans3. .. This vector was then used for allelic exchange by introducing it into the S. meliloti strains GR4, and the fadD mutant QS77 via triparental mating, and selecting putative mutants by streptomycin/spectinomycin resistance and sensitivity to sucrose.

Article Title: Unique secreted–surface protein complex of Lactobacillus rhamnosus, identified by phage display
Article Snippet: Next, the origin of replication in pDJ08 (colEI ) was replaced with the colD origin of replication from the vector pGZ119EH ( ). .. This was achieved by cleavage of the psp -pelB -c-myc-gIIIC cassette and origin of replication from pDJ08 using PstI-ScaI followed by removal of 3′ protruding end from PstI by T4 DNA polymerase (Roche, Basel, Switzerland) and ligation of the resulting fragment to pGZ119EH EcoRV-ScaI fragment carrying colD origin of replication.

Article Title: Human antibody responses against non-covalently cell wall-bound Staphylococcus aureus proteins
Article Snippet: .. Briefly, Swa I-digested pRE-USP plasmid and PCR fragments were treated with T4 DNA polymerase (20 °C, 20 min; 75 °C, 20 min; Roche Diagnostics) before incubation for 5 min at room temperature (3:1 vector:insert). .. Z-Competent E. coli MC1061 cells (Zymo Research, Orange, CA, USA) were transformed with the plasmid:vector mixtures.

Article Title: Context-Dependent Functional Divergence of the Notch Ligands DLL1 and DLL4 In Vivo
Article Snippet: .. Dll1-attB , Dll4-attB and chimeric Dll1-4-attB The eGFP gene was removed from pNC-attB vector [ ] by AfeI/HindIII digest, blunting ends with T4 DNA-Polymerase (Roche) and religation; Flag-tagged Dll1 and Dll4 ORFs were released from pTracer-CMV and inserted into pNC-attB-deltaGFP using restriction enzymes EcoRI/BamHI. .. To generate an alternative construct with HA-tagged DLL4, the HA-tag was added to the Dll4 ORF by PCR with primer pair Dll4.up (EcoRI) GAA TTC ACC ATG ACG CCT GCG TCC CGG AGC G / Dll4.lowHA (NotI) GCG GCC GCT TAT TAT TAA GCG TAG TCT GGA ACG TCG TAT GGG TAT ACC TCT GTG GCA ATC ACA CAC TCG.

Article Title: Mutations in the β-Subunit of the RNA Polymerase Impair the Surface-Associated Motility and Virulence of Acinetobacter baumannii
Article Snippet: .. The plasmid pBAV1K-T5-gfp was used to complement the Rifr 3, Rifr 5, and Rifr 8 mutants by blunt-end cloning the wild-type rpoB gene from the A. baumannii ATCC 17978 wild-type genome into the XbaI site of the vector, whose overhangs were filled with T4 DNA polymerase (Roche). .. As this plasmid carries a gene encoding kanamycin resistance, complementation of the mutants constructed using the pCR-BluntII-TOPO vector, which also encodes kanamycin resistance, was carried out using a variant of the pBAV1K-T5-gfp vector (pBAV1TIC-T5-gfp), constructed in this work as follows.

Article Title: Human antibody responses against non-covalently cell wall-bound Staphylococcus aureus proteins
Article Snippet: .. Briefly, Swa I-digested pRE-USP plasmid and PCR fragments were treated with T4 DNA polymerase (20 °C, 20 min; 75 °C, 20 min; Roche Diagnostics) before incubation for 5 min at room temperature (3:1 vector:insert). .. Z-Competent E. coli MC1061 cells (Zymo Research, Orange, CA, USA) were transformed with the plasmid:vector mixtures.

Concentration Assay:

Article Title: Dynamic remodeling of histone modifications in response to osmotic stress in Saccharomyces cerevisiae
Article Snippet: DNA purification and annealed linkers ligation Proteins were degraded by adding 50 μg of proteinase K and SDS at a final concentration of 2.5% with incubation at 37°C for 1 h. DNA was purified by phenol/chloroform/isoamylic alcohol extraction. .. Immunoprecipitated DNA was blunted by T4 phage DNA polymerase in a reaction volume of 124 μL (T4 DNA Pol buffer, 40 μg/μL BSA, 80 μM dNTPs, 0.6 U T4 DNA Polymerase from Roche).

DNA Purification:

Article Title: The Sas3p and Gcn5p histone acetyltransferases are recruited to similar genes
Article Snippet: Paragraph title: DNA purification and annealed linker ligation ... The immunoprecipitated DNA was blunted by T4 phage DNA polymerase in a reaction volume of 124 μL (T4 DNA Pol buffer, 40 μg/μl BSA, 80 μM dNTPs, 0.6 U T4 DNA Polymerase from Roche).

Article Title: Dynamic remodeling of histone modifications in response to osmotic stress in Saccharomyces cerevisiae
Article Snippet: Paragraph title: DNA purification and annealed linkers ligation ... Immunoprecipitated DNA was blunted by T4 phage DNA polymerase in a reaction volume of 124 μL (T4 DNA Pol buffer, 40 μg/μL BSA, 80 μM dNTPs, 0.6 U T4 DNA Polymerase from Roche).

Gel Extraction:

Article Title: A Robust High Throughput Platform to Generate Functional Recombinant Monoclonal Antibodies Using Rabbit B Cells from Peripheral Blood
Article Snippet: Subsequently, the linearized plasmid DNA was purified by preparative agarose electrophoresis and extracted from the gel (QIAquick Gel Extraction Kit, Qiagen). .. The purified linear vector and the insert were treated with 0.5 U T4 DNA polymerase (Roche Applied Sciences, Mannheim, Germany) per 1 µg DNA for 45 min at 25°C in the absence of dNTPs to generate matching overhangs.

Variant Assay:

Article Title: Mutations in the β-Subunit of the RNA Polymerase Impair the Surface-Associated Motility and Virulence of Acinetobacter baumannii
Article Snippet: The plasmid pBAV1K-T5-gfp was used to complement the Rifr 3, Rifr 5, and Rifr 8 mutants by blunt-end cloning the wild-type rpoB gene from the A. baumannii ATCC 17978 wild-type genome into the XbaI site of the vector, whose overhangs were filled with T4 DNA polymerase (Roche). .. As this plasmid carries a gene encoding kanamycin resistance, complementation of the mutants constructed using the pCR-BluntII-TOPO vector, which also encodes kanamycin resistance, was carried out using a variant of the pBAV1K-T5-gfp vector (pBAV1TIC-T5-gfp), constructed in this work as follows.

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  • 99
    Roche dnase i
    Effects of <t>DNase</t> I on endothelial activation. ( a ) Aortas were collected from rats that underwent cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) at the end of surgery. Relative expression levels of intercellular adhesion molecule-1 ( ICAM-1) , vascular cell adhesion molecule-1 ( VCAM-1) , inducible NO synthase ( iNOS) , IL-6 , TNF-α , and IL-10 in aortic tissue were analyzed by quantitative real-time PCR. 18 S rRNA was used to normalize the data. Two-time DNase I administration (before CPB and before reperfusion) significantly reduced the aortic expression of ICAM-1 and VCAM-1 . * P
    Dnase I, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i/product/Roche
    Average 99 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    dnase i - by Bioz Stars, 2020-01
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    99
    Roche recombinant dnase i
    Effects of <t>DNase</t> I on endothelial activation. ( a ) Aortas were collected from rats that underwent cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) at the end of surgery. Relative expression levels of intercellular adhesion molecule-1 ( ICAM-1) , vascular cell adhesion molecule-1 ( VCAM-1) , inducible NO synthase ( iNOS) , IL-6 , TNF-α , and IL-10 in aortic tissue were analyzed by quantitative real-time PCR. 18 S rRNA was used to normalize the data. Two-time DNase I administration (before CPB and before reperfusion) significantly reduced the aortic expression of ICAM-1 and VCAM-1 . * P
    Recombinant Dnase I, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 109 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant dnase i/product/Roche
    Average 99 stars, based on 109 article reviews
    Price from $9.99 to $1999.99
    recombinant dnase i - by Bioz Stars, 2020-01
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    90
    Roche dna digestion
    Physical properties of <t>HBV</t> particles released from HepG2.2.15 cells after apoptosis induction. (a and b) Characterization of HBV particles released due to their density profiles. (a) Twenty-four hours after UV-C irradiation (20 mJ/cm 2 ), anti-CD95 MAb treatment (1 μg/ml), or a combination of both, cell culture media were subjected to CsCl gradient centrifugation, and collected fractions (F) were analyzed by dot blotting using a 32 P-labeled HBV <t>DNA</t> probe. The pictogram illustrates CsCl densities and expected distributions of naked HBV DNA, naked capsids, and virions. (b) Density profile of concentrated culture medium 24 h after UV-C irradiation. (c) Immunoprecipitation by HBc (IPc) or HBs (IPs) antigen of the indicated density fractions followed by absolute quantification of the isolated nucleic acid contents using HBV DNA-specific primers. (d) HBV DNA Southern blot analysis of the indicated density fractions. To differentiate between circular and linear HBV DNA genomes, XhoI digestion was performed. The obtained fragments are marked by arrows: the 3.2-kb fragment was derived from circular DNA, and the 1.8- and 1.4-kb fragments were derived from linear DNA. M, molecular weight marker.
    Dna Digestion, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of DNase I on endothelial activation. ( a ) Aortas were collected from rats that underwent cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) at the end of surgery. Relative expression levels of intercellular adhesion molecule-1 ( ICAM-1) , vascular cell adhesion molecule-1 ( VCAM-1) , inducible NO synthase ( iNOS) , IL-6 , TNF-α , and IL-10 in aortic tissue were analyzed by quantitative real-time PCR. 18 S rRNA was used to normalize the data. Two-time DNase I administration (before CPB and before reperfusion) significantly reduced the aortic expression of ICAM-1 and VCAM-1 . * P

    Journal: Scientific Reports

    Article Title: Targeting of cell-free DNA by DNase I diminishes endothelial dysfunction and inflammation in a rat model of cardiopulmonary bypass

    doi: 10.1038/s41598-019-55863-8

    Figure Lengend Snippet: Effects of DNase I on endothelial activation. ( a ) Aortas were collected from rats that underwent cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) at the end of surgery. Relative expression levels of intercellular adhesion molecule-1 ( ICAM-1) , vascular cell adhesion molecule-1 ( VCAM-1) , inducible NO synthase ( iNOS) , IL-6 , TNF-α , and IL-10 in aortic tissue were analyzed by quantitative real-time PCR. 18 S rRNA was used to normalize the data. Two-time DNase I administration (before CPB and before reperfusion) significantly reduced the aortic expression of ICAM-1 and VCAM-1 . * P

    Article Snippet: 24 male Wistar rats weighing between 510 and 525 g (Janvier Breeding Center, France) were randomly assigned to three experimental groups: one control group undergoing CPB with DHCA (group 1), one group undergoing CPB with DHCA that received an i.v . bolus of DNase I (Pulmozyme®, Roche, 5 mg/kg body weight) before CPB (group 2), and one group undergoing CPB with DHCA that received DNase I before CPB and a second bolus after rewarming/before reperfusion, respectively (group 3).

    Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction

    Effects of DNase I on vascular function. Rat aortas were removed at the end of surgical procedure and immediately used for ex vivo functional analyses. ( a ) Aortic rings (4 mm width) were pre-constricted with 0.1 µM Phenylephrin (PE) and endothelial-dependent vasorelaxation was achieved by the addition of different concentrations of Acethylcholin (ACh, range 0 nM-10 µM). Pre-constriction was defined as 100%. Improved vasorelaxation was found in aortic vessels of rats treated with two doses of DNase I . * P

    Journal: Scientific Reports

    Article Title: Targeting of cell-free DNA by DNase I diminishes endothelial dysfunction and inflammation in a rat model of cardiopulmonary bypass

    doi: 10.1038/s41598-019-55863-8

    Figure Lengend Snippet: Effects of DNase I on vascular function. Rat aortas were removed at the end of surgical procedure and immediately used for ex vivo functional analyses. ( a ) Aortic rings (4 mm width) were pre-constricted with 0.1 µM Phenylephrin (PE) and endothelial-dependent vasorelaxation was achieved by the addition of different concentrations of Acethylcholin (ACh, range 0 nM-10 µM). Pre-constriction was defined as 100%. Improved vasorelaxation was found in aortic vessels of rats treated with two doses of DNase I . * P

    Article Snippet: 24 male Wistar rats weighing between 510 and 525 g (Janvier Breeding Center, France) were randomly assigned to three experimental groups: one control group undergoing CPB with DHCA (group 1), one group undergoing CPB with DHCA that received an i.v . bolus of DNase I (Pulmozyme®, Roche, 5 mg/kg body weight) before CPB (group 2), and one group undergoing CPB with DHCA that received DNase I before CPB and a second bolus after rewarming/before reperfusion, respectively (group 3).

    Techniques: Ex Vivo, Functional Assay

    Assessment of DNase I effects on plasma cytokine levels. Rats were subjected to cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) as described in the Methods section. Rats received DNase I treatment before CPB (1 × DNase , n = 7) or a second DNase I dose before reperfusion (2 × DNase , n = 8). Animals without DNase I treatment served as control (n = 7). Plasma levels of IL-6, TNF-α, IFN-ɣ and IL-10 were quantified using Procartaplex Multiplex Assays before CPB (T1), before (T2) and after (T3) reperfusion. Rats that received two doses of DNase I showed significant reduction of IL-6 at the end of reperfusion (T3).* P

    Journal: Scientific Reports

    Article Title: Targeting of cell-free DNA by DNase I diminishes endothelial dysfunction and inflammation in a rat model of cardiopulmonary bypass

    doi: 10.1038/s41598-019-55863-8

    Figure Lengend Snippet: Assessment of DNase I effects on plasma cytokine levels. Rats were subjected to cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) as described in the Methods section. Rats received DNase I treatment before CPB (1 × DNase , n = 7) or a second DNase I dose before reperfusion (2 × DNase , n = 8). Animals without DNase I treatment served as control (n = 7). Plasma levels of IL-6, TNF-α, IFN-ɣ and IL-10 were quantified using Procartaplex Multiplex Assays before CPB (T1), before (T2) and after (T3) reperfusion. Rats that received two doses of DNase I showed significant reduction of IL-6 at the end of reperfusion (T3).* P

    Article Snippet: 24 male Wistar rats weighing between 510 and 525 g (Janvier Breeding Center, France) were randomly assigned to three experimental groups: one control group undergoing CPB with DHCA (group 1), one group undergoing CPB with DHCA that received an i.v . bolus of DNase I (Pulmozyme®, Roche, 5 mg/kg body weight) before CPB (group 2), and one group undergoing CPB with DHCA that received DNase I before CPB and a second bolus after rewarming/before reperfusion, respectively (group 3).

    Techniques: Multiplex Assay

    Impact of DNase I on leukocyte extravasation. Rats with or without DNase I treatment that underwent cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) were sacrificed and lung tissue was collected at the end of surgery. ( a ) Immunofluorescence detection of CD45 in lung tissue of control rats (Ctrl) and those treated with DNase I (1 × DNase I , 2 × DNase I ). Three animals per group were examined. Representative images are depicted. Scale bar: 50 µm. ( b ) Quantification of CD45 fluorescence expressed as mean fluorescence intensity. Ten random fields were examined from each specimen at 400 × magnification. DNase I treatment significantly reduced the number of CD45-positive cells.* P

    Journal: Scientific Reports

    Article Title: Targeting of cell-free DNA by DNase I diminishes endothelial dysfunction and inflammation in a rat model of cardiopulmonary bypass

    doi: 10.1038/s41598-019-55863-8

    Figure Lengend Snippet: Impact of DNase I on leukocyte extravasation. Rats with or without DNase I treatment that underwent cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) were sacrificed and lung tissue was collected at the end of surgery. ( a ) Immunofluorescence detection of CD45 in lung tissue of control rats (Ctrl) and those treated with DNase I (1 × DNase I , 2 × DNase I ). Three animals per group were examined. Representative images are depicted. Scale bar: 50 µm. ( b ) Quantification of CD45 fluorescence expressed as mean fluorescence intensity. Ten random fields were examined from each specimen at 400 × magnification. DNase I treatment significantly reduced the number of CD45-positive cells.* P

    Article Snippet: 24 male Wistar rats weighing between 510 and 525 g (Janvier Breeding Center, France) were randomly assigned to three experimental groups: one control group undergoing CPB with DHCA (group 1), one group undergoing CPB with DHCA that received an i.v . bolus of DNase I (Pulmozyme®, Roche, 5 mg/kg body weight) before CPB (group 2), and one group undergoing CPB with DHCA that received DNase I before CPB and a second bolus after rewarming/before reperfusion, respectively (group 3).

    Techniques: Immunofluorescence, Fluorescence

    Evaluation of plasma cell-free DNA (cfDNA) levels and DNase activity. Rats underwent cardiopulmonary bypass (CPB) with deep hypothermic circulatory arrest (DHCA) as described in the Methods section. Plasma samples were collected from control rats without DNase I therapy (n = 7), rats receiving DNase I before CPB (1 × DNase , n = 7) and those receiving a second DNase I dose before reperfusion (2 × DNase , n = 8) at following times: before CPB (T1), before reperfusion (T2) and at the end of reperfusion (T3). ( a ) Plasma cfDNA levels were quantified by PicoGreen staining and were found to be significantly decreased upon DNase I administration. ( b ) Additionally, relative plasma DNase activity was determined and significantly increased in rats that received DNase I . ** P

    Journal: Scientific Reports

    Article Title: Targeting of cell-free DNA by DNase I diminishes endothelial dysfunction and inflammation in a rat model of cardiopulmonary bypass

    doi: 10.1038/s41598-019-55863-8

    Figure Lengend Snippet: Evaluation of plasma cell-free DNA (cfDNA) levels and DNase activity. Rats underwent cardiopulmonary bypass (CPB) with deep hypothermic circulatory arrest (DHCA) as described in the Methods section. Plasma samples were collected from control rats without DNase I therapy (n = 7), rats receiving DNase I before CPB (1 × DNase , n = 7) and those receiving a second DNase I dose before reperfusion (2 × DNase , n = 8) at following times: before CPB (T1), before reperfusion (T2) and at the end of reperfusion (T3). ( a ) Plasma cfDNA levels were quantified by PicoGreen staining and were found to be significantly decreased upon DNase I administration. ( b ) Additionally, relative plasma DNase activity was determined and significantly increased in rats that received DNase I . ** P

    Article Snippet: 24 male Wistar rats weighing between 510 and 525 g (Janvier Breeding Center, France) were randomly assigned to three experimental groups: one control group undergoing CPB with DHCA (group 1), one group undergoing CPB with DHCA that received an i.v . bolus of DNase I (Pulmozyme®, Roche, 5 mg/kg body weight) before CPB (group 2), and one group undergoing CPB with DHCA that received DNase I before CPB and a second bolus after rewarming/before reperfusion, respectively (group 3).

    Techniques: Activity Assay, Staining

    Visual summary of DNase I -mediated vasoprotection during CPB. Systemic application of DNase I during CPB efficiently degraded cfDNA/NETs released by activated neutrophils and prevented reperfusion-induced cellular damage. DNase I improved endothelial function, reduced endothelial activation and leukocyte extravasation.

    Journal: Scientific Reports

    Article Title: Targeting of cell-free DNA by DNase I diminishes endothelial dysfunction and inflammation in a rat model of cardiopulmonary bypass

    doi: 10.1038/s41598-019-55863-8

    Figure Lengend Snippet: Visual summary of DNase I -mediated vasoprotection during CPB. Systemic application of DNase I during CPB efficiently degraded cfDNA/NETs released by activated neutrophils and prevented reperfusion-induced cellular damage. DNase I improved endothelial function, reduced endothelial activation and leukocyte extravasation.

    Article Snippet: 24 male Wistar rats weighing between 510 and 525 g (Janvier Breeding Center, France) were randomly assigned to three experimental groups: one control group undergoing CPB with DHCA (group 1), one group undergoing CPB with DHCA that received an i.v . bolus of DNase I (Pulmozyme®, Roche, 5 mg/kg body weight) before CPB (group 2), and one group undergoing CPB with DHCA that received DNase I before CPB and a second bolus after rewarming/before reperfusion, respectively (group 3).

    Techniques: Activation Assay

    Real-time PCR quantitation of DNase I-resistant genomes after incubation with nuclear extract or cytoplasmic extract. A total of 10 10 particles of purified AAV2/2-hF.IX vector were incubated for 30 min at 37°C with 50 μg of nuclear extract, cytoplasmic extract, or the same volume of buffer alone, before digesting with DNase I for a further hour at 37°C (final volume, 100 μl). (A) DNase I-resistant VG in 5 μl were quantified by real-time PCR. Error bars show the standard error of the mean of four samples per group. (B) Twenty microliters of the remaining sample was analyzed by Western blotting with the anti-VP1,2,3 antibody to detect the integrity of the capsid proteins following incubation with nuclear or cytoplasmic extract. Lanes 1 and 2, AAV particles incubated with buffer only; lanes 3 to 6, AAV particles incubated with cytoplasmic extract; lanes 7 to 10, AAV particles incubated with nuclear extract.

    Journal: Journal of Virology

    Article Title: Rapid Uncoating of Vector Genomes Is the Key to Efficient Liver Transduction with Pseudotyped Adeno-Associated Virus Vectors

    doi: 10.1128/JVI.78.6.3110-3122.2004

    Figure Lengend Snippet: Real-time PCR quantitation of DNase I-resistant genomes after incubation with nuclear extract or cytoplasmic extract. A total of 10 10 particles of purified AAV2/2-hF.IX vector were incubated for 30 min at 37°C with 50 μg of nuclear extract, cytoplasmic extract, or the same volume of buffer alone, before digesting with DNase I for a further hour at 37°C (final volume, 100 μl). (A) DNase I-resistant VG in 5 μl were quantified by real-time PCR. Error bars show the standard error of the mean of four samples per group. (B) Twenty microliters of the remaining sample was analyzed by Western blotting with the anti-VP1,2,3 antibody to detect the integrity of the capsid proteins following incubation with nuclear or cytoplasmic extract. Lanes 1 and 2, AAV particles incubated with buffer only; lanes 3 to 6, AAV particles incubated with cytoplasmic extract; lanes 7 to 10, AAV particles incubated with nuclear extract.

    Article Snippet: To assess the sensitivity of nuclear-localized AAV genomes to DNase I, a second aliquot of nuclei from each mouse was digested with 50 U of DNase I (10 U/μl; Roche, Basel, Switzerland) for 5 h, with occasional gentle vortexing, before digesting with proteinase K and extracting with phenol-chloroform and chloroform as described above.

    Techniques: Real-time Polymerase Chain Reaction, Quantitation Assay, Incubation, Purification, Plasmid Preparation, Western Blot

    Immunoprecipitation of intact AAV2/2 particles from purified liver nuclei. Liver nuclei purified at different time points after intraportal injection of 5 × 10 11 VG of AAV2/2-hF.IX16 into C57BL/6 mice were incubated with DNase I for 1 h at 37°C. (A and B) Intact AAV capsids were immunoprecipitated from solubilized, DNase I-treated nuclei using the A20 antibody (A) or heparin-Sepharose beads (B). Immunoprecipitated capsids were boiled for 5 min in alkaline buffer, and the released VG were separated on a 1% alkaline agarose gel, blotted, and probed with a sequence-specific probe. (C) Control immunoprecipitations were performed with the anti-VP1,2,3 antibody, which recognizes dissociated, but not intact, capsid proteins. Positive and negative controls were performed for immunoprecipitations with all antibodies. For the positive control, purified nuclei were spiked with 10 9 VG of AAV2/2-hF.IX.16 prior to solubilization (lane 6). For the negative control, solubilized nuclei were spiked with 10 10 VG of AAV-hF.IX16 DNA extracted from purified AAV2/2-hF.IX16 particles (lane 5). Lanes 1 to 4 show copy number standards (purified AAV2/2-hF.IX particles boiled in alkaline buffer prior to loading). Lanes 7 to 15 represent individual mice. (D) Ten microliters of supernatant from solubilized nuclei (from a total volume of 1 ml for each mouse) removed prior to immunoprecipitation was also boiled in alkaline buffer and loaded on a gel.

    Journal: Journal of Virology

    Article Title: Rapid Uncoating of Vector Genomes Is the Key to Efficient Liver Transduction with Pseudotyped Adeno-Associated Virus Vectors

    doi: 10.1128/JVI.78.6.3110-3122.2004

    Figure Lengend Snippet: Immunoprecipitation of intact AAV2/2 particles from purified liver nuclei. Liver nuclei purified at different time points after intraportal injection of 5 × 10 11 VG of AAV2/2-hF.IX16 into C57BL/6 mice were incubated with DNase I for 1 h at 37°C. (A and B) Intact AAV capsids were immunoprecipitated from solubilized, DNase I-treated nuclei using the A20 antibody (A) or heparin-Sepharose beads (B). Immunoprecipitated capsids were boiled for 5 min in alkaline buffer, and the released VG were separated on a 1% alkaline agarose gel, blotted, and probed with a sequence-specific probe. (C) Control immunoprecipitations were performed with the anti-VP1,2,3 antibody, which recognizes dissociated, but not intact, capsid proteins. Positive and negative controls were performed for immunoprecipitations with all antibodies. For the positive control, purified nuclei were spiked with 10 9 VG of AAV2/2-hF.IX.16 prior to solubilization (lane 6). For the negative control, solubilized nuclei were spiked with 10 10 VG of AAV-hF.IX16 DNA extracted from purified AAV2/2-hF.IX16 particles (lane 5). Lanes 1 to 4 show copy number standards (purified AAV2/2-hF.IX particles boiled in alkaline buffer prior to loading). Lanes 7 to 15 represent individual mice. (D) Ten microliters of supernatant from solubilized nuclei (from a total volume of 1 ml for each mouse) removed prior to immunoprecipitation was also boiled in alkaline buffer and loaded on a gel.

    Article Snippet: To assess the sensitivity of nuclear-localized AAV genomes to DNase I, a second aliquot of nuclei from each mouse was digested with 50 U of DNase I (10 U/μl; Roche, Basel, Switzerland) for 5 h, with occasional gentle vortexing, before digesting with proteinase K and extracting with phenol-chloroform and chloroform as described above.

    Techniques: Immunoprecipitation, Purification, Injection, Mouse Assay, Incubation, Agarose Gel Electrophoresis, Sequencing, Positive Control, Negative Control

    Southern blot analysis of rAAV VG extracted from purified liver nuclei without preincubation with DNase I (A), or after incubation with DNase I for 5 h at 37°C (B). Vector forms in DNA samples extracted 3 weeks after injection of pseudotyped AAV-hF.IX16 vectors are shown. Forty micrograms of undigested total DNA (A) or the equivalent volume of DNase I-treated sample (B) was separated on a 1% agarose gel, blotted, and probed with a vector sequence-specific probe. Lanes 1 and 2, 1- and 0.1-VG/DGE standards, respectively (40 μg of DNA extracted from naïve mice was spiked with a 6.4-kb plasmid containing the AAVhF.IX16 sequence and then digested with Sac I prior to loading). A total of 10 7 VG of AAV-hF.IX16 extracted from purified vector stock was denatured by boiling for 5 min in the presence of formamide and loaded in lane 3 as a size marker for ss AAV-hF.IX genomes. Lanes 5 to 15 represent individual mice. Arrowheads indicate the different molecular forms of the AAV-hF.IX16 genome. ds indicates either relaxed circular, supercoiled circular, or linear ds forms. c, concatemers.

    Journal: Journal of Virology

    Article Title: Rapid Uncoating of Vector Genomes Is the Key to Efficient Liver Transduction with Pseudotyped Adeno-Associated Virus Vectors

    doi: 10.1128/JVI.78.6.3110-3122.2004

    Figure Lengend Snippet: Southern blot analysis of rAAV VG extracted from purified liver nuclei without preincubation with DNase I (A), or after incubation with DNase I for 5 h at 37°C (B). Vector forms in DNA samples extracted 3 weeks after injection of pseudotyped AAV-hF.IX16 vectors are shown. Forty micrograms of undigested total DNA (A) or the equivalent volume of DNase I-treated sample (B) was separated on a 1% agarose gel, blotted, and probed with a vector sequence-specific probe. Lanes 1 and 2, 1- and 0.1-VG/DGE standards, respectively (40 μg of DNA extracted from naïve mice was spiked with a 6.4-kb plasmid containing the AAVhF.IX16 sequence and then digested with Sac I prior to loading). A total of 10 7 VG of AAV-hF.IX16 extracted from purified vector stock was denatured by boiling for 5 min in the presence of formamide and loaded in lane 3 as a size marker for ss AAV-hF.IX genomes. Lanes 5 to 15 represent individual mice. Arrowheads indicate the different molecular forms of the AAV-hF.IX16 genome. ds indicates either relaxed circular, supercoiled circular, or linear ds forms. c, concatemers.

    Article Snippet: To assess the sensitivity of nuclear-localized AAV genomes to DNase I, a second aliquot of nuclei from each mouse was digested with 50 U of DNase I (10 U/μl; Roche, Basel, Switzerland) for 5 h, with occasional gentle vortexing, before digesting with proteinase K and extracting with phenol-chloroform and chloroform as described above.

    Techniques: Southern Blot, Purification, Incubation, Plasmid Preparation, Injection, Agarose Gel Electrophoresis, Sequencing, Mouse Assay, Marker

    Real-time PCR quantitation of the proportions of DNase I-resistant AAV genomes localized within the nucleus over time. Numbers of AAV genomes were quantified in total DNA extracted from purified liver nuclei prepared at different time points after intraportal injection of 10 11  VG of pseudotyped AAV-hF.IX16 vectors into C57BL/6 mice. Solid black bars indicate the total number of nuclear-localized AAV genomes at each time point (expressed as copy numbers per DGE), and open bars indicate the relative number of DNase I-resistant AAV genomes. The ratio of DNase I-resistant to total nuclear-localized VG at each time point is expressed as a percentage above each graph.  n  = 4 mice per vector per time point. Error bars show standard errors of the means.

    Journal: Journal of Virology

    Article Title: Rapid Uncoating of Vector Genomes Is the Key to Efficient Liver Transduction with Pseudotyped Adeno-Associated Virus Vectors

    doi: 10.1128/JVI.78.6.3110-3122.2004

    Figure Lengend Snippet: Real-time PCR quantitation of the proportions of DNase I-resistant AAV genomes localized within the nucleus over time. Numbers of AAV genomes were quantified in total DNA extracted from purified liver nuclei prepared at different time points after intraportal injection of 10 11 VG of pseudotyped AAV-hF.IX16 vectors into C57BL/6 mice. Solid black bars indicate the total number of nuclear-localized AAV genomes at each time point (expressed as copy numbers per DGE), and open bars indicate the relative number of DNase I-resistant AAV genomes. The ratio of DNase I-resistant to total nuclear-localized VG at each time point is expressed as a percentage above each graph. n = 4 mice per vector per time point. Error bars show standard errors of the means.

    Article Snippet: To assess the sensitivity of nuclear-localized AAV genomes to DNase I, a second aliquot of nuclei from each mouse was digested with 50 U of DNase I (10 U/μl; Roche, Basel, Switzerland) for 5 h, with occasional gentle vortexing, before digesting with proteinase K and extracting with phenol-chloroform and chloroform as described above.

    Techniques: Real-time Polymerase Chain Reaction, Quantitation Assay, Purification, Injection, Mouse Assay, Plasmid Preparation

    Effects of DNase I on endothelial activation. ( a ) Aortas were collected from rats that underwent cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) at the end of surgery. Relative expression levels of intercellular adhesion molecule-1 ( ICAM-1) , vascular cell adhesion molecule-1 ( VCAM-1) , inducible NO synthase ( iNOS) , IL-6 , TNF-α , and IL-10 in aortic tissue were analyzed by quantitative real-time PCR. 18 S rRNA was used to normalize the data. Two-time DNase I administration (before CPB and before reperfusion) significantly reduced the aortic expression of ICAM-1 and VCAM-1 . * P

    Journal: Scientific Reports

    Article Title: Targeting of cell-free DNA by DNase I diminishes endothelial dysfunction and inflammation in a rat model of cardiopulmonary bypass

    doi: 10.1038/s41598-019-55863-8

    Figure Lengend Snippet: Effects of DNase I on endothelial activation. ( a ) Aortas were collected from rats that underwent cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) at the end of surgery. Relative expression levels of intercellular adhesion molecule-1 ( ICAM-1) , vascular cell adhesion molecule-1 ( VCAM-1) , inducible NO synthase ( iNOS) , IL-6 , TNF-α , and IL-10 in aortic tissue were analyzed by quantitative real-time PCR. 18 S rRNA was used to normalize the data. Two-time DNase I administration (before CPB and before reperfusion) significantly reduced the aortic expression of ICAM-1 and VCAM-1 . * P

    Article Snippet: In parallel experiments, cells were incubated in the presence of NETs pre-digested with recombinant DNase I (100 U/ml, Roche) or DNase I alone.

    Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction

    Effects of DNase I on vascular function. Rat aortas were removed at the end of surgical procedure and immediately used for ex vivo functional analyses. ( a ) Aortic rings (4 mm width) were pre-constricted with 0.1 µM Phenylephrin (PE) and endothelial-dependent vasorelaxation was achieved by the addition of different concentrations of Acethylcholin (ACh, range 0 nM-10 µM). Pre-constriction was defined as 100%. Improved vasorelaxation was found in aortic vessels of rats treated with two doses of DNase I . * P

    Journal: Scientific Reports

    Article Title: Targeting of cell-free DNA by DNase I diminishes endothelial dysfunction and inflammation in a rat model of cardiopulmonary bypass

    doi: 10.1038/s41598-019-55863-8

    Figure Lengend Snippet: Effects of DNase I on vascular function. Rat aortas were removed at the end of surgical procedure and immediately used for ex vivo functional analyses. ( a ) Aortic rings (4 mm width) were pre-constricted with 0.1 µM Phenylephrin (PE) and endothelial-dependent vasorelaxation was achieved by the addition of different concentrations of Acethylcholin (ACh, range 0 nM-10 µM). Pre-constriction was defined as 100%. Improved vasorelaxation was found in aortic vessels of rats treated with two doses of DNase I . * P

    Article Snippet: In parallel experiments, cells were incubated in the presence of NETs pre-digested with recombinant DNase I (100 U/ml, Roche) or DNase I alone.

    Techniques: Ex Vivo, Functional Assay

    Assessment of DNase I effects on plasma cytokine levels. Rats were subjected to cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) as described in the Methods section. Rats received DNase I treatment before CPB (1 × DNase , n = 7) or a second DNase I dose before reperfusion (2 × DNase , n = 8). Animals without DNase I treatment served as control (n = 7). Plasma levels of IL-6, TNF-α, IFN-ɣ and IL-10 were quantified using Procartaplex Multiplex Assays before CPB (T1), before (T2) and after (T3) reperfusion. Rats that received two doses of DNase I showed significant reduction of IL-6 at the end of reperfusion (T3).* P

    Journal: Scientific Reports

    Article Title: Targeting of cell-free DNA by DNase I diminishes endothelial dysfunction and inflammation in a rat model of cardiopulmonary bypass

    doi: 10.1038/s41598-019-55863-8

    Figure Lengend Snippet: Assessment of DNase I effects on plasma cytokine levels. Rats were subjected to cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) as described in the Methods section. Rats received DNase I treatment before CPB (1 × DNase , n = 7) or a second DNase I dose before reperfusion (2 × DNase , n = 8). Animals without DNase I treatment served as control (n = 7). Plasma levels of IL-6, TNF-α, IFN-ɣ and IL-10 were quantified using Procartaplex Multiplex Assays before CPB (T1), before (T2) and after (T3) reperfusion. Rats that received two doses of DNase I showed significant reduction of IL-6 at the end of reperfusion (T3).* P

    Article Snippet: In parallel experiments, cells were incubated in the presence of NETs pre-digested with recombinant DNase I (100 U/ml, Roche) or DNase I alone.

    Techniques: Multiplex Assay

    Impact of DNase I on leukocyte extravasation. Rats with or without DNase I treatment that underwent cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) were sacrificed and lung tissue was collected at the end of surgery. ( a ) Immunofluorescence detection of CD45 in lung tissue of control rats (Ctrl) and those treated with DNase I (1 × DNase I , 2 × DNase I ). Three animals per group were examined. Representative images are depicted. Scale bar: 50 µm. ( b ) Quantification of CD45 fluorescence expressed as mean fluorescence intensity. Ten random fields were examined from each specimen at 400 × magnification. DNase I treatment significantly reduced the number of CD45-positive cells.* P

    Journal: Scientific Reports

    Article Title: Targeting of cell-free DNA by DNase I diminishes endothelial dysfunction and inflammation in a rat model of cardiopulmonary bypass

    doi: 10.1038/s41598-019-55863-8

    Figure Lengend Snippet: Impact of DNase I on leukocyte extravasation. Rats with or without DNase I treatment that underwent cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) were sacrificed and lung tissue was collected at the end of surgery. ( a ) Immunofluorescence detection of CD45 in lung tissue of control rats (Ctrl) and those treated with DNase I (1 × DNase I , 2 × DNase I ). Three animals per group were examined. Representative images are depicted. Scale bar: 50 µm. ( b ) Quantification of CD45 fluorescence expressed as mean fluorescence intensity. Ten random fields were examined from each specimen at 400 × magnification. DNase I treatment significantly reduced the number of CD45-positive cells.* P

    Article Snippet: In parallel experiments, cells were incubated in the presence of NETs pre-digested with recombinant DNase I (100 U/ml, Roche) or DNase I alone.

    Techniques: Immunofluorescence, Fluorescence

    Evaluation of plasma cell-free DNA (cfDNA) levels and DNase activity. Rats underwent cardiopulmonary bypass (CPB) with deep hypothermic circulatory arrest (DHCA) as described in the Methods section. Plasma samples were collected from control rats without DNase I therapy (n = 7), rats receiving DNase I before CPB (1 × DNase , n = 7) and those receiving a second DNase I dose before reperfusion (2 × DNase , n = 8) at following times: before CPB (T1), before reperfusion (T2) and at the end of reperfusion (T3). ( a ) Plasma cfDNA levels were quantified by PicoGreen staining and were found to be significantly decreased upon DNase I administration. ( b ) Additionally, relative plasma DNase activity was determined and significantly increased in rats that received DNase I . ** P

    Journal: Scientific Reports

    Article Title: Targeting of cell-free DNA by DNase I diminishes endothelial dysfunction and inflammation in a rat model of cardiopulmonary bypass

    doi: 10.1038/s41598-019-55863-8

    Figure Lengend Snippet: Evaluation of plasma cell-free DNA (cfDNA) levels and DNase activity. Rats underwent cardiopulmonary bypass (CPB) with deep hypothermic circulatory arrest (DHCA) as described in the Methods section. Plasma samples were collected from control rats without DNase I therapy (n = 7), rats receiving DNase I before CPB (1 × DNase , n = 7) and those receiving a second DNase I dose before reperfusion (2 × DNase , n = 8) at following times: before CPB (T1), before reperfusion (T2) and at the end of reperfusion (T3). ( a ) Plasma cfDNA levels were quantified by PicoGreen staining and were found to be significantly decreased upon DNase I administration. ( b ) Additionally, relative plasma DNase activity was determined and significantly increased in rats that received DNase I . ** P

    Article Snippet: In parallel experiments, cells were incubated in the presence of NETs pre-digested with recombinant DNase I (100 U/ml, Roche) or DNase I alone.

    Techniques: Activity Assay, Staining

    Visual summary of DNase I -mediated vasoprotection during CPB. Systemic application of DNase I during CPB efficiently degraded cfDNA/NETs released by activated neutrophils and prevented reperfusion-induced cellular damage. DNase I improved endothelial function, reduced endothelial activation and leukocyte extravasation.

    Journal: Scientific Reports

    Article Title: Targeting of cell-free DNA by DNase I diminishes endothelial dysfunction and inflammation in a rat model of cardiopulmonary bypass

    doi: 10.1038/s41598-019-55863-8

    Figure Lengend Snippet: Visual summary of DNase I -mediated vasoprotection during CPB. Systemic application of DNase I during CPB efficiently degraded cfDNA/NETs released by activated neutrophils and prevented reperfusion-induced cellular damage. DNase I improved endothelial function, reduced endothelial activation and leukocyte extravasation.

    Article Snippet: In parallel experiments, cells were incubated in the presence of NETs pre-digested with recombinant DNase I (100 U/ml, Roche) or DNase I alone.

    Techniques: Activation Assay

    Physical properties of HBV particles released from HepG2.2.15 cells after apoptosis induction. (a and b) Characterization of HBV particles released due to their density profiles. (a) Twenty-four hours after UV-C irradiation (20 mJ/cm 2 ), anti-CD95 MAb treatment (1 μg/ml), or a combination of both, cell culture media were subjected to CsCl gradient centrifugation, and collected fractions (F) were analyzed by dot blotting using a 32 P-labeled HBV DNA probe. The pictogram illustrates CsCl densities and expected distributions of naked HBV DNA, naked capsids, and virions. (b) Density profile of concentrated culture medium 24 h after UV-C irradiation. (c) Immunoprecipitation by HBc (IPc) or HBs (IPs) antigen of the indicated density fractions followed by absolute quantification of the isolated nucleic acid contents using HBV DNA-specific primers. (d) HBV DNA Southern blot analysis of the indicated density fractions. To differentiate between circular and linear HBV DNA genomes, XhoI digestion was performed. The obtained fragments are marked by arrows: the 3.2-kb fragment was derived from circular DNA, and the 1.8- and 1.4-kb fragments were derived from linear DNA. M, molecular weight marker.

    Journal: Journal of Virology

    Article Title: Apoptosis of Hepatitis B Virus-Infected Hepatocytes Prevents Release of Infectious Virus ▿

    doi: 10.1128/JVI.00653-10

    Figure Lengend Snippet: Physical properties of HBV particles released from HepG2.2.15 cells after apoptosis induction. (a and b) Characterization of HBV particles released due to their density profiles. (a) Twenty-four hours after UV-C irradiation (20 mJ/cm 2 ), anti-CD95 MAb treatment (1 μg/ml), or a combination of both, cell culture media were subjected to CsCl gradient centrifugation, and collected fractions (F) were analyzed by dot blotting using a 32 P-labeled HBV DNA probe. The pictogram illustrates CsCl densities and expected distributions of naked HBV DNA, naked capsids, and virions. (b) Density profile of concentrated culture medium 24 h after UV-C irradiation. (c) Immunoprecipitation by HBc (IPc) or HBs (IPs) antigen of the indicated density fractions followed by absolute quantification of the isolated nucleic acid contents using HBV DNA-specific primers. (d) HBV DNA Southern blot analysis of the indicated density fractions. To differentiate between circular and linear HBV DNA genomes, XhoI digestion was performed. The obtained fragments are marked by arrows: the 3.2-kb fragment was derived from circular DNA, and the 1.8- and 1.4-kb fragments were derived from linear DNA. M, molecular weight marker.

    Article Snippet: To quantify HBV RNA, DNA digestion (DNase I; Roche, Mannheim, Germany) was performed for 1 h at room temperature before nucleic acids were quantified as described above.

    Techniques: Irradiation, Cell Culture, Gradient Centrifugation, Labeling, Immunoprecipitation, Isolation, Southern Blot, Derivative Assay, Molecular Weight, Marker

    Sensitivity of HBV-producing HepG2 cells to apoptosis. HepG2, HepG2-H1.3, HepG2-H1.3x−, and HepAD38 cells were treated with UV-C light (UV) or anti-CD95 monoclonal Ab (MAb). (a and b) Phase-contrast microscopy (top) showed morphological changes of HepAD38 cells without (+Tet) (a) and with (−Tet) (b) HBV replication 24 h after UV-C irradiation (20 mJ/cm 2 ). Dead cells were visualized by ethidium homodimer (EthD-1) staining (bottom). (c) Cell viabilities of the indicated cell lines after UV-C irradiation were determined by an XTT assay. Data are expressed as percentages of untreated cells (means ± standard deviations [SD]; n = 3). (d) Detection of caspase-3/7 activity by cleavage of Ac-DEVD-AFC fluorogenic substrates. Cell lysates of HepG2.2.15 cells were prepared 4 h after treatment with anti-CD95 Ab (1 μg/ml), UV-C irradiation, or a combination of both. Unexposed cells cultured in parallel served as a control. Data show the relative light units (RLU) per μg of protein (mean ± SD; n = 3). (e) Analysis of DNA fragmentation of HepG2.2.15 cells 30 h after treatment. Low-molecular-weight DNA was separated on a 1.4% agarose gel and visualized by ethidium bromide staining.

    Journal: Journal of Virology

    Article Title: Apoptosis of Hepatitis B Virus-Infected Hepatocytes Prevents Release of Infectious Virus ▿

    doi: 10.1128/JVI.00653-10

    Figure Lengend Snippet: Sensitivity of HBV-producing HepG2 cells to apoptosis. HepG2, HepG2-H1.3, HepG2-H1.3x−, and HepAD38 cells were treated with UV-C light (UV) or anti-CD95 monoclonal Ab (MAb). (a and b) Phase-contrast microscopy (top) showed morphological changes of HepAD38 cells without (+Tet) (a) and with (−Tet) (b) HBV replication 24 h after UV-C irradiation (20 mJ/cm 2 ). Dead cells were visualized by ethidium homodimer (EthD-1) staining (bottom). (c) Cell viabilities of the indicated cell lines after UV-C irradiation were determined by an XTT assay. Data are expressed as percentages of untreated cells (means ± standard deviations [SD]; n = 3). (d) Detection of caspase-3/7 activity by cleavage of Ac-DEVD-AFC fluorogenic substrates. Cell lysates of HepG2.2.15 cells were prepared 4 h after treatment with anti-CD95 Ab (1 μg/ml), UV-C irradiation, or a combination of both. Unexposed cells cultured in parallel served as a control. Data show the relative light units (RLU) per μg of protein (mean ± SD; n = 3). (e) Analysis of DNA fragmentation of HepG2.2.15 cells 30 h after treatment. Low-molecular-weight DNA was separated on a 1.4% agarose gel and visualized by ethidium bromide staining.

    Article Snippet: To quantify HBV RNA, DNA digestion (DNase I; Roche, Mannheim, Germany) was performed for 1 h at room temperature before nucleic acids were quantified as described above.

    Techniques: Microscopy, Irradiation, Ethidium Homodimer Assay, Staining, XTT Assay, Activity Assay, Cell Culture, Molecular Weight, Agarose Gel Electrophoresis

    Structural characterization and infectivity of HBV particles released from apoptotic PHH. Thirty hours after anti-CD95 MAb (130 ng/ml) treatment and mock treatment of HBV-infected PHH, cell culture media were collected. (a) HBV particles contained were sedimented into a CsCl gradient. Shown are data from analyses of fractions (F) collected by dot blotting using a 32 P-labeled HBV DNA probe. (b) Culture media of HBV-infected PHH undergoing anti-CD95 or mock treatment were transferred to PHH of a different donor. At the indicated time points, the establishment of a productive HBV infection was analyzed by progeny HBV DNA dot blot analysis. From the signal detected, the input MOI (HBV DNA-containing particles per cell used for infection) was calculated relative to an external standard. In addition, the number of progeny HBV was calculated from the amount of HBV DNA-containing particles released per cell at day 10 p.i. (c) Quantification of HBV cccDNA in PHH at day 10 p.i. using a specific qPCR. Results were normalized to mitochondrial DNA content. HBV cccDNA copies per cell are given (mean ± SD; n = 3). P values were determined by a Student's t test.

    Journal: Journal of Virology

    Article Title: Apoptosis of Hepatitis B Virus-Infected Hepatocytes Prevents Release of Infectious Virus ▿

    doi: 10.1128/JVI.00653-10

    Figure Lengend Snippet: Structural characterization and infectivity of HBV particles released from apoptotic PHH. Thirty hours after anti-CD95 MAb (130 ng/ml) treatment and mock treatment of HBV-infected PHH, cell culture media were collected. (a) HBV particles contained were sedimented into a CsCl gradient. Shown are data from analyses of fractions (F) collected by dot blotting using a 32 P-labeled HBV DNA probe. (b) Culture media of HBV-infected PHH undergoing anti-CD95 or mock treatment were transferred to PHH of a different donor. At the indicated time points, the establishment of a productive HBV infection was analyzed by progeny HBV DNA dot blot analysis. From the signal detected, the input MOI (HBV DNA-containing particles per cell used for infection) was calculated relative to an external standard. In addition, the number of progeny HBV was calculated from the amount of HBV DNA-containing particles released per cell at day 10 p.i. (c) Quantification of HBV cccDNA in PHH at day 10 p.i. using a specific qPCR. Results were normalized to mitochondrial DNA content. HBV cccDNA copies per cell are given (mean ± SD; n = 3). P values were determined by a Student's t test.

    Article Snippet: To quantify HBV RNA, DNA digestion (DNase I; Roche, Mannheim, Germany) was performed for 1 h at room temperature before nucleic acids were quantified as described above.

    Techniques: Infection, Cell Culture, Labeling, Dot Blot, Real-time Polymerase Chain Reaction

    Analysis of the infectivity of HBV released from HepG2.2.15 cells. (a) PHH cultures were infected with HBV particles (left) or particles preincubated with neutralizing anti-HBs antibodies (right). Productive infection was monitored by progeny HBV DNA dot blot analysis (filled area) and HBeAg secretion (continuous line) up to 10 days p.i. HBV DNA-containing particles (viral particles [vp]) per cell were quantified by using a PhosphorImager apparatus (mean ± SD; n = 3). (b) Quantification of HBV cccDNA at 10 days p.i. using qPCR relative to the mitochondrial DNA content. Data are expressed as the number of copies of cccDNA per 10 cells (mean ± SD; n = 3). (c) Culture media of HepG2.2.15 cells collected 24 h after apoptosis induction with anti-CD95 MAb (1 μg/ml), UV-C irradiation (20 mJ/cm 2 ), or combined treatments were transferred onto PHH. At the indicated time points, the establishment of infection was monitored by HBV progeny release using HBV DNA dot blot analysis of PHH culture media. The input was calculated from the amount of HBV DNA-containing particles per cell used for infection. In addition, the number of HBV progeny was calculated from the amount of HBV DNA-containing particles per cell released at day 10 p.i. (d) At day 10 p.i., with media from apoptosis-treated or mock-treated cells, PHH were lysed, and HBV cccDNA was quantified by qPCR. The values obtained were normalized to mitochondrial DNA content. Numbers of HBV cccDNA copies per cell are given (mean ± SD; n = 3). P values were analyzed by a Student's t test.

    Journal: Journal of Virology

    Article Title: Apoptosis of Hepatitis B Virus-Infected Hepatocytes Prevents Release of Infectious Virus ▿

    doi: 10.1128/JVI.00653-10

    Figure Lengend Snippet: Analysis of the infectivity of HBV released from HepG2.2.15 cells. (a) PHH cultures were infected with HBV particles (left) or particles preincubated with neutralizing anti-HBs antibodies (right). Productive infection was monitored by progeny HBV DNA dot blot analysis (filled area) and HBeAg secretion (continuous line) up to 10 days p.i. HBV DNA-containing particles (viral particles [vp]) per cell were quantified by using a PhosphorImager apparatus (mean ± SD; n = 3). (b) Quantification of HBV cccDNA at 10 days p.i. using qPCR relative to the mitochondrial DNA content. Data are expressed as the number of copies of cccDNA per 10 cells (mean ± SD; n = 3). (c) Culture media of HepG2.2.15 cells collected 24 h after apoptosis induction with anti-CD95 MAb (1 μg/ml), UV-C irradiation (20 mJ/cm 2 ), or combined treatments were transferred onto PHH. At the indicated time points, the establishment of infection was monitored by HBV progeny release using HBV DNA dot blot analysis of PHH culture media. The input was calculated from the amount of HBV DNA-containing particles per cell used for infection. In addition, the number of HBV progeny was calculated from the amount of HBV DNA-containing particles per cell released at day 10 p.i. (d) At day 10 p.i., with media from apoptosis-treated or mock-treated cells, PHH were lysed, and HBV cccDNA was quantified by qPCR. The values obtained were normalized to mitochondrial DNA content. Numbers of HBV cccDNA copies per cell are given (mean ± SD; n = 3). P values were analyzed by a Student's t test.

    Article Snippet: To quantify HBV RNA, DNA digestion (DNase I; Roche, Mannheim, Germany) was performed for 1 h at room temperature before nucleic acids were quantified as described above.

    Techniques: Infection, Dot Blot, Real-time Polymerase Chain Reaction, Irradiation

    Characterization of HBV particles released from apoptotic PHH. Supernatants of HBV-infected PHH cultures were collected 24 h after anti-CD95 MAb (130 ng/ml) or mock treatment and subjected to CsCl gradient centrifugation. Twelve fractions (F) with decreasing densities (F1 to F12) were collected; F3 to F10 were used for further characterization. (a) HBsAg ELISA indicating enveloped HBV particles. Average values from three experiments ± standard errors of the means are given. (b) Qualitative and quantitative analysis of encapsidated viral nucleic acids using one-step RT-PCR. To discriminate between DNA and RNA, DNase digestion was performed. (Left) Mock treatment. (Right) Anti-CD95 treatment. The total amount of HBV nucleic acids (DNA plus RNA) (gray bars) detected in F8 of mock-treated cells was set to 100%. HBV RNA levels (continued line) of all samples are reported as parts per thousand. (c) Amplification products of qPCR runs of F4 to F6 with the indicated treatments were separated on a 2% agarose gel and visualized by ethidium bromide staining.

    Journal: Journal of Virology

    Article Title: Apoptosis of Hepatitis B Virus-Infected Hepatocytes Prevents Release of Infectious Virus ▿

    doi: 10.1128/JVI.00653-10

    Figure Lengend Snippet: Characterization of HBV particles released from apoptotic PHH. Supernatants of HBV-infected PHH cultures were collected 24 h after anti-CD95 MAb (130 ng/ml) or mock treatment and subjected to CsCl gradient centrifugation. Twelve fractions (F) with decreasing densities (F1 to F12) were collected; F3 to F10 were used for further characterization. (a) HBsAg ELISA indicating enveloped HBV particles. Average values from three experiments ± standard errors of the means are given. (b) Qualitative and quantitative analysis of encapsidated viral nucleic acids using one-step RT-PCR. To discriminate between DNA and RNA, DNase digestion was performed. (Left) Mock treatment. (Right) Anti-CD95 treatment. The total amount of HBV nucleic acids (DNA plus RNA) (gray bars) detected in F8 of mock-treated cells was set to 100%. HBV RNA levels (continued line) of all samples are reported as parts per thousand. (c) Amplification products of qPCR runs of F4 to F6 with the indicated treatments were separated on a 2% agarose gel and visualized by ethidium bromide staining.

    Article Snippet: To quantify HBV RNA, DNA digestion (DNase I; Roche, Mannheim, Germany) was performed for 1 h at room temperature before nucleic acids were quantified as described above.

    Techniques: Infection, Gradient Centrifugation, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining