Structured Review

Boehringer Mannheim klenow fragment
The effect of Ku on the 3′→5′ exonuclease activity of WRN, <t>Klenow</t> and exo <t>III</t> on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.
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Images

1) Product Images from "A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA"

Article Title: A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA

Journal: Nucleic Acids Research

doi:

The effect of Ku on the 3′→5′ exonuclease activity of WRN, Klenow and exo III on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.
Figure Legend Snippet: The effect of Ku on the 3′→5′ exonuclease activity of WRN, Klenow and exo III on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.

Techniques Used: Activity Assay, Incubation, Labeling

2) Product Images from "Structure of the Herpes Simplex Virus 1 Genome: Manipulation of Nicks and Gaps Can Abrogate Infectivity and Alter the Cellular DNA Damage Response"

Article Title: Structure of the Herpes Simplex Virus 1 Genome: Manipulation of Nicks and Gaps Can Abrogate Infectivity and Alter the Cellular DNA Damage Response

Journal: Journal of Virology

doi: 10.1128/JVI.01723-14

Incorporation of labeled nucleotides into HSV-DNA by Klenow fragment polymerase alone, Klenow and ligase together, or T4 DNA polymerase. Purified HSV-1 DNA was incubated with labeled nucleotides and the following enzymes, as described in Materials and
Figure Legend Snippet: Incorporation of labeled nucleotides into HSV-DNA by Klenow fragment polymerase alone, Klenow and ligase together, or T4 DNA polymerase. Purified HSV-1 DNA was incubated with labeled nucleotides and the following enzymes, as described in Materials and

Techniques Used: Labeling, Purification, Incubation

Virion DNA before and after treatment with Klenow or Klenow and ligase. Biotinylated nucleotides were incorporated into HSV DNA using Klenow alone or Klenow and ligase. Pulsed-field gel electrophoresis was performed with untreated (WT), Klenow-treated
Figure Legend Snippet: Virion DNA before and after treatment with Klenow or Klenow and ligase. Biotinylated nucleotides were incorporated into HSV DNA using Klenow alone or Klenow and ligase. Pulsed-field gel electrophoresis was performed with untreated (WT), Klenow-treated

Techniques Used: Pulsed-Field Gel, Electrophoresis

Addition of 5′ flaps to virion DNA increases hyperphosphorylation of RPA32. (A) Vero cells were transfected with purified KOS DNA that was either left untreated (NT) or treated with Klenow (K) or Klenow and ligase (KL). Samples were harvested
Figure Legend Snippet: Addition of 5′ flaps to virion DNA increases hyperphosphorylation of RPA32. (A) Vero cells were transfected with purified KOS DNA that was either left untreated (NT) or treated with Klenow (K) or Klenow and ligase (KL). Samples were harvested

Techniques Used: Transfection, Purification

Transfection efficiency of treated and untreated KOS-GFP DNA. Vero cells were transfected with untreated (black), Klenow-treated (red), or Klenow-ligase-treated (blue) KOS-GFP DNA. The graph depicts overlaid histograms of cells sorted by FACS and gated
Figure Legend Snippet: Transfection efficiency of treated and untreated KOS-GFP DNA. Vero cells were transfected with untreated (black), Klenow-treated (red), or Klenow-ligase-treated (blue) KOS-GFP DNA. The graph depicts overlaid histograms of cells sorted by FACS and gated

Techniques Used: Transfection, FACS

Purified HSV-1 DNA contains gaps than can be filled by DNA polymerase. (A) The incorporation of labeled nucleotides by the Klenow fragment of E. coli DNA polymerase I into 200 ng of HSV-1 DNA was performed as described in Materials and Methods. For lanes
Figure Legend Snippet: Purified HSV-1 DNA contains gaps than can be filled by DNA polymerase. (A) The incorporation of labeled nucleotides by the Klenow fragment of E. coli DNA polymerase I into 200 ng of HSV-1 DNA was performed as described in Materials and Methods. For lanes

Techniques Used: Purification, Labeling

Cotransfection of ICP0 with untreated and Klenow-treated HSV-1 DNA dramatically improves infectivity.
Figure Legend Snippet: Cotransfection of ICP0 with untreated and Klenow-treated HSV-1 DNA dramatically improves infectivity.

Techniques Used: Cotransfection, Infection

Infectivity of Klenow-treated DNA is rescued in the absence of DNA-PK. HCT-116 cells and DNA-PK −/− cells were transfected with 500 ng of untreated or Klenow-treated virion DNA. Samples were harvested at 48 h following serum addition and
Figure Legend Snippet: Infectivity of Klenow-treated DNA is rescued in the absence of DNA-PK. HCT-116 cells and DNA-PK −/− cells were transfected with 500 ng of untreated or Klenow-treated virion DNA. Samples were harvested at 48 h following serum addition and

Techniques Used: Infection, Transfection

Related Articles

Hybridization:

Article Title: A regulatory role for Sec tRNA[Ser]Sec in selenoprotein synthesis
Article Snippet: Double-stranded complementary tRNASer (GenBank accession number ) was generated by annealing two complementary oligonucleotides, one of which corresponded to 24 nt of the 5′ end of tRNASer with three additional non-base-pairing guanosines at the 5′ end: (5′-GGGCAGGTTCGAATCCTGCC GACTACG-3′) and a second oligonucleotide, complementary to the first (5′-CGTAGTCGGCAGGATTCGAACCTG-3′). .. A hybridization probe was generated by annealing the oligonucleotides and the extension of the protruding 3′ end with Klenow fragment (Boehringer Mannheim), 80 μCi α32 P-dCTP (Perkin Elmer, 3000 Ci/mmole), and 200 μmoles/L each dATP, dGTP, dTTP (Invitro-gen). .. Washing conditions for the tRNA[Ser]Sec hybridization were 0.1 × SSC, 0.1% SDS for 3 h at 65°C.

Generated:

Article Title: A regulatory role for Sec tRNA[Ser]Sec in selenoprotein synthesis
Article Snippet: Double-stranded complementary tRNASer (GenBank accession number ) was generated by annealing two complementary oligonucleotides, one of which corresponded to 24 nt of the 5′ end of tRNASer with three additional non-base-pairing guanosines at the 5′ end: (5′-GGGCAGGTTCGAATCCTGCC GACTACG-3′) and a second oligonucleotide, complementary to the first (5′-CGTAGTCGGCAGGATTCGAACCTG-3′). .. A hybridization probe was generated by annealing the oligonucleotides and the extension of the protruding 3′ end with Klenow fragment (Boehringer Mannheim), 80 μCi α32 P-dCTP (Perkin Elmer, 3000 Ci/mmole), and 200 μmoles/L each dATP, dGTP, dTTP (Invitro-gen). .. Washing conditions for the tRNA[Ser]Sec hybridization were 0.1 × SSC, 0.1% SDS for 3 h at 65°C.

Sequencing:

Article Title: ?-Fetoprotein gene sequences mediating Afr2 regulation during liver regeneration
Article Snippet: Therefore, Afr2 regulation of AFP gene expression requires the sequence between −1,010 and −838 bp and is independent of the other sources of regulation observed for the AFP gene. .. pAFP 5′ with 7.6 kb of AFP 5′-flanking sequence was digested with Bam HI (New England Biolabs), and the 5′-overhangs were filled in by the Klenow fragment of DNA polymerase I (Boehringer Mannheim). .. Xba I linkers (New England Biolabs) were ligated, and the DNA was digested with both Xba I and BsteII (New England Biolabs).

Labeling:

Article Title: CD30 Is a CD40-Inducible Molecule that Negatively Regulates CD40-Mediated Immunoglobulin Class Switching in Non-Antigen-Selected Human B Cells
Article Snippet: .. This probe was labeled with [α-32 P]dCTP and [α-32 P]dGTP using the Klenow fragment of DNA polymerase I (Boehringer Mannheim, Indianapolis, IN). .. DNA binding reactions and shift assays were performed as described ( ).

Article Title: Effect of Altered Spacing between uhpT Promoter Elements on Transcription Activation
Article Snippet: The activity of β-galactosidase expressed from uhpT-lacZ fusions was measured as previously described ( ). .. DNA was isolated as Eco RI- Bam HI fragments released from the respective plasmids and labeled at the 3′ end of the bottom strand by incubation with [α-32 P]dATP (1 μCi/ml; 3,000 Ci/mmol; ICN) and Klenow fragment (40 U/ml; Boehringer-Mannheim). .. Nucleotide precursors were removed by gel filtration through G-50 QuickSpin columns (Boehringer-Mannheim).

Isolation:

Article Title: Effect of Altered Spacing between uhpT Promoter Elements on Transcription Activation
Article Snippet: The activity of β-galactosidase expressed from uhpT-lacZ fusions was measured as previously described ( ). .. DNA was isolated as Eco RI- Bam HI fragments released from the respective plasmids and labeled at the 3′ end of the bottom strand by incubation with [α-32 P]dATP (1 μCi/ml; 3,000 Ci/mmol; ICN) and Klenow fragment (40 U/ml; Boehringer-Mannheim). .. Nucleotide precursors were removed by gel filtration through G-50 QuickSpin columns (Boehringer-Mannheim).

Incubation:

Article Title: Effect of Altered Spacing between uhpT Promoter Elements on Transcription Activation
Article Snippet: The activity of β-galactosidase expressed from uhpT-lacZ fusions was measured as previously described ( ). .. DNA was isolated as Eco RI- Bam HI fragments released from the respective plasmids and labeled at the 3′ end of the bottom strand by incubation with [α-32 P]dATP (1 μCi/ml; 3,000 Ci/mmol; ICN) and Klenow fragment (40 U/ml; Boehringer-Mannheim). .. Nucleotide precursors were removed by gel filtration through G-50 QuickSpin columns (Boehringer-Mannheim).

Activity Assay:

Article Title: Translesional synthesis on DNA templates containing the 2?-deoxyribonolactone lesion
Article Snippet: Oligonucleotides were labeled using [γ-32 P]ATP (specific activity 3000 Ci/mmol) (NENTM Life Science) and T4 polynucleotide kinase purchased either from Boehringer Mannheim or MBI Fermentas. .. The Klenow fragments of Escherichia coli DNA polymerase I proficient (KF exo+ ) and deficient (KF exo– ) in 3′→5′ proofreading exonuclease activity were obtained from Boehringer Mannheim and MBI Fermentas, respectively. .. M-MuLV RT and nucleotide triphosphates (dNTPs) were purchased from Eurogentec and MBI Fermentas.

other:

Article Title: Activation of Transcription of the Human Cytomegalovirus Early UL4 Promoter by the Ets Transcription Factor Binding Element
Article Snippet: T4 DNA ligase, the Klenow fragment of Escherichia coli DNA polymerase I, and calf intestinal alkaline phosphatase were obtained from Boehringer Mannheim Biochemicals (Indianapolis, Ind.).

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    Boehringer Mannheim dnase i
    Regulatory loci downstream of human Cα1 and Cα2. Lines A and C , based on this study, show an expanded map of the region downstream of Cα1 and Cα2, respectively, as well as available DNA clones, which are shown above (α1) or below (α2) the line: phage clones are marked with diagrammatic phage heads, while the subclones of PCR-amplified segments A1-HS3-12 and A1-HS4-3′ are drawn with hatched lines; and a BAC clone is drawn as a double line, containing a deletion ( dashed box ). Vertical ovals mark <t>DNase</t> I sites demonstrating enhancer activity and named according to the homologous murine HS sites. A series of small triangles identifies the 20-bp repeats located downstream from human Cα genes. X marks the position of a DNase I site which shows human/mouse sequence conservation. The position of a CpG island previously identified by Southern blotting is also shown ( oval ). The arrow under HS12 in line A indicates the orientation of this sequence, which is the same as that of the homologous mouse HS site, but opposite from the orientation of HS12 in the α2 locus (line C ). The thick black lines under the maps of lines A and C ( single lower case letters ) represent hybridization probes used in this study.
    Dnase I, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Boehringer Mannheim hpa i restriction enzyme
    RFLP analysis. (A) Hybridization of Hpa I-digested total Borrelia <t>DNA</t> with an rrl -directed probe. (B) PCR-amplified rrf-rrl intergenic spacer fragments digested with Mse I and separated by PAGE.
    Hpa I Restriction Enzyme, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Boehringer Mannheim endonuclease free kornberg dna polymerase i
    The incorporation of dig-dUTP by <t>Kornberg</t> <t>DNA</t> <t>polymerase-I</t> is dependent on SSB bearing 3′-OH ends. The incorporation of dig-dUTP to 3′-OH termini (white fluores-cent signal) in the sham-operated control brains (one of four is presented here) was tested using only Fpg protein ( A ), Kornberg DNA polymerase-I ( B ), or DNase I and then Kornberg DNA polymerase-I ( C ). The fluorescent signal that can be observed in panels A and B comes mostly from the background of the cytoplasm. Similar results were noted in another set of experiments when Kornberg enzyme was replaced with Klenow or terminal transferase (not shown). Bar = 35 μm.
    Endonuclease Free Kornberg Dna Polymerase I, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/endonuclease free kornberg dna polymerase i/product/Boehringer Mannheim
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    86
    Boehringer Mannheim klenow fragment
    The effect of Ku on the 3′→5′ exonuclease activity of WRN, <t>Klenow</t> and exo <t>III</t> on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.
    Klenow Fragment, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Regulatory loci downstream of human Cα1 and Cα2. Lines A and C , based on this study, show an expanded map of the region downstream of Cα1 and Cα2, respectively, as well as available DNA clones, which are shown above (α1) or below (α2) the line: phage clones are marked with diagrammatic phage heads, while the subclones of PCR-amplified segments A1-HS3-12 and A1-HS4-3′ are drawn with hatched lines; and a BAC clone is drawn as a double line, containing a deletion ( dashed box ). Vertical ovals mark DNase I sites demonstrating enhancer activity and named according to the homologous murine HS sites. A series of small triangles identifies the 20-bp repeats located downstream from human Cα genes. X marks the position of a DNase I site which shows human/mouse sequence conservation. The position of a CpG island previously identified by Southern blotting is also shown ( oval ). The arrow under HS12 in line A indicates the orientation of this sequence, which is the same as that of the homologous mouse HS site, but opposite from the orientation of HS12 in the α2 locus (line C ). The thick black lines under the maps of lines A and C ( single lower case letters ) represent hybridization probes used in this study.

    Journal: The Journal of Experimental Medicine

    Article Title: Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes

    doi:

    Figure Lengend Snippet: Regulatory loci downstream of human Cα1 and Cα2. Lines A and C , based on this study, show an expanded map of the region downstream of Cα1 and Cα2, respectively, as well as available DNA clones, which are shown above (α1) or below (α2) the line: phage clones are marked with diagrammatic phage heads, while the subclones of PCR-amplified segments A1-HS3-12 and A1-HS4-3′ are drawn with hatched lines; and a BAC clone is drawn as a double line, containing a deletion ( dashed box ). Vertical ovals mark DNase I sites demonstrating enhancer activity and named according to the homologous murine HS sites. A series of small triangles identifies the 20-bp repeats located downstream from human Cα genes. X marks the position of a DNase I site which shows human/mouse sequence conservation. The position of a CpG island previously identified by Southern blotting is also shown ( oval ). The arrow under HS12 in line A indicates the orientation of this sequence, which is the same as that of the homologous mouse HS site, but opposite from the orientation of HS12 in the α2 locus (line C ). The thick black lines under the maps of lines A and C ( single lower case letters ) represent hybridization probes used in this study.

    Article Snippet: Although this probe hybridizes to both the α1 and α2 loci, in this DNA the α1 band is ∼23 kb, so large that any fragments from this locus that were generated by HindIII and DNase I and which hybridized to the probe would be larger than the 12-kb band representing the α2 locus.

    Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, BAC Assay, Activity Assay, Sequencing, Southern Blot, Hybridization

    Sequence similarities between human and mouse 3′ α elements. Human-mouse alignments are shown between 3′α enhancers (H12, HS3, and HS4), as well as for the X DNase I site. Nucleotide matches between human α1 and α2 sequences and between one or both human sequences and mouse are indicated by shading. Core homology regions are indicated by a thick line above the sequences. Boxes denote motifs shown to function in mouse as transcription factor binding sites. For HS12, HS3, and HS4, 50–100 bp of sequence flanking the core homology regions are shown. Mouse sequence numbering is 5′ to 3′ with regard to the coding strand of the mouse heavy chain locus. Numbering for mouse HS12, HS3A, HS3B, and X segments is according to reference 13 (EMBL/GenBank/DDBJ accession numbers X96607 and X96608 ), while numbering for mouse HS4 is according to reference 12 (EMBL/DDBJ/GenBank accession number S74166 ). ( A ) HS12 sequences (α2 sequence inverted). Overlining highlights the striking 135-bp core segment which is 90% homologous between human and mouse. The sequence alignment has been extended downstream from the core to include additional transcription factor motifs which are functional in mouse. Vertical lines indicate the boundaries of the GC-rich 59-bp repeat units. ( B ) HS3. Comparison of the nearly identical human α1 and α2 HS3 sequences with mouse HS3A and HS3B sequences, which are also nearly identical, shows that there is a 200-bp core segment which is 74% homologous between the mouse and human sequences. ( C ) HS4. Excluding the 25-bp gap containing the mouse HS4 BSAP site, the 145 core HS4 region is 76% homologous between human and mouse. ( D ) X site. Near the center of a 61-bp segment which has 70% human-mouse homology, there is a 20-bp sequence which matches at 19 positions between humans and mice. In both mice and humans this segment contains a consensus HSE ( 41 , 42 ). The sequences of the human enhancers and X sites are available from EMBL/GenBank/DDBJ under accession numbers AF013718 (α1HS3), AF013719 (α2HS3), AF013720 (α1X), AF013721 (α2X), AF013722 (α1HS12T), AF013723 (α1HS12B), AF013724 (α2HS12), AF013725 (α1HS4), and AF013726 (α2HS4).

    Journal: The Journal of Experimental Medicine

    Article Title: Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes

    doi:

    Figure Lengend Snippet: Sequence similarities between human and mouse 3′ α elements. Human-mouse alignments are shown between 3′α enhancers (H12, HS3, and HS4), as well as for the X DNase I site. Nucleotide matches between human α1 and α2 sequences and between one or both human sequences and mouse are indicated by shading. Core homology regions are indicated by a thick line above the sequences. Boxes denote motifs shown to function in mouse as transcription factor binding sites. For HS12, HS3, and HS4, 50–100 bp of sequence flanking the core homology regions are shown. Mouse sequence numbering is 5′ to 3′ with regard to the coding strand of the mouse heavy chain locus. Numbering for mouse HS12, HS3A, HS3B, and X segments is according to reference 13 (EMBL/GenBank/DDBJ accession numbers X96607 and X96608 ), while numbering for mouse HS4 is according to reference 12 (EMBL/DDBJ/GenBank accession number S74166 ). ( A ) HS12 sequences (α2 sequence inverted). Overlining highlights the striking 135-bp core segment which is 90% homologous between human and mouse. The sequence alignment has been extended downstream from the core to include additional transcription factor motifs which are functional in mouse. Vertical lines indicate the boundaries of the GC-rich 59-bp repeat units. ( B ) HS3. Comparison of the nearly identical human α1 and α2 HS3 sequences with mouse HS3A and HS3B sequences, which are also nearly identical, shows that there is a 200-bp core segment which is 74% homologous between the mouse and human sequences. ( C ) HS4. Excluding the 25-bp gap containing the mouse HS4 BSAP site, the 145 core HS4 region is 76% homologous between human and mouse. ( D ) X site. Near the center of a 61-bp segment which has 70% human-mouse homology, there is a 20-bp sequence which matches at 19 positions between humans and mice. In both mice and humans this segment contains a consensus HSE ( 41 , 42 ). The sequences of the human enhancers and X sites are available from EMBL/GenBank/DDBJ under accession numbers AF013718 (α1HS3), AF013719 (α2HS3), AF013720 (α1X), AF013721 (α2X), AF013722 (α1HS12T), AF013723 (α1HS12B), AF013724 (α2HS12), AF013725 (α1HS4), and AF013726 (α2HS4).

    Article Snippet: Although this probe hybridizes to both the α1 and α2 loci, in this DNA the α1 band is ∼23 kb, so large that any fragments from this locus that were generated by HindIII and DNase I and which hybridized to the probe would be larger than the 12-kb band representing the α2 locus.

    Techniques: Sequencing, Binding Assay, Functional Assay, Mouse Assay

    Comparison of IgsH loci of mouse and human. Line A shows a map of the murine IgH locus, from which the region downstream from Cα is expanded in line B. The murine enhancers designated Cα3′E ( 11 ) and 3′αE ( 9 ) are shown as vertical ovals, along with the DNase I hypersensitivity site designations ( 12 ). We have distinguished the two copies of HS3 sequence as HS3A and HS3B; these are included in a large palindrome ( arrows ) that flanks HS12, according to the sequence analysis of Chauveau and Cogné ( 13 ). Line C shows the human IgH locus, illustrating the γ-γ-ε-α duplication units ( brackets ) and the possibility of two regions homologous to the murine LCR.

    Journal: The Journal of Experimental Medicine

    Article Title: Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes

    doi:

    Figure Lengend Snippet: Comparison of IgsH loci of mouse and human. Line A shows a map of the murine IgH locus, from which the region downstream from Cα is expanded in line B. The murine enhancers designated Cα3′E ( 11 ) and 3′αE ( 9 ) are shown as vertical ovals, along with the DNase I hypersensitivity site designations ( 12 ). We have distinguished the two copies of HS3 sequence as HS3A and HS3B; these are included in a large palindrome ( arrows ) that flanks HS12, according to the sequence analysis of Chauveau and Cogné ( 13 ). Line C shows the human IgH locus, illustrating the γ-γ-ε-α duplication units ( brackets ) and the possibility of two regions homologous to the murine LCR.

    Article Snippet: Although this probe hybridizes to both the α1 and α2 loci, in this DNA the α1 band is ∼23 kb, so large that any fragments from this locus that were generated by HindIII and DNase I and which hybridized to the probe would be larger than the 12-kb band representing the α2 locus.

    Techniques: Sequencing

    Mapping of DNase I hypersensitive sites in the regions 3′ of the human Cα genes. ( A ) DNase I hypersensitive sites lie downstream from the human Cα genes in the HS Sultan plasmacytoma. DNA samples prepared from DNase I–digested nuclei isolated from K562 promyeloid and HS Sultan myeloma cells were digested with BglII, electrophoresed, blotted, and hybridized with probe a (αm, Fig.1). No DNase I hypersensitive sites are seen in the K562 samples. In contrast, at least seven DNase I hypersensitive sites are observed in samples from HS Sultan plasmacytoma cells. The size of each DNase I–generated band corresponds to its distance from the BglII sites located ∼1 kb 5′ of each α membrane exon ( αm ). This mapping strategy does not distinguish between sites in the α1 versus α2 loci; sites are labeled according to their subsequent assignment (see B and C , and sequence analyses). Due to their large size, bands resulting from DNase I cutting at the α1 and α2 HS4 sites are not resolved in this analysis. ( B ) HS4 sites are accessible to nuclease in both α1 and α2 loci. HS Sultan nuclei were digested with DNase I or SspI restriction enzyme (both the α1 and α2 HS4 sequences contain an SspI site). Purified DNA was digested with EcoRI and hybridized with probe b′, yielding two closely spaced DNase I HS bands, whose sizes correspond to the expected distance between the HS4 enhancers and the downstream EcoRI sites. Furthermore, there are two similarly positioned bands in the samples from SspI-digested nuclei, indicating that both the α1 and α2 HS4 sites are accessible to SspI. ( C ) Assignment of DNase I hypersensitive sites to the 3′ Cα2 region. HS Sultan DNA samples were digested with HindIII and hybridized with probe g (α2 HS12, Fig. 1 ). Because DNAse I–generated bands from the α1 region which hybridize to this probe are expected to be larger than the 12-kb α2 HindIII fragment, all bands

    Journal: The Journal of Experimental Medicine

    Article Title: Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes

    doi:

    Figure Lengend Snippet: Mapping of DNase I hypersensitive sites in the regions 3′ of the human Cα genes. ( A ) DNase I hypersensitive sites lie downstream from the human Cα genes in the HS Sultan plasmacytoma. DNA samples prepared from DNase I–digested nuclei isolated from K562 promyeloid and HS Sultan myeloma cells were digested with BglII, electrophoresed, blotted, and hybridized with probe a (αm, Fig.1). No DNase I hypersensitive sites are seen in the K562 samples. In contrast, at least seven DNase I hypersensitive sites are observed in samples from HS Sultan plasmacytoma cells. The size of each DNase I–generated band corresponds to its distance from the BglII sites located ∼1 kb 5′ of each α membrane exon ( αm ). This mapping strategy does not distinguish between sites in the α1 versus α2 loci; sites are labeled according to their subsequent assignment (see B and C , and sequence analyses). Due to their large size, bands resulting from DNase I cutting at the α1 and α2 HS4 sites are not resolved in this analysis. ( B ) HS4 sites are accessible to nuclease in both α1 and α2 loci. HS Sultan nuclei were digested with DNase I or SspI restriction enzyme (both the α1 and α2 HS4 sequences contain an SspI site). Purified DNA was digested with EcoRI and hybridized with probe b′, yielding two closely spaced DNase I HS bands, whose sizes correspond to the expected distance between the HS4 enhancers and the downstream EcoRI sites. Furthermore, there are two similarly positioned bands in the samples from SspI-digested nuclei, indicating that both the α1 and α2 HS4 sites are accessible to SspI. ( C ) Assignment of DNase I hypersensitive sites to the 3′ Cα2 region. HS Sultan DNA samples were digested with HindIII and hybridized with probe g (α2 HS12, Fig. 1 ). Because DNAse I–generated bands from the α1 region which hybridize to this probe are expected to be larger than the 12-kb α2 HindIII fragment, all bands

    Article Snippet: Although this probe hybridizes to both the α1 and α2 loci, in this DNA the α1 band is ∼23 kb, so large that any fragments from this locus that were generated by HindIII and DNase I and which hybridized to the probe would be larger than the 12-kb band representing the α2 locus.

    Techniques: Isolation, Generated, Labeling, Sequencing, Purification

    Enhancer activity of selected regions downstream of human Cα1 and Cα2 genes. ( A ) Analysis of the locus downstream of α2, which was studied in detail. The map shows the position of DNase I sites; below this are diagrammed the restriction sites defining the boundaries of each fragment tested for enhancer activity by insertion into pGL3-Vκ, transfection into the human myeloma HS Sultan, and assay of resulting luciferase activity, as described in the text. The enhancer activities are given for constructs in the A orientation (the same orientation with respect to transcribed strands of immunoglobulin and luciferase) or the opposite B orientation, where examined. The luciferase activities were normalized to β-galactosidase activity encoded by a cotransfected plasmid, and expressed as fold-increase over the activity of an enhancerless control plasmid. For fragments showing enhancer activity, assays were performed at least in triplicate, and standard deviations are given. ( B ) Comparable analysis of selected fragments amplified from the homologous locus downstream from Cα1.

    Journal: The Journal of Experimental Medicine

    Article Title: Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes

    doi:

    Figure Lengend Snippet: Enhancer activity of selected regions downstream of human Cα1 and Cα2 genes. ( A ) Analysis of the locus downstream of α2, which was studied in detail. The map shows the position of DNase I sites; below this are diagrammed the restriction sites defining the boundaries of each fragment tested for enhancer activity by insertion into pGL3-Vκ, transfection into the human myeloma HS Sultan, and assay of resulting luciferase activity, as described in the text. The enhancer activities are given for constructs in the A orientation (the same orientation with respect to transcribed strands of immunoglobulin and luciferase) or the opposite B orientation, where examined. The luciferase activities were normalized to β-galactosidase activity encoded by a cotransfected plasmid, and expressed as fold-increase over the activity of an enhancerless control plasmid. For fragments showing enhancer activity, assays were performed at least in triplicate, and standard deviations are given. ( B ) Comparable analysis of selected fragments amplified from the homologous locus downstream from Cα1.

    Article Snippet: Although this probe hybridizes to both the α1 and α2 loci, in this DNA the α1 band is ∼23 kb, so large that any fragments from this locus that were generated by HindIII and DNase I and which hybridized to the probe would be larger than the 12-kb band representing the α2 locus.

    Techniques: Activity Assay, Transfection, Luciferase, Construct, Plasmid Preparation, Amplification

    RFLP analysis. (A) Hybridization of Hpa I-digested total Borrelia DNA with an rrl -directed probe. (B) PCR-amplified rrf-rrl intergenic spacer fragments digested with Mse I and separated by PAGE.

    Journal: Journal of Clinical Microbiology

    Article Title: Isolation of Lyme Disease Borrelia from Puffins (Fratercula arctica) and Seabird Ticks (Ixodes uriae) on the Faeroe Islands

    doi:

    Figure Lengend Snippet: RFLP analysis. (A) Hybridization of Hpa I-digested total Borrelia DNA with an rrl -directed probe. (B) PCR-amplified rrf-rrl intergenic spacer fragments digested with Mse I and separated by PAGE.

    Article Snippet: The DNA was digested with the Hpa I restriction enzyme (Boehringer Mannheim), and the fragments were separated on a 0.7% agarose gel in TBE (89 mM Tris-borate, 89 mM boric acid, 2 mM EDTA) and blotted onto a Hybond N nylon filter.

    Techniques: Hybridization, Polymerase Chain Reaction, Amplification, Polyacrylamide Gel Electrophoresis

    The incorporation of dig-dUTP by Kornberg DNA polymerase-I is dependent on SSB bearing 3′-OH ends. The incorporation of dig-dUTP to 3′-OH termini (white fluores-cent signal) in the sham-operated control brains (one of four is presented here) was tested using only Fpg protein ( A ), Kornberg DNA polymerase-I ( B ), or DNase I and then Kornberg DNA polymerase-I ( C ). The fluorescent signal that can be observed in panels A and B comes mostly from the background of the cytoplasm. Similar results were noted in another set of experiments when Kornberg enzyme was replaced with Klenow or terminal transferase (not shown). Bar = 35 μm.

    Journal: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology

    Article Title: Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain

    doi:

    Figure Lengend Snippet: The incorporation of dig-dUTP by Kornberg DNA polymerase-I is dependent on SSB bearing 3′-OH ends. The incorporation of dig-dUTP to 3′-OH termini (white fluores-cent signal) in the sham-operated control brains (one of four is presented here) was tested using only Fpg protein ( A ), Kornberg DNA polymerase-I ( B ), or DNase I and then Kornberg DNA polymerase-I ( C ). The fluorescent signal that can be observed in panels A and B comes mostly from the background of the cytoplasm. Similar results were noted in another set of experiments when Kornberg enzyme was replaced with Klenow or terminal transferase (not shown). Bar = 35 μm.

    Article Snippet: Both groups were incubated at 37°C for 1 h. After extensive washes in 10 mM Tris HCl (pH 7.4), the tissue sections were treated with endonuclease-free Kornberg DNA polymerase I (10 U per section, Boehringer Mannheim, Indianapolis, Ind.) with dNTP minus dTTP (each at 200 μM) plus dig-dUTP (20 μM), DTT (1 mM), 50 mM Tris HCl (pH 7.8), and 2 mM MgCl2 for 1 h at 37°C.

    Techniques:

    The ability of E. coli DNA polymerase-I (Kornberg pol-I) to extend synthesis of the 3′-end generated by E. coli Fpg protein. A synthetic DNA sequence of c- fos gene (5′-CATCATGGTC Z TGGTTTGGGCA-3′, where Z is the oh8dG) was labeled on the 5′ end using [γ]- 32 ), then was hybridized to the complementary strand, which C was opposite Z. The double-stranded DNA (7.5 × 10 6 cpm/pmol, 100 fmol) was treated with buffer (lanes 1, 2) or with Fpg protein (0.15 μg, lanes 3–10) in 37°C for 10 min, followed by heating at 80°C for 10 min. All of the reaction products were then incubated with dNTP (40 μM) (lanes 1–10) and additional endonuclease-free DNA polymerase-I (2.5 U, two different preparations of Kornberg enzyme [lanes 5, 6, 9, 10] or endonuclease-free Klenow enzyme [lanes 7, 8]) at 37°C for 5 min. The reaction was stopped by heating and then resolved in 10% sequencing PAGE gel to analyze single-strand DNA.

    Journal: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology

    Article Title: Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain

    doi:

    Figure Lengend Snippet: The ability of E. coli DNA polymerase-I (Kornberg pol-I) to extend synthesis of the 3′-end generated by E. coli Fpg protein. A synthetic DNA sequence of c- fos gene (5′-CATCATGGTC Z TGGTTTGGGCA-3′, where Z is the oh8dG) was labeled on the 5′ end using [γ]- 32 ), then was hybridized to the complementary strand, which C was opposite Z. The double-stranded DNA (7.5 × 10 6 cpm/pmol, 100 fmol) was treated with buffer (lanes 1, 2) or with Fpg protein (0.15 μg, lanes 3–10) in 37°C for 10 min, followed by heating at 80°C for 10 min. All of the reaction products were then incubated with dNTP (40 μM) (lanes 1–10) and additional endonuclease-free DNA polymerase-I (2.5 U, two different preparations of Kornberg enzyme [lanes 5, 6, 9, 10] or endonuclease-free Klenow enzyme [lanes 7, 8]) at 37°C for 5 min. The reaction was stopped by heating and then resolved in 10% sequencing PAGE gel to analyze single-strand DNA.

    Article Snippet: Both groups were incubated at 37°C for 1 h. After extensive washes in 10 mM Tris HCl (pH 7.4), the tissue sections were treated with endonuclease-free Kornberg DNA polymerase I (10 U per section, Boehringer Mannheim, Indianapolis, Ind.) with dNTP minus dTTP (each at 200 μM) plus dig-dUTP (20 μM), DTT (1 mM), 50 mM Tris HCl (pH 7.8), and 2 mM MgCl2 for 1 h at 37°C.

    Techniques: Generated, Sequencing, Labeling, Incubation, Polyacrylamide Gel Electrophoresis

    The effect of Ku on the 3′→5′ exonuclease activity of WRN, Klenow and exo III on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.

    Journal: Nucleic Acids Research

    Article Title: A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA

    doi:

    Figure Lengend Snippet: The effect of Ku on the 3′→5′ exonuclease activity of WRN, Klenow and exo III on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.

    Article Snippet: Exonuclease III (exo III) and the Klenow fragment of Escherichia coli were purchased from Boehringer Mannheim and T4 polynucleotide kinase was from New England Biolabs (Beverly, MA).

    Techniques: Activity Assay, Incubation, Labeling