vent dna polymerase  (New England Biolabs)


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    Name:
    Vent DNA Polymerase
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    Vent DNA Polymerase 1 000 units
    Catalog Number:
    m0254l
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    288
    Size:
    1 000 units
    Category:
    Thermostable DNA Polymerases
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    New England Biolabs vent dna polymerase
    Vent DNA Polymerase
    Vent DNA Polymerase 1 000 units
    https://www.bioz.com/result/vent dna polymerase/product/New England Biolabs
    Average 99 stars, based on 73 article reviews
    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "Tissue distribution of products of the mouse decay-accelerating factor (DAF) genes. Exploitation of a Daf1 knock-out mouse and site-specific monoclonal antibodies"

    Article Title: Tissue distribution of products of the mouse decay-accelerating factor (DAF) genes. Exploitation of a Daf1 knock-out mouse and site-specific monoclonal antibodies

    Journal: Immunology

    doi: 10.1046/j.0019-2805.2001.01287.x

    Targeting strategy for inactivation of the mouse Daf genes. The tandem Daf1 and Daf2 genes are shown diagrammatically (not drawn to scale). The black-filled boxes represent exons and the open boxes represent selection marker genes as marked. As the figure shows, the pDAFup construct integrates to the Daf1 gene first to introduce a loxP site and a TK gene within exon 3, then the pDAFdown construct may integrate into its homologous region in either the Daf1 or Daf2 gene together with another loxP site and TK gene. Upon recombination induced by Cre recombinase, the DNA fragment flanked by the two loxP sites is deleted together with the two TK genes to generate either Daf1 or Daf2 knock-out ES cells.
    Figure Legend Snippet: Targeting strategy for inactivation of the mouse Daf genes. The tandem Daf1 and Daf2 genes are shown diagrammatically (not drawn to scale). The black-filled boxes represent exons and the open boxes represent selection marker genes as marked. As the figure shows, the pDAFup construct integrates to the Daf1 gene first to introduce a loxP site and a TK gene within exon 3, then the pDAFdown construct may integrate into its homologous region in either the Daf1 or Daf2 gene together with another loxP site and TK gene. Upon recombination induced by Cre recombinase, the DNA fragment flanked by the two loxP sites is deleted together with the two TK genes to generate either Daf1 or Daf2 knock-out ES cells.

    Techniques Used: Selection, Marker, Construct, Introduce, Knock-Out

    PCR analysis of recombined ES cells. (a1) PCR with Neo and Hph-specific primers P3 and P4 yielded a ∼500-bp fragment verifying that the two markers were brought together by Cre/ loxP recombination. M, 1 kb ladder; C, PCR with the wild-type DNA as template; K/O, recombined DNA as template. (a2) PCR with primers P5 , P6 ( Daf1 -specific) and P7 ( Daf2 -specific) showing that the pDAFdown construct integrated into exon 5 of the Daf1 gene and that the Daf1 gene thus was selectively inactivated. (a3) RT-PCR with primers P8 and P10 of the Daf1 mRNA product in the Daf1 −/− mice. A truncated product corresponding to sequence for CCP1,4 is seen. (b) A diagram of the mouse Daf1 and Daf2 genes is shown. B, Bam HI; E, Eco RI; and S, Sac I. The position of the 1·5-kb Sac I probe is indicated and the hybridized Eco RI and Bam HI fragments are shown by the brackets. (c) Southern blot analyses of Eco RI- and Bam HI-digested genomic DNA from parental and K/O mice. DNA from wild-type, heterozygous ( Daf1 +/− ) and homozygous ( Daf1 −/− ) knock-out mice were hybridized with the Sac I fragment of the Daf1 gene (panel B). The pattern corresponded to the expected deletion from Daf1 exon 3 to exon 5. The high M r band corresponds to the homologous Eco RI fragment in the Daf2 gene.
    Figure Legend Snippet: PCR analysis of recombined ES cells. (a1) PCR with Neo and Hph-specific primers P3 and P4 yielded a ∼500-bp fragment verifying that the two markers were brought together by Cre/ loxP recombination. M, 1 kb ladder; C, PCR with the wild-type DNA as template; K/O, recombined DNA as template. (a2) PCR with primers P5 , P6 ( Daf1 -specific) and P7 ( Daf2 -specific) showing that the pDAFdown construct integrated into exon 5 of the Daf1 gene and that the Daf1 gene thus was selectively inactivated. (a3) RT-PCR with primers P8 and P10 of the Daf1 mRNA product in the Daf1 −/− mice. A truncated product corresponding to sequence for CCP1,4 is seen. (b) A diagram of the mouse Daf1 and Daf2 genes is shown. B, Bam HI; E, Eco RI; and S, Sac I. The position of the 1·5-kb Sac I probe is indicated and the hybridized Eco RI and Bam HI fragments are shown by the brackets. (c) Southern blot analyses of Eco RI- and Bam HI-digested genomic DNA from parental and K/O mice. DNA from wild-type, heterozygous ( Daf1 +/− ) and homozygous ( Daf1 −/− ) knock-out mice were hybridized with the Sac I fragment of the Daf1 gene (panel B). The pattern corresponded to the expected deletion from Daf1 exon 3 to exon 5. The high M r band corresponds to the homologous Eco RI fragment in the Daf2 gene.

    Techniques Used: Polymerase Chain Reaction, Construct, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Sequencing, Southern Blot, Knock-Out

    2) Product Images from "The Plasmid-Encoded Signal Peptidase SipP Can Functionally Replace the Major Signal Peptidases SipS and SipT of Bacillus subtilis"

    Article Title: The Plasmid-Encoded Signal Peptidase SipP Can Functionally Replace the Major Signal Peptidases SipS and SipT of Bacillus subtilis

    Journal: Journal of Bacteriology

    doi:

    Cotranscription of ORF1 and sipP . (A) Schematic presentation of the ORF1- sipP module of pTA1015. The positions of primers used for RT-PCR are indicated. Primers O1 (5′-GAT GGC GCT ACT CTG GG-3′) and O2 (5′-ACT ATC TAC AAT CGG GAC TCC-3′) were used to detect ORF1-specific transcripts; primers P1 (5′-TAG AAA TGA AGA ATG ACC-3′) and P2 (5′-TCG CAT ATT ACT AAA TGG-3′) were used to detect sipP -specific transcripts; primers O1 and P2 were used to detect transcripts of ORF1 and sipP ). (B) DNA fragments amplified by RT-PCR with the primer sets O1-O2 (ORF1), P1-P2 ( sipP ), and O1-P2 (ORF1- sipP ) were separated on a 2% agarose gel containing ethidium bromide (1 μg/ml) and visualized by UV illumination (lanes labeled +RT). As a negative control, reactions were also performed in the absence of reverse transcriptase (lanes labeled −RT). The positions of amplified DNA fragments corresponding to ORF1 (180 bp), sipP (270 bp), and ORF1- sipP (650 bp) are indicated.
    Figure Legend Snippet: Cotranscription of ORF1 and sipP . (A) Schematic presentation of the ORF1- sipP module of pTA1015. The positions of primers used for RT-PCR are indicated. Primers O1 (5′-GAT GGC GCT ACT CTG GG-3′) and O2 (5′-ACT ATC TAC AAT CGG GAC TCC-3′) were used to detect ORF1-specific transcripts; primers P1 (5′-TAG AAA TGA AGA ATG ACC-3′) and P2 (5′-TCG CAT ATT ACT AAA TGG-3′) were used to detect sipP -specific transcripts; primers O1 and P2 were used to detect transcripts of ORF1 and sipP ). (B) DNA fragments amplified by RT-PCR with the primer sets O1-O2 (ORF1), P1-P2 ( sipP ), and O1-P2 (ORF1- sipP ) were separated on a 2% agarose gel containing ethidium bromide (1 μg/ml) and visualized by UV illumination (lanes labeled +RT). As a negative control, reactions were also performed in the absence of reverse transcriptase (lanes labeled −RT). The positions of amplified DNA fragments corresponding to ORF1 (180 bp), sipP (270 bp), and ORF1- sipP (650 bp) are indicated.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Activated Clotting Time Assay, CTG Assay, Amplification, Agarose Gel Electrophoresis, Labeling, Negative Control

    Temporally controlled transcription of ORF1 and sipP . (A) Schematic presentation of the ORF1- sipP regions of pTAB-OL and pTAB-PL, containing transcriptional ORF1- lacZ and sipP-lacZ gene fusions, respectively. The ORF1- and sipP-lacZ ), an integration plasmid for B. subtilis containing a promoterless spoVG-lacZ gene fusion (see Materials and Methods for details). In pTAB-OL, the transcription of lacZ is directed by the promoter(s) of ORF1. In pTAB-PL, the transcription of lacZ is directed by the promoter(s) of ORF1 and/or sipP . DNA fragments amplified by PCR are indicated with black bars. Only restriction sites relevant for the constructions are shown (Ba, Bam HI; Ec, Eco RI; Sc, Sac I). ‘ rep , 5′ truncated rep gene; ORF1’, 3′ truncated ORF1 gene; sipP ’, 3′ truncated sipP gene; ‘ORF1, 5′ truncated ORF1 gene; ori pBR322, replication functions of pBR322; SL, potential stem-loop structures. (B) Time course of the transcription of sip-lacZ gene fusions were determined in cells growing in TY medium at 37°C. β-Galactosidase activities (in units per OD 600 ) were determined for B. subtilis 8G5(pTAB-PL) (■) ( sipP-lacZ ), B. subtilis 8G5::pGDE22 (○) ( sipS-lacZ ), and B. subtilis ) ( sipV-lacZ ). Time zero indicates the transition point between the exponential and post-exponential growth phases. (C) The time course of transcription of the ORF1- lacZ gene fusion was determined as for panel B, using B. subtilis 8G5(pTAB-OL) (□).
    Figure Legend Snippet: Temporally controlled transcription of ORF1 and sipP . (A) Schematic presentation of the ORF1- sipP regions of pTAB-OL and pTAB-PL, containing transcriptional ORF1- lacZ and sipP-lacZ gene fusions, respectively. The ORF1- and sipP-lacZ ), an integration plasmid for B. subtilis containing a promoterless spoVG-lacZ gene fusion (see Materials and Methods for details). In pTAB-OL, the transcription of lacZ is directed by the promoter(s) of ORF1. In pTAB-PL, the transcription of lacZ is directed by the promoter(s) of ORF1 and/or sipP . DNA fragments amplified by PCR are indicated with black bars. Only restriction sites relevant for the constructions are shown (Ba, Bam HI; Ec, Eco RI; Sc, Sac I). ‘ rep , 5′ truncated rep gene; ORF1’, 3′ truncated ORF1 gene; sipP ’, 3′ truncated sipP gene; ‘ORF1, 5′ truncated ORF1 gene; ori pBR322, replication functions of pBR322; SL, potential stem-loop structures. (B) Time course of the transcription of sip-lacZ gene fusions were determined in cells growing in TY medium at 37°C. β-Galactosidase activities (in units per OD 600 ) were determined for B. subtilis 8G5(pTAB-PL) (■) ( sipP-lacZ ), B. subtilis 8G5::pGDE22 (○) ( sipS-lacZ ), and B. subtilis ) ( sipV-lacZ ). Time zero indicates the transition point between the exponential and post-exponential growth phases. (C) The time course of transcription of the ORF1- lacZ gene fusion was determined as for panel B, using B. subtilis 8G5(pTAB-OL) (□).

    Techniques Used: Plasmid Preparation, Amplification, Polymerase Chain Reaction

    3) Product Images from "Catabolite Repression of the Citrate Fermentation Genes in Klebsiella pneumoniae: Evidence for Involvement of the Cyclic AMP Receptor Protein"

    Article Title: Catabolite Repression of the Citrate Fermentation Genes in Klebsiella pneumoniae: Evidence for Involvement of the Cyclic AMP Receptor Protein

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.183.18.5248-5256.2001

    Organization of the citrate-specific fermentation genes in K. pneumoniae . The 13-kb DNA cluster encompassing 11 genes involved in citrate fermentation is shown in panel A. The lower part shows an enlargement of the citC - citS intergenic region. The arrows indicate transcription start sites. The positions of the CitB binding sites as deduced from DNase I footprints and of putative CRP binding sites are indicated. The sequence of the citC - citS ), the −10 regions, the CitB binding sites, the putative CRP binding sites, and the hypersensitive sites observed in DNase I footprints (asterisks).
    Figure Legend Snippet: Organization of the citrate-specific fermentation genes in K. pneumoniae . The 13-kb DNA cluster encompassing 11 genes involved in citrate fermentation is shown in panel A. The lower part shows an enlargement of the citC - citS intergenic region. The arrows indicate transcription start sites. The positions of the CitB binding sites as deduced from DNase I footprints and of putative CRP binding sites are indicated. The sequence of the citC - citS ), the −10 regions, the CitB binding sites, the putative CRP binding sites, and the hypersensitive sites observed in DNase I footprints (asterisks).

    Techniques Used: Binding Assay, Sequencing

    4) Product Images from "The Glycan Moieties and the N-Terminal Polypeptide Backbone of a Fimbria-Associated Adhesin, Fap1, Play Distinct Roles in the Biofilm Development of Streptococcus parasanguinis ▿"

    Article Title: The Glycan Moieties and the N-Terminal Polypeptide Backbone of a Fimbria-Associated Adhesin, Fap1, Play Distinct Roles in the Biofilm Development of Streptococcus parasanguinis ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01544-06

    Diagram of gly, nss, galT1, galT2 , and fap1 gene loci. (A) Restriction map of the fap1 -positive recombinant phage DNA. A 3.9-kb fragment was PCR amplified from the recombinant phage DNA using a λEMBL3 left-arm-specific sequence (F1) and a fap1 5′-end-specific sequence (800bp2) as primers. The left and right arms represent λEMBL3 cloning vectors. (B) Organization of the fap1 upstream region. Genes that share homology with glycosyltransferase ( gly ), nucleotide-sugar synthetase ( nss ), and galactosyltransferase genes ( galT1 and galT2 ) are located upstream of the fap1 locus. The fap1 gene is 618 bp from the stop codon of galT2 . The HaeII genomic DNA fragment was amplified by inverse PCR using nss 5′-1 and nss 5′-2 primers.
    Figure Legend Snippet: Diagram of gly, nss, galT1, galT2 , and fap1 gene loci. (A) Restriction map of the fap1 -positive recombinant phage DNA. A 3.9-kb fragment was PCR amplified from the recombinant phage DNA using a λEMBL3 left-arm-specific sequence (F1) and a fap1 5′-end-specific sequence (800bp2) as primers. The left and right arms represent λEMBL3 cloning vectors. (B) Organization of the fap1 upstream region. Genes that share homology with glycosyltransferase ( gly ), nucleotide-sugar synthetase ( nss ), and galactosyltransferase genes ( galT1 and galT2 ) are located upstream of the fap1 locus. The fap1 gene is 618 bp from the stop codon of galT2 . The HaeII genomic DNA fragment was amplified by inverse PCR using nss 5′-1 and nss 5′-2 primers.

    Techniques Used: Recombinant, Polymerase Chain Reaction, Amplification, Sequencing, Clone Assay, Inverse PCR

    5) Product Images from "Purification and In Vitro Characterization of the Serratia marcescens NucC Protein, a Zinc-Binding Transcription Factor Homologous to P2 Ogr"

    Article Title: Purification and In Vitro Characterization of the Serratia marcescens NucC Protein, a Zinc-Binding Transcription Factor Homologous to P2 Ogr

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.185.6.1808-1816.2003

    Comparison of NucC binding to DNA fragments containing point mutations in the P2 P F activator binding site. Labeled DNA fragments of 154 bp were generated by PCR amplification from pFWT, pF51A, or pF64G as indicated. The triangles designate increasing NucC concentrations (0, 0.3, 0.6, 1.8, and 3.0 mM) in each set of five lanes. Complexes were resolved by electrophoresis in 0.5× TBE on a 6% polyacrylamide gel. This figure was compiled by using Adobe Photoshop and Microsoft PowerPoint.
    Figure Legend Snippet: Comparison of NucC binding to DNA fragments containing point mutations in the P2 P F activator binding site. Labeled DNA fragments of 154 bp were generated by PCR amplification from pFWT, pF51A, or pF64G as indicated. The triangles designate increasing NucC concentrations (0, 0.3, 0.6, 1.8, and 3.0 mM) in each set of five lanes. Complexes were resolved by electrophoresis in 0.5× TBE on a 6% polyacrylamide gel. This figure was compiled by using Adobe Photoshop and Microsoft PowerPoint.

    Techniques Used: Binding Assay, Labeling, Generated, Polymerase Chain Reaction, Amplification, Electrophoresis

    DNA bending analysis. (A) NucC electrophoretic mobility shift assays using the 153-bp DNA binding site fragments released from pBendF51 by digestion with Mlu I (a), Bgl II (b), Nhe I (c), Spe I (d), Eco RV (e), Pvu II (f), Stu I (g), Nru I (h), Kpn I (i), and Bam HI (j). (B) Plot of the relative mobility of NucC-DNA complexes as a function of the relative location of the NucC binding site within the 153-bp fragments. This figure was compiled by using Adobe Photoshop and Microsoft PowerPoint.
    Figure Legend Snippet: DNA bending analysis. (A) NucC electrophoretic mobility shift assays using the 153-bp DNA binding site fragments released from pBendF51 by digestion with Mlu I (a), Bgl II (b), Nhe I (c), Spe I (d), Eco RV (e), Pvu II (f), Stu I (g), Nru I (h), Kpn I (i), and Bam HI (j). (B) Plot of the relative mobility of NucC-DNA complexes as a function of the relative location of the NucC binding site within the 153-bp fragments. This figure was compiled by using Adobe Photoshop and Microsoft PowerPoint.

    Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay

    Specificity of NucC activation in a coupled in vitro transcription-translation assay. The DNA template is indicated by the promoter fused to lacZ . Reaction mixtures 1 to 4 contained P4 sid promoter plasmid pSidZT (0.2 pmol), and reaction mixtures 5 to 8 contained lacUV5 promoter plasmid pRS229 (0.1 pmol). Purified NucC (30 pmol) was added as indicated to reaction mixtures 2 to 5. Competitor DNA (1 pmol, reaction mixtures 3 and 7, or 10 pmol, reaction mixtures 4 and 8) was a 154-bp fragment generated by PCR amplification of P2 P F promoter variant 64G 51A.
    Figure Legend Snippet: Specificity of NucC activation in a coupled in vitro transcription-translation assay. The DNA template is indicated by the promoter fused to lacZ . Reaction mixtures 1 to 4 contained P4 sid promoter plasmid pSidZT (0.2 pmol), and reaction mixtures 5 to 8 contained lacUV5 promoter plasmid pRS229 (0.1 pmol). Purified NucC (30 pmol) was added as indicated to reaction mixtures 2 to 5. Competitor DNA (1 pmol, reaction mixtures 3 and 7, or 10 pmol, reaction mixtures 4 and 8) was a 154-bp fragment generated by PCR amplification of P2 P F promoter variant 64G 51A.

    Techniques Used: Activation Assay, In Vitro, Plasmid Preparation, Purification, Generated, Polymerase Chain Reaction, Amplification, Variant Assay

    Titration of DNA binding by NucC. The DNA template was a 154-bp P F fragment generated by PCR amplification from pFWT. Various amounts of purified NucC protein, as indicated, were incubated with approximately 1 ng of the labeled probe and resolved by electrophoresis in 0.5× TBE on a 6% polyacrylamide gel (19:1) containing 5% glycerol at 4°C for 2.5 h at 100 V. This figure was compiled by using Adobe Photoshop and Microsoft PowerPoint.
    Figure Legend Snippet: Titration of DNA binding by NucC. The DNA template was a 154-bp P F fragment generated by PCR amplification from pFWT. Various amounts of purified NucC protein, as indicated, were incubated with approximately 1 ng of the labeled probe and resolved by electrophoresis in 0.5× TBE on a 6% polyacrylamide gel (19:1) containing 5% glycerol at 4°C for 2.5 h at 100 V. This figure was compiled by using Adobe Photoshop and Microsoft PowerPoint.

    Techniques Used: Titration, Binding Assay, Generated, Polymerase Chain Reaction, Amplification, Purification, Incubation, Labeling, Electrophoresis

    6) Product Images from "Polymerase Amplification, Cloning, and Gene Expression of Benzo-homologous "yDNA" Base Pairs"

    Article Title: Polymerase Amplification, Cloning, and Gene Expression of Benzo-homologous "yDNA" Base Pairs

    Journal: Chembiochem : a European journal of chemical biology

    doi: 10.1002/cbic.200800339

    Survey of selectivity of enzymatic nucleotide incorporation opposite yDNA bases. A and C show yDNA template bases; B and D show natural bases as controls. Enzymes are Thermococcus litoralis DNA polymerase (Vent exo−) and Klenow fragment of DNA
    Figure Legend Snippet: Survey of selectivity of enzymatic nucleotide incorporation opposite yDNA bases. A and C show yDNA template bases; B and D show natural bases as controls. Enzymes are Thermococcus litoralis DNA polymerase (Vent exo−) and Klenow fragment of DNA

    Techniques Used:

    7) Product Images from "Identification of a New Class of Cytochrome P450 from a Rhodococcus sp."

    Article Title: Identification of a New Class of Cytochrome P450 from a Rhodococcus sp.

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.184.14.3898-3908.2002

    Restriction map of a 4.4-kbp segment of chromosomal DNA from Rhodococcus sp. strain NCIMB 9784 containing the P450RhF gene. ORFs are denoted by arrows, which indicate the direction of transcription. The relative positions of the three overlapping clones are shown by the thick bars below the restriction map. P350 represents the PCR product obtained by using a degenerate set of primers designed from the oxygen- and heme-binding motifs of cytochrome P450 enzymes. The asterisks denote the site from which an oligonucleotide hybridization probe (P45) was designed. This probe was then used to clone both Sm3.0 and Bc3.5 from a sublibrary of genomic DNA. Only the restriction sites mentioned in the text are shown.
    Figure Legend Snippet: Restriction map of a 4.4-kbp segment of chromosomal DNA from Rhodococcus sp. strain NCIMB 9784 containing the P450RhF gene. ORFs are denoted by arrows, which indicate the direction of transcription. The relative positions of the three overlapping clones are shown by the thick bars below the restriction map. P350 represents the PCR product obtained by using a degenerate set of primers designed from the oxygen- and heme-binding motifs of cytochrome P450 enzymes. The asterisks denote the site from which an oligonucleotide hybridization probe (P45) was designed. This probe was then used to clone both Sm3.0 and Bc3.5 from a sublibrary of genomic DNA. Only the restriction sites mentioned in the text are shown.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Binding Assay, Hybridization

    DNA sequence of the 4,411-bp Sma I- Bcl I region encoding the cytochrome P450 (P450RhF) from Rhodococcus sp. strain NCIMB 9784. The deduced ORFs are labeled, and the direction of transcription is indicated by arrowheads. Stop codons are denoted by asterisks, and potential ribosome binding sites are shown in italics. The sites of annealing of the PCR primers used to amplify the segment of DNA between the oxygen- and heme-binding sites are underlined.
    Figure Legend Snippet: DNA sequence of the 4,411-bp Sma I- Bcl I region encoding the cytochrome P450 (P450RhF) from Rhodococcus sp. strain NCIMB 9784. The deduced ORFs are labeled, and the direction of transcription is indicated by arrowheads. Stop codons are denoted by asterisks, and potential ribosome binding sites are shown in italics. The sites of annealing of the PCR primers used to amplify the segment of DNA between the oxygen- and heme-binding sites are underlined.

    Techniques Used: Sequencing, Labeling, Binding Assay, Polymerase Chain Reaction

    8) Product Images from "Genome Sequences of Two Closely Related Vibrio parahaemolyticus Phages, VP16T and VP16C †"

    Article Title: Genome Sequences of Two Closely Related Vibrio parahaemolyticus Phages, VP16T and VP16C †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.185.21.6434-6447.2003

    Restriction analysis of phage genomic DNA isolated from the VP16 lysates. Phage DNA was purified from the original lysate (mixed) and from lysates made from clear and turbid plaques from the original lysate after three successive plaque purifications on LB medium containing 2.5% NaCl. The phage DNA was restricted with Hpa I or Xmn I. After restriction digestion at 37°C, the digests were either kept at 37°C before they were loaded on the agarose gel or incubated for 20 min at 65°C and then loaded on the agarose gel.
    Figure Legend Snippet: Restriction analysis of phage genomic DNA isolated from the VP16 lysates. Phage DNA was purified from the original lysate (mixed) and from lysates made from clear and turbid plaques from the original lysate after three successive plaque purifications on LB medium containing 2.5% NaCl. The phage DNA was restricted with Hpa I or Xmn I. After restriction digestion at 37°C, the digests were either kept at 37°C before they were loaded on the agarose gel or incubated for 20 min at 65°C and then loaded on the agarose gel.

    Techniques Used: Isolation, Purification, Agarose Gel Electrophoresis, Incubation

    9) Product Images from "Alternative Excision Repair of Ultraviolet B- and C-Induced DNA Damage in Dormant and Developing Spores of Bacillus subtilis"

    Article Title: Alternative Excision Repair of Ultraviolet B- and C-Induced DNA Damage in Dormant and Developing Spores of Bacillus subtilis

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01340-12

    (A to C) Levels of β-galactosidase from B. subtilis wild-type (A) and Δσ G (B) strains containing a ywjD-lacZ fusion and RT-PCR analysis of ywjD transcription (C). (A and B) B. subtilis strains PERM557 ( ywjD-lacZ ) (A) and PERM755 ( sigGΔ1 ywjD-lacZ ). (C) RNA samples (∼1 μg) isolated from a B. subtilis 168 DSM culture at the times indicated were processed for RT-PCR analysis as described in Materials and Methods. The arrowhead shows the size of the expected RT-PCR products. Lanes: M, DNA markers, 1-kb Plus ladder; Veg, logarithmic growth; T 0 , the time when the slopes of the logarithmic and stationary phases of growth intersected; T 1 to T 9 , times in hours after T 0 .
    Figure Legend Snippet: (A to C) Levels of β-galactosidase from B. subtilis wild-type (A) and Δσ G (B) strains containing a ywjD-lacZ fusion and RT-PCR analysis of ywjD transcription (C). (A and B) B. subtilis strains PERM557 ( ywjD-lacZ ) (A) and PERM755 ( sigGΔ1 ywjD-lacZ ). (C) RNA samples (∼1 μg) isolated from a B. subtilis 168 DSM culture at the times indicated were processed for RT-PCR analysis as described in Materials and Methods. The arrowhead shows the size of the expected RT-PCR products. Lanes: M, DNA markers, 1-kb Plus ladder; Veg, logarithmic growth; T 0 , the time when the slopes of the logarithmic and stationary phases of growth intersected; T 1 to T 9 , times in hours after T 0 .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation

    10) Product Images from "The Conserved Carboxy Terminus of the Capsid Domain of Human Immunodeficiency Virus Type 1 Gag Protein Is Important for Virion Assembly and Release"

    Article Title: The Conserved Carboxy Terminus of the Capsid Domain of Human Immunodeficiency Virus Type 1 Gag Protein Is Important for Virion Assembly and Release

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.18.9675-9688.2004

    Confocal analysis of Gag protein mutants. HeLa cells were transfected with 1 μg of pHIVgptSVPA DNA expressing wild-type (WT; upper panel), G355A (middle panel), or V355A (lower panel) Gag proteins. Two days later the cells were fixed, permeabilized, and stained with a monoclonal anti-HIV-1 capsid antibody and a secondary indocarbocyanine-conjugated antibody. Cells were examined with a confocal laser scan microscope. Fluorescence is shown on the left, and Nomarski images of the same field are shown on the right. Note the selective staining of only a portion of the cells. Similarly, no membrane staining was observed in control cells that were transfected without plasmid DNA (data not shown). Bars represent the indicated length.
    Figure Legend Snippet: Confocal analysis of Gag protein mutants. HeLa cells were transfected with 1 μg of pHIVgptSVPA DNA expressing wild-type (WT; upper panel), G355A (middle panel), or V355A (lower panel) Gag proteins. Two days later the cells were fixed, permeabilized, and stained with a monoclonal anti-HIV-1 capsid antibody and a secondary indocarbocyanine-conjugated antibody. Cells were examined with a confocal laser scan microscope. Fluorescence is shown on the left, and Nomarski images of the same field are shown on the right. Note the selective staining of only a portion of the cells. Similarly, no membrane staining was observed in control cells that were transfected without plasmid DNA (data not shown). Bars represent the indicated length.

    Techniques Used: Transfection, Expressing, Staining, Microscopy, Fluorescence, Plasmid Preparation

    VLP production by wild-type and mutant Gag proteins. Cell extracts from 293T cells transiently transfected with 10 μg of the indicated pHIVgptSVPA-based clone (A) and purified VLPs from the culture medium (B) were analyzed for Gag protein expression and cleavage by Western blot with an anti-CA monoclonal antibody. Mock samples indicate cells transfected without plasmid DNA. The wild type (wt) was the HIVgptSVPA clone expressing wild-type Gag proteins. Re wt designates an HIVgptSVPA clone into which the wild-type Gag sequence was introduced with the same cloning procedure that was used to construct the clones with the mutated gag sequences. This clone was used to ensure the absence of secondary mutations in the HIV sequence and the plasmid backbone that might affect viral protein expression. The migration positions of the Gag polyprotein (Pr55 gag ) and Gag cleavage products (p41, p25, and p24) are indicated with arrows. (C) VLP production by Gag protein mutants relative to that by the wild-type proteins was calculated after densitometry scanning and analysis of Gag protein bands (B). The data are presented as mean ± standard deviation ( n = 2).
    Figure Legend Snippet: VLP production by wild-type and mutant Gag proteins. Cell extracts from 293T cells transiently transfected with 10 μg of the indicated pHIVgptSVPA-based clone (A) and purified VLPs from the culture medium (B) were analyzed for Gag protein expression and cleavage by Western blot with an anti-CA monoclonal antibody. Mock samples indicate cells transfected without plasmid DNA. The wild type (wt) was the HIVgptSVPA clone expressing wild-type Gag proteins. Re wt designates an HIVgptSVPA clone into which the wild-type Gag sequence was introduced with the same cloning procedure that was used to construct the clones with the mutated gag sequences. This clone was used to ensure the absence of secondary mutations in the HIV sequence and the plasmid backbone that might affect viral protein expression. The migration positions of the Gag polyprotein (Pr55 gag ) and Gag cleavage products (p41, p25, and p24) are indicated with arrows. (C) VLP production by Gag protein mutants relative to that by the wild-type proteins was calculated after densitometry scanning and analysis of Gag protein bands (B). The data are presented as mean ± standard deviation ( n = 2).

    Techniques Used: Mutagenesis, Transfection, Purification, Expressing, Western Blot, Plasmid Preparation, Sequencing, Clone Assay, Construct, Migration, Standard Deviation

    Mutations of the conserved sequence at the CA C terminus reduce virus infectivity. (A) 293T cells were transfected with 7.5 μg of wild-type pHIVgptSVPA DNA or the indicated mutant together with 10 μg of HIV-1-based vector expressing the GFP marker (pHR′-CMV-GFP) and 2.5 μg of plasmid DNA expressing the vesicular stomatitis virus G envelope protein (pMD.G). Two days posttransfection, culture supernatants were used to infect naïve 293T cell. FACS analysis of transfected and infected cells was used to calculate and normalize the infectivity of the pseudotyped particles (Materials and Methods). Values are expressed as a percentage of wild-type infectivity. GagPol- indicates transfection of pHR′-CMV-GFP and pMD.G without pHIVgptSVPA plasmid DNA to control for transduction-independent expression of GFP in infected cells made by carryover of pHR′-CMV-GFP DNA. The data are presented as mean ± standard deviation ( n = 3).
    Figure Legend Snippet: Mutations of the conserved sequence at the CA C terminus reduce virus infectivity. (A) 293T cells were transfected with 7.5 μg of wild-type pHIVgptSVPA DNA or the indicated mutant together with 10 μg of HIV-1-based vector expressing the GFP marker (pHR′-CMV-GFP) and 2.5 μg of plasmid DNA expressing the vesicular stomatitis virus G envelope protein (pMD.G). Two days posttransfection, culture supernatants were used to infect naïve 293T cell. FACS analysis of transfected and infected cells was used to calculate and normalize the infectivity of the pseudotyped particles (Materials and Methods). Values are expressed as a percentage of wild-type infectivity. GagPol- indicates transfection of pHR′-CMV-GFP and pMD.G without pHIVgptSVPA plasmid DNA to control for transduction-independent expression of GFP in infected cells made by carryover of pHR′-CMV-GFP DNA. The data are presented as mean ± standard deviation ( n = 3).

    Techniques Used: Sequencing, Infection, Transfection, Mutagenesis, Plasmid Preparation, Expressing, Marker, FACS, Transduction, Standard Deviation

    11) Product Images from "Leukotriene B4 receptor transgenic mice reveal novel protective roles for lipoxins and aspirin-triggered lipoxins in reperfusion"

    Article Title: Leukotriene B4 receptor transgenic mice reveal novel protective roles for lipoxins and aspirin-triggered lipoxins in reperfusion

    Journal: Journal of Clinical Investigation

    doi:

    Construction of hBLTR transgene and screening of hBLTR TG mice. ( a ) Illustration of the pGL-CD11b-hBLTR construct. Restriction sites used for cloning, and sense (right-facing arrows) and antisense (left-facing arrows) primers used for PCR, are indicated. Nucleotide sequences of the primers corresponding to the hBLTR are underlined. ( b ) pGL-CD11b-hBLTR and mock (pGL) plasmids were transiently transfected into HEK293 cells, and intracellular calcium was mobilized by addition of LTB 4 (100 nM). Genomic DNA isolated from potential founder mice ( c ) and F1 generations ( d ) were analyzed by PCR using primers specific for the CD11b-hBLTR transgene construct. Molecular sizes of expected PCR products are indicated by arrows.
    Figure Legend Snippet: Construction of hBLTR transgene and screening of hBLTR TG mice. ( a ) Illustration of the pGL-CD11b-hBLTR construct. Restriction sites used for cloning, and sense (right-facing arrows) and antisense (left-facing arrows) primers used for PCR, are indicated. Nucleotide sequences of the primers corresponding to the hBLTR are underlined. ( b ) pGL-CD11b-hBLTR and mock (pGL) plasmids were transiently transfected into HEK293 cells, and intracellular calcium was mobilized by addition of LTB 4 (100 nM). Genomic DNA isolated from potential founder mice ( c ) and F1 generations ( d ) were analyzed by PCR using primers specific for the CD11b-hBLTR transgene construct. Molecular sizes of expected PCR products are indicated by arrows.

    Techniques Used: Mouse Assay, Construct, Clone Assay, Polymerase Chain Reaction, Transfection, Isolation

    12) Product Images from "The winged helix transcription factor HFH-4 is expressed during choroid plexus epithelial development in the mouse embryo"

    Article Title: The winged helix transcription factor HFH-4 is expressed during choroid plexus epithelial development in the mouse embryo

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

    doi:

    ) and truncated HFH-4 proteins synthesized by deleted HFH-4 cDNA constructs. Numbers represent the amino acid positions of the potential transcriptional activation motifs and the winged helix DNA-binding domain. Summarized in the bar graph is the normalized transcriptional activity of the HFH-4 protein deletions relative to the wild-type activity and the error bars represent the SD from three separate experiments. Western blots using an HFH-4 antibody specific to the N terminus of the protein (amino acids 1–100) showed that HFH-4 protein was expressed in nuclear extracts (data not shown).
    Figure Legend Snippet: ) and truncated HFH-4 proteins synthesized by deleted HFH-4 cDNA constructs. Numbers represent the amino acid positions of the potential transcriptional activation motifs and the winged helix DNA-binding domain. Summarized in the bar graph is the normalized transcriptional activity of the HFH-4 protein deletions relative to the wild-type activity and the error bars represent the SD from three separate experiments. Western blots using an HFH-4 antibody specific to the N terminus of the protein (amino acids 1–100) showed that HFH-4 protein was expressed in nuclear extracts (data not shown).

    Techniques Used: Synthesized, Construct, Activation Assay, Binding Assay, Activity Assay, Western Blot

    13) Product Images from "Structure of the sporulation-specific transcription factor Ndt80 bound to DNA"

    Article Title: Structure of the sporulation-specific transcription factor Ndt80 bound to DNA

    Journal: The EMBO Journal

    doi: 10.1093/emboj/cdf572

    Fig. 7. 5′-YpG-3′ recognition by Ndt80. ( A ) View down the DNA helix axis showing base stacking between the C–G base pair at position 5 and the A–T pair at position 6. van der Waals surface representations of R177 (yellow), P57 (red) and T6′ (gray) are displayed and hydrogen bonding interactions are indicated in green. ( B ). The view is such that the C–G base pairs in both panels are in identical orientations.
    Figure Legend Snippet: Fig. 7. 5′-YpG-3′ recognition by Ndt80. ( A ) View down the DNA helix axis showing base stacking between the C–G base pair at position 5 and the A–T pair at position 6. van der Waals surface representations of R177 (yellow), P57 (red) and T6′ (gray) are displayed and hydrogen bonding interactions are indicated in green. ( B ). The view is such that the C–G base pairs in both panels are in identical orientations.

    Techniques Used:

    Fig. 1. Definition of the Ndt80 DNA-binding domain. ( A ) Purified recombinant MBP–Ndt80, Ndt80(1–340) or Ndt80(59–340) was bound to 32 P-labeled MSE DNA and challenged with either the unlabeled MSE DNA as a specific competitor or poly(dI–dC) as non-specific competitor at a variety of molar ratios of competitor to 32 P-labeled MSE DNA, as indicated. Note that an ∼10-fold lower concentration of Ndt80(1–340) than MBP–Ndt80 was used to bind 32 P-labeled MSE DNA. As a result, ∼10-fold less of either cold DNA is required to compete the Ndt80(1–340)–MSE complex, compared with the MBP–Ndt80–MSE complex. ( B ) Summary of the in vivo N-terminal truncation mutants. A series of mutants encoding the indicated Ndt80 fragments were transformed into yeast cells lacking endogenous Ndt80. Six hours after shift to sporulation medium, MSE-dependent transcription was assayed using a lacZ gene under the control of the SPS4 upstream sequence containing an MSE. The gray bars represent β-galactosidase activity in Miller units. After 24 h, sporulation was monitored by microscopic examination of the culture to score for ascus formation (black bars).
    Figure Legend Snippet: Fig. 1. Definition of the Ndt80 DNA-binding domain. ( A ) Purified recombinant MBP–Ndt80, Ndt80(1–340) or Ndt80(59–340) was bound to 32 P-labeled MSE DNA and challenged with either the unlabeled MSE DNA as a specific competitor or poly(dI–dC) as non-specific competitor at a variety of molar ratios of competitor to 32 P-labeled MSE DNA, as indicated. Note that an ∼10-fold lower concentration of Ndt80(1–340) than MBP–Ndt80 was used to bind 32 P-labeled MSE DNA. As a result, ∼10-fold less of either cold DNA is required to compete the Ndt80(1–340)–MSE complex, compared with the MBP–Ndt80–MSE complex. ( B ) Summary of the in vivo N-terminal truncation mutants. A series of mutants encoding the indicated Ndt80 fragments were transformed into yeast cells lacking endogenous Ndt80. Six hours after shift to sporulation medium, MSE-dependent transcription was assayed using a lacZ gene under the control of the SPS4 upstream sequence containing an MSE. The gray bars represent β-galactosidase activity in Miller units. After 24 h, sporulation was monitored by microscopic examination of the culture to score for ascus formation (black bars).

    Techniques Used: Binding Assay, Purification, Recombinant, Labeling, Concentration Assay, In Vivo, Transformation Assay, Sequencing, Activity Assay

    Fig. 4. Structure of Ndt80(1–340) bound to DNA. ( A ) Overview of the Ndt80(1–340)–MSE complex. The regions that contact DNA are rainbow- colored from N- to C-terminus, a color scheme that is maintained throughout the paper. Only the core Ig-fold β-strands and α-helices are labeled. Chain breaks are indicated by the thin coil. The DNA sequence is shown aligned with the structure and the MSE is highlighted. ( B ) Topology diagram of Ndt80. The conserved s-type Ig-fold strands are indicated in black, while the peripheral strands, in gray, are named according to their order in the primary sequence. ( C ) Secondary structure and sequence alignment of the DNA-binding domain of Ndt80. The secondary structure includes regions that form 3 10 helices. Saccharomyces cerevisiae Ndt80 is aligned with sequences from other fungi that share high sequence similarity. The regions highlighted in green share ≥75% sequence identity. Residues with stars above them are involved in DNA contacts. The red lines within the alignment indicate amino acid insertions that were omitted in order to maintain an ungapped sequence for S.cerevisiae Ndt80. The numbering along the bottom refers to the Ndt80 numbering, while the numbers on either side are for the indicated reading frame.
    Figure Legend Snippet: Fig. 4. Structure of Ndt80(1–340) bound to DNA. ( A ) Overview of the Ndt80(1–340)–MSE complex. The regions that contact DNA are rainbow- colored from N- to C-terminus, a color scheme that is maintained throughout the paper. Only the core Ig-fold β-strands and α-helices are labeled. Chain breaks are indicated by the thin coil. The DNA sequence is shown aligned with the structure and the MSE is highlighted. ( B ) Topology diagram of Ndt80. The conserved s-type Ig-fold strands are indicated in black, while the peripheral strands, in gray, are named according to their order in the primary sequence. ( C ) Secondary structure and sequence alignment of the DNA-binding domain of Ndt80. The secondary structure includes regions that form 3 10 helices. Saccharomyces cerevisiae Ndt80 is aligned with sequences from other fungi that share high sequence similarity. The regions highlighted in green share ≥75% sequence identity. Residues with stars above them are involved in DNA contacts. The red lines within the alignment indicate amino acid insertions that were omitted in order to maintain an ungapped sequence for S.cerevisiae Ndt80. The numbering along the bottom refers to the Ndt80 numbering, while the numbers on either side are for the indicated reading frame.

    Techniques Used: Labeling, Sequencing, Binding Assay

    Fig. 5. Ndt80–DNA interface. ( A ) Stereo image of the major-groove interactions between Ndt80 and DNA. Involved in this interface are the a–b, c–c′ and e–f loops, and the C-terminal tail. ( B ) Stereo image of the minor-groove interactions between Ndt80 and DNA. This interface involves the N-terminal tail (or β2–β3 loop) and the c′–e loop (HLH).
    Figure Legend Snippet: Fig. 5. Ndt80–DNA interface. ( A ) Stereo image of the major-groove interactions between Ndt80 and DNA. Involved in this interface are the a–b, c–c′ and e–f loops, and the C-terminal tail. ( B ) Stereo image of the minor-groove interactions between Ndt80 and DNA. This interface involves the N-terminal tail (or β2–β3 loop) and the c′–e loop (HLH).

    Techniques Used:

    14) Product Images from "Identification by In Vivo Genomic Footprinting of a Transcriptional Switch Containing NF-?B and Sp1 That Regulates the I?B? Promoter"

    Article Title: Identification by In Vivo Genomic Footprinting of a Transcriptional Switch Containing NF-?B and Sp1 That Regulates the I?B? Promoter

    Journal: Molecular and Cellular Biology

    doi:

    In vivo footprinting of the proximal IκBα gene promoter in Jurkat T cells. (A) Noncoding strand analysis; (B) coding strand analysis. Naked DNA was treated in vitro with DMS (lane 1). Cells were either nonstimulated (lane 2) or treated with PMA-PHA (lane 3) or by TNF-α (lane 4) for 40 min and then were treated with DMS. Genomic DNA was extracted and treated with piperidine. All DNA samples were amplified by LM-PCR and visualized on a Long-Ranger sequencing gel. (C) Sequence of the IκBα promoter. Major consensus sites for protein binding such as the NF-κB sites and sites of Ets-1, Sp1, and AP2 are enclosed in boxes. The mRNA start site and TATA box are also shown. Arrows correspond to primers used in genomic footprinting. Primers 1, 2, and 3 were designed to characterize the noncoding strand; primers 1B, 2B, and 3B were used for the coding strand.
    Figure Legend Snippet: In vivo footprinting of the proximal IκBα gene promoter in Jurkat T cells. (A) Noncoding strand analysis; (B) coding strand analysis. Naked DNA was treated in vitro with DMS (lane 1). Cells were either nonstimulated (lane 2) or treated with PMA-PHA (lane 3) or by TNF-α (lane 4) for 40 min and then were treated with DMS. Genomic DNA was extracted and treated with piperidine. All DNA samples were amplified by LM-PCR and visualized on a Long-Ranger sequencing gel. (C) Sequence of the IκBα promoter. Major consensus sites for protein binding such as the NF-κB sites and sites of Ets-1, Sp1, and AP2 are enclosed in boxes. The mRNA start site and TATA box are also shown. Arrows correspond to primers used in genomic footprinting. Primers 1, 2, and 3 were designed to characterize the noncoding strand; primers 1B, 2B, and 3B were used for the coding strand.

    Techniques Used: In Vivo, Footprinting, In Vitro, Amplification, Polymerase Chain Reaction, Sequencing, Protein Binding

    15) Product Images from "Telomere-binding and Stn1p-interacting activities are required for the essential function of Saccharomyces cerevisiae Cdc13p"

    Article Title: Telomere-binding and Stn1p-interacting activities are required for the essential function of Saccharomyces cerevisiae Cdc13p

    Journal: Nucleic Acids Research

    doi:

    Single-stranded TG 1–3 -binding domain of Cdc13p. ( A ) Schematic representation of Cdc13p deletion mutants. The wild-type protein is 924 amino acids in length. The relative locations of the fragments (thick line) and the first as well as the last amino acids are indicated. All these mutants were fused in-frame to a GST protein on their N terminus. On the right are the single-stranded TG22-binding activities of mutants that were analyzed by EMSA. ( B ) Cdc13(451–693)p contains the single-stranded TG 1–3 -binding domain of Cdc13p. Escherichia coli extracts (15 µg) were mixed with 32 P-labeled TG22 in 15-µl reaction mixtures. The reactions were incubated at room temperature for 10 min. A 3-µl volume of 80% glycerol was added to each reaction before analysis of the reaction products on an 8% polyacrylamide gel. Extracts carrying the GST fusion of Cdc13p deletion mutants are indicated. The first lane has no extracts. Migration of free DNA (TG22), Cdc13(451–693)p, Cdc13(451–871)p, Cdc13(451–924)p and Cdc13p are indicated.
    Figure Legend Snippet: Single-stranded TG 1–3 -binding domain of Cdc13p. ( A ) Schematic representation of Cdc13p deletion mutants. The wild-type protein is 924 amino acids in length. The relative locations of the fragments (thick line) and the first as well as the last amino acids are indicated. All these mutants were fused in-frame to a GST protein on their N terminus. On the right are the single-stranded TG22-binding activities of mutants that were analyzed by EMSA. ( B ) Cdc13(451–693)p contains the single-stranded TG 1–3 -binding domain of Cdc13p. Escherichia coli extracts (15 µg) were mixed with 32 P-labeled TG22 in 15-µl reaction mixtures. The reactions were incubated at room temperature for 10 min. A 3-µl volume of 80% glycerol was added to each reaction before analysis of the reaction products on an 8% polyacrylamide gel. Extracts carrying the GST fusion of Cdc13p deletion mutants are indicated. The first lane has no extracts. Migration of free DNA (TG22), Cdc13(451–693)p, Cdc13(451–871)p, Cdc13(451–924)p and Cdc13p are indicated.

    Techniques Used: Binding Assay, Labeling, Incubation, Migration

    Purified Cdc13(451–693)p binds single-stranded TG 1–3 telomeric DNA. ( A ) Purification of Cdc13(451–693)p. A 6× His-tagged Cdc13(451–693)p was purified from E.coli using a Ni-NTA–agarose column (see Materials and Methods). A Coomassie blue-stained 10% SDS–polyacrylamide gel is given. Lane 1 shows the molecular weight marker; lanes 2 and 3 were 50 µl of E.coli cultures harboring the pET6H-CDC13(451–693) plasmid grown without and with IPTG induction, respectively; lane 4 was 5 µg of purified Cdc13(451–693)p. ( B ) Cdc13(451–693)p contains the single-stranded TG 1–3 -binding domain of Cdc13p. Approximately 27 nM each of 32 P-labeled TG10 (lanes 1–6), TG15 (lanes 7–12), TG20 (lanes 13–18), TG25 (lanes 19–24), TG30 (lanes 25–30) and TG35 (lanes 31–36) were mixed with several concentrations of the purified Cdc13(451–693)p and then gel shift assay was carried out. Cdc13(451–693)p used in each set of experiments were 0, 7, 22, 66, 200 and 600 nM. Autoradiograms are shown.
    Figure Legend Snippet: Purified Cdc13(451–693)p binds single-stranded TG 1–3 telomeric DNA. ( A ) Purification of Cdc13(451–693)p. A 6× His-tagged Cdc13(451–693)p was purified from E.coli using a Ni-NTA–agarose column (see Materials and Methods). A Coomassie blue-stained 10% SDS–polyacrylamide gel is given. Lane 1 shows the molecular weight marker; lanes 2 and 3 were 50 µl of E.coli cultures harboring the pET6H-CDC13(451–693) plasmid grown without and with IPTG induction, respectively; lane 4 was 5 µg of purified Cdc13(451–693)p. ( B ) Cdc13(451–693)p contains the single-stranded TG 1–3 -binding domain of Cdc13p. Approximately 27 nM each of 32 P-labeled TG10 (lanes 1–6), TG15 (lanes 7–12), TG20 (lanes 13–18), TG25 (lanes 19–24), TG30 (lanes 25–30) and TG35 (lanes 31–36) were mixed with several concentrations of the purified Cdc13(451–693)p and then gel shift assay was carried out. Cdc13(451–693)p used in each set of experiments were 0, 7, 22, 66, 200 and 600 nM. Autoradiograms are shown.

    Techniques Used: Purification, Staining, Molecular Weight, Marker, Plasmid Preparation, Binding Assay, Labeling, Electrophoretic Mobility Shift Assay

    16) Product Images from "In Vivo Genomic Footprinting of the Human T-Cell Leukemia Virus Type 1 (HTLV-1) Long Terminal Repeat Enhancer Sequences in HTLV-1-Infected Human T-Cell Lines with Different Levels of Tax I Activity"

    Article Title: In Vivo Genomic Footprinting of the Human T-Cell Leukemia Virus Type 1 (HTLV-1) Long Terminal Repeat Enhancer Sequences in HTLV-1-Infected Human T-Cell Lines with Different Levels of Tax I Activity

    Journal: Journal of Virology

    doi:

    In vivo DMS footprinting of the lower strand of the TRE-1s. DNAs from in vivo DMS-treated MT-2 and MT-4 cells were piperidine cleaved and subjected to LMPCR as described in Materials and Methods. The reaction products were separated on polyacrylamide gels and exposed to film. Autoradiograms corresponding to the three TRE-1s are shown. (A) Promoter-distal TRE-1. A band is missing in the 5′ GC-rich flank most likely because the proviral DNA contains a base other than the predicted guanine residue at that position. (B) Promoter-central TRE-1. (C) Promoter-proximal TRE-1. Arrowheads pointing toward bands indicate hypersensitive residues; arrowheads pointing away from bands indicate protected residues. Larger arrowheads indicate major protections or hypersensitive sites.
    Figure Legend Snippet: In vivo DMS footprinting of the lower strand of the TRE-1s. DNAs from in vivo DMS-treated MT-2 and MT-4 cells were piperidine cleaved and subjected to LMPCR as described in Materials and Methods. The reaction products were separated on polyacrylamide gels and exposed to film. Autoradiograms corresponding to the three TRE-1s are shown. (A) Promoter-distal TRE-1. A band is missing in the 5′ GC-rich flank most likely because the proviral DNA contains a base other than the predicted guanine residue at that position. (B) Promoter-central TRE-1. (C) Promoter-proximal TRE-1. Arrowheads pointing toward bands indicate hypersensitive residues; arrowheads pointing away from bands indicate protected residues. Larger arrowheads indicate major protections or hypersensitive sites.

    Techniques Used: In Vivo, Footprinting

    17) Product Images from "Control of Sporulation Gene Expression in Bacillus subtilis by the Chromosome Partitioning Proteins Soj (ParA) and Spo0J (ParB)"

    Article Title: Control of Sporulation Gene Expression in Bacillus subtilis by the Chromosome Partitioning Proteins Soj (ParA) and Spo0J (ParB)

    Journal: Journal of Bacteriology

    doi:

    Association of Soj with the spo0A promoter region in vivo. Wild-type (JH642), Δ spo0J (AG1468), and Δ( soj-spo0J ) (AG1505) strains were grown in 2×SG medium at 37°C. Two hours after the end of exponential growth, formaldehyde was added to cross-link the protein and DNA. Protein-DNA complexes were immunoprecipitated by using anti-Soj antibodies. The presence of a given promoter region was assayed by PCR amplification with primers designed to amplify the promoter regions of spo0A , and codV and recA were used as controls. Lanes labeled IP DNA are DNA products from PCR assays performed on a dilution series (4, 2, 1, 0.5, and 0.25 μl) of the immunoprecipitated material. Lanes labeled Total DNA are DNA products from PCR assays performed on a dilution series (the equivalent of 1/250, 1/500, 1/1,000, 1/2,000, and 1/4,000 μl) of sample DNA taken prior to immunoprecipitation.
    Figure Legend Snippet: Association of Soj with the spo0A promoter region in vivo. Wild-type (JH642), Δ spo0J (AG1468), and Δ( soj-spo0J ) (AG1505) strains were grown in 2×SG medium at 37°C. Two hours after the end of exponential growth, formaldehyde was added to cross-link the protein and DNA. Protein-DNA complexes were immunoprecipitated by using anti-Soj antibodies. The presence of a given promoter region was assayed by PCR amplification with primers designed to amplify the promoter regions of spo0A , and codV and recA were used as controls. Lanes labeled IP DNA are DNA products from PCR assays performed on a dilution series (4, 2, 1, 0.5, and 0.25 μl) of the immunoprecipitated material. Lanes labeled Total DNA are DNA products from PCR assays performed on a dilution series (the equivalent of 1/250, 1/500, 1/1,000, 1/2,000, and 1/4,000 μl) of sample DNA taken prior to immunoprecipitation.

    Techniques Used: In Vivo, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Labeling

    18) Product Images from "Genetic basis for retention of a critical virulence plasmid of Borrelia burgdorferi"

    Article Title: Genetic basis for retention of a critical virulence plasmid of Borrelia burgdorferi

    Journal: Molecular Microbiology

    doi: 10.1111/j.1365-2958.2007.05969.x

    A. PCR analysis of genomic DNA from B. burgdorferi clones transformed with gene inactivation constructs targeting genes in the bbb26–27 region. Template DNAs from transformants are identified below the lanes and PCR amplification targets above the lanes. Template DNA from B31-A illustrates the PCR products from the wild-type alleles of bbb26–27 , bbb26 and bbb27 (lanes 2–5, 15 and 19), whereas template DNAs from the gene inactivation constructs (XL-BBB26–27Δ, XL-BBB26Δ and XL-BBB27Δ) depict the PCR profiles of the mutated alleles (lanes 6–9, 16 and 20). The PCR products resulting from the clones transformed with the allelic exchange inactivation plasmids are illustrated in lanes 10–13, 17 and 21. The 1 kbp-plus size standards (Invitrogen) were run in lanes 1, 14 and 18 and sizes (base pairs) are indicated to the left of the panel. B. Graphical representation of the bbb26–27 region on cp26 (B31-A) and the cloned pieces of DNA used for the allelic exchange constructs (XL-BBB26–27Δ, XL-BBB26Δ and XL-BBB27Δ). The 1168 bp region of cp26 between nucleotides 21923 and 23091 was replaced with the 1146 bp flaB p – aadA resistance cassette to create XL-BBB26–27Δ for disruption of both bbb26 and bbb27 . The 742 bp region of cp26 between nucleotides 21923 and 22579 was replaced with the 1100 bp flgB p – aacC1 resistance cassette to create XL-BBB26Δ for disruption of bbb26 . The 438 bp region of cp26 between nucleotides 22653 and 23091 was replaced with the 1100 bp flgB p – aacC1 resistance cassette to create XL-BBB27Δ for disruption of bbb27 . Locations of the primers used for analysis in (A) are indicated and the sequences are listed in Table S1 .
    Figure Legend Snippet: A. PCR analysis of genomic DNA from B. burgdorferi clones transformed with gene inactivation constructs targeting genes in the bbb26–27 region. Template DNAs from transformants are identified below the lanes and PCR amplification targets above the lanes. Template DNA from B31-A illustrates the PCR products from the wild-type alleles of bbb26–27 , bbb26 and bbb27 (lanes 2–5, 15 and 19), whereas template DNAs from the gene inactivation constructs (XL-BBB26–27Δ, XL-BBB26Δ and XL-BBB27Δ) depict the PCR profiles of the mutated alleles (lanes 6–9, 16 and 20). The PCR products resulting from the clones transformed with the allelic exchange inactivation plasmids are illustrated in lanes 10–13, 17 and 21. The 1 kbp-plus size standards (Invitrogen) were run in lanes 1, 14 and 18 and sizes (base pairs) are indicated to the left of the panel. B. Graphical representation of the bbb26–27 region on cp26 (B31-A) and the cloned pieces of DNA used for the allelic exchange constructs (XL-BBB26–27Δ, XL-BBB26Δ and XL-BBB27Δ). The 1168 bp region of cp26 between nucleotides 21923 and 23091 was replaced with the 1146 bp flaB p – aadA resistance cassette to create XL-BBB26–27Δ for disruption of both bbb26 and bbb27 . The 742 bp region of cp26 between nucleotides 21923 and 22579 was replaced with the 1100 bp flgB p – aacC1 resistance cassette to create XL-BBB26Δ for disruption of bbb26 . The 438 bp region of cp26 between nucleotides 22653 and 23091 was replaced with the 1100 bp flgB p – aacC1 resistance cassette to create XL-BBB27Δ for disruption of bbb27 . Locations of the primers used for analysis in (A) are indicated and the sequences are listed in Table S1 .

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Transformation Assay, Construct, Amplification

    19) Product Images from "Retrohoming of a Mobile Group II Intron in Human Cells Suggests How Eukaryotes Limit Group II Intron Proliferation"

    Article Title: Retrohoming of a Mobile Group II Intron in Human Cells Suggests How Eukaryotes Limit Group II Intron Proliferation

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005422

    Selection of Ll.LtrB group II intron for retrohoming within HEK-293 cells at different MgCl 2 concentrations. (A) Diagram of plasmid-based selection for retrohoming in human cells. The three Ll.LtrB expression plasmids, including a derivative of pLl.LtrB in which the expressed intron carries a phage T7 promoter sequence in DIVb, were transfected into HEK-293 cells along with recipient plasmid pBRRQ, which contains the wild-type Ll.LtrB target site cloned upstream of a promoterless tet R gene. After incubating the cells in culture medium supplemented with 80 or 40 mM Mg 2+ for 24 h, plasmids were isolated and electroporated into E . coli HMS174(λDE3), which was then plated on LB-agar containing tetracycline. Plasmids were isolated from scraped E . coli colonies, and introns that had retrohomed into the target site were amplified by PCR using primers that flank the intron and recloned into pLl.LtrB for the next round of selection. (B) Ll.LtrB introns carrying a phage T7 promoter in DIVb have ~70% wild-type retrohoming efficiency in plasmid targeting assays in HEK-293 cells. The bar graphs show retrohoming frequencies assayed by Taqman qPCR of 5’- (blue) or 3’- (red) integration junctions in DNA extracted from adherent HEK-293 cells after 24-h incubation in culture medium supplemented with 80 mM Mg 2+ . Values are the mean for two or three separate transfections on the same day, with the error bars indicating the SEM. (C) The Ll.LtrB intron was evolved for retrohoming into plasmid targets within HEK-293 cells via eight cycles of selection at 80 mM MgCl 2 with addition of three new mutations per kb between each cycle (rounds 1–8). After round 8, intron variants were selected for an additional four cycles in HEK-293 cells in culture medium supplemented 40 mM MgCl 2 without mutagenesis (rounds 9–12) to enrich for variants that enhance retrohoming within HEK-293 cells. The retrohoming frequencies for the wild-type Ll.LtrB intron and libraries for rounds 1 to 12 were assayed in parallel by Taqman qPCR for three separate transfections on the same day. The values plotted are the mean with the error bars indicating the SEM.
    Figure Legend Snippet: Selection of Ll.LtrB group II intron for retrohoming within HEK-293 cells at different MgCl 2 concentrations. (A) Diagram of plasmid-based selection for retrohoming in human cells. The three Ll.LtrB expression plasmids, including a derivative of pLl.LtrB in which the expressed intron carries a phage T7 promoter sequence in DIVb, were transfected into HEK-293 cells along with recipient plasmid pBRRQ, which contains the wild-type Ll.LtrB target site cloned upstream of a promoterless tet R gene. After incubating the cells in culture medium supplemented with 80 or 40 mM Mg 2+ for 24 h, plasmids were isolated and electroporated into E . coli HMS174(λDE3), which was then plated on LB-agar containing tetracycline. Plasmids were isolated from scraped E . coli colonies, and introns that had retrohomed into the target site were amplified by PCR using primers that flank the intron and recloned into pLl.LtrB for the next round of selection. (B) Ll.LtrB introns carrying a phage T7 promoter in DIVb have ~70% wild-type retrohoming efficiency in plasmid targeting assays in HEK-293 cells. The bar graphs show retrohoming frequencies assayed by Taqman qPCR of 5’- (blue) or 3’- (red) integration junctions in DNA extracted from adherent HEK-293 cells after 24-h incubation in culture medium supplemented with 80 mM Mg 2+ . Values are the mean for two or three separate transfections on the same day, with the error bars indicating the SEM. (C) The Ll.LtrB intron was evolved for retrohoming into plasmid targets within HEK-293 cells via eight cycles of selection at 80 mM MgCl 2 with addition of three new mutations per kb between each cycle (rounds 1–8). After round 8, intron variants were selected for an additional four cycles in HEK-293 cells in culture medium supplemented 40 mM MgCl 2 without mutagenesis (rounds 9–12) to enrich for variants that enhance retrohoming within HEK-293 cells. The retrohoming frequencies for the wild-type Ll.LtrB intron and libraries for rounds 1 to 12 were assayed in parallel by Taqman qPCR for three separate transfections on the same day. The values plotted are the mean with the error bars indicating the SEM.

    Techniques Used: Selection, Plasmid Preparation, Expressing, Sequencing, Transfection, Clone Assay, Isolation, Amplification, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Incubation, Mutagenesis

    20) Product Images from "Altered Growth Characteristics of Recombinant Respiratory Syncytial Viruses Which Do Not Produce NS2 Protein"

    Article Title: Altered Growth Characteristics of Recombinant Respiratory Syncytial Viruses Which Do Not Produce NS2 Protein

    Journal: Journal of Virology

    doi:

    Mutation of the NS2 gene to ablate expression of its encoded protein. (a) Site-directed mutagenesis was used to alter codons 21 and 22 in the NS2 ORF of the antigenome cDNA (sequences in positive sense). D51 is a cDNA containing nt 1 to 4623 of the RSV antigenome, which was an intermediate in the engineering (see Materials and Methods). Mutated bases are shown above the wild-type sequence. A novel Xho I restriction site (underlined) was added as a marker for mutants. Restriction enzyme sites used in DNA manipulation are shown (see Materials and Methods). (b) The entire NS2 gene was deleted by PCR, as described in Materials and Methods, such that the NS1 gene end signal was fused to the NS2-N intergenic region. The sequence is shown in positive sense, and the point of deletion is indicated by an open triangle.
    Figure Legend Snippet: Mutation of the NS2 gene to ablate expression of its encoded protein. (a) Site-directed mutagenesis was used to alter codons 21 and 22 in the NS2 ORF of the antigenome cDNA (sequences in positive sense). D51 is a cDNA containing nt 1 to 4623 of the RSV antigenome, which was an intermediate in the engineering (see Materials and Methods). Mutated bases are shown above the wild-type sequence. A novel Xho I restriction site (underlined) was added as a marker for mutants. Restriction enzyme sites used in DNA manipulation are shown (see Materials and Methods). (b) The entire NS2 gene was deleted by PCR, as described in Materials and Methods, such that the NS1 gene end signal was fused to the NS2-N intergenic region. The sequence is shown in positive sense, and the point of deletion is indicated by an open triangle.

    Techniques Used: Mutagenesis, Expressing, Sequencing, Marker, Polymerase Chain Reaction

    21) Product Images from "Sequence analysis of old and new strains of porcine circovirus associated with congenital tremors in pigs and their comparison with strains involved with postweaning multisystemic wasting syndrome"

    Article Title: Sequence analysis of old and new strains of porcine circovirus associated with congenital tremors in pigs and their comparison with strains involved with postweaning multisystemic wasting syndrome

    Journal: Canadian Journal of Veterinary Research

    doi:

    DNA isolation and PCR
    Figure Legend Snippet: DNA isolation and PCR

    Techniques Used: DNA Extraction, Polymerase Chain Reaction

    22) Product Images from "Alternative Excision Repair of Ultraviolet B- and C-Induced DNA Damage in Dormant and Developing Spores of Bacillus subtilis"

    Article Title: Alternative Excision Repair of Ultraviolet B- and C-Induced DNA Damage in Dormant and Developing Spores of Bacillus subtilis

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01340-12

    (A to C) Levels of β-galactosidase from B. subtilis wild-type (A) and Δσ G (B) strains containing a ywjD-lacZ fusion and RT-PCR analysis of ywjD transcription (C). (A and B) B. subtilis strains PERM557 ( ywjD-lacZ ) (A) and PERM755 ( sigGΔ1 ywjD-lacZ ). (C) RNA samples (∼1 μg) isolated from a B. subtilis 168 DSM culture at the times indicated were processed for RT-PCR analysis as described in Materials and Methods. The arrowhead shows the size of the expected RT-PCR products. Lanes: M, DNA markers, 1-kb Plus ladder; Veg, logarithmic growth; T 0 , the time when the slopes of the logarithmic and stationary phases of growth intersected; T 1 to T 9 , times in hours after T 0 .
    Figure Legend Snippet: (A to C) Levels of β-galactosidase from B. subtilis wild-type (A) and Δσ G (B) strains containing a ywjD-lacZ fusion and RT-PCR analysis of ywjD transcription (C). (A and B) B. subtilis strains PERM557 ( ywjD-lacZ ) (A) and PERM755 ( sigGΔ1 ywjD-lacZ ). (C) RNA samples (∼1 μg) isolated from a B. subtilis 168 DSM culture at the times indicated were processed for RT-PCR analysis as described in Materials and Methods. The arrowhead shows the size of the expected RT-PCR products. Lanes: M, DNA markers, 1-kb Plus ladder; Veg, logarithmic growth; T 0 , the time when the slopes of the logarithmic and stationary phases of growth intersected; T 1 to T 9 , times in hours after T 0 .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation

    23) Product Images from "Biochemical Interactions between Proteins and mat1 cis-Acting Sequences Required for Imprinting in Fission Yeast"

    Article Title: Biochemical Interactions between Proteins and mat1 cis-Acting Sequences Required for Imprinting in Fission Yeast

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.22.9813-9822.2004

    Localization of Swi1p and Swi3p to the mat1 locus. (A) A schematic diagram of mat1 and its MPS and RTS1 regions amplified by PCR is shown. Primer sets for PCR (see Materials and Methods) are represented by filled-in arrows and were used to amplify 234-bp and 473-bp fragments of MPS and RTS1 , respectively. (B) Chromatin immunoprecipitation analysis. Related SP976 (untagged swi1 ) and BSP4 ( swi1-myc ) strains were used to determine the localization of Swi1p-myc to the MPS and RTS1 regions. (C) Chromatin immunoprecipitation analysis of related strains SP982 (untagged swi3 ) and BSP16 ( swi3-myc ) was used to determine the localization of Swi3p-myc to the MPS and RTS1 regions. DNA recovered from each chromatin precipitate was analyzed by hot PCR with the indicated primer sets (see panel A) and deoxynucleoside triphosphates, including [α- 32 p]dCTP. Inp (input) denotes DNA extracted from the total cell extract that was subjected to immunoprecipitation experiments. It was loaded after manyfold dilution; −, mock precipitation without antibody; +, precipitation with anti-c-myc antibody. The lys11 and trp5 primer sets (see Materials and Methods) were used to amplify fragments of 345 bp for lys11 and 370 bp for trp5 for nonspecific DNA binding controls in both panels B and C.
    Figure Legend Snippet: Localization of Swi1p and Swi3p to the mat1 locus. (A) A schematic diagram of mat1 and its MPS and RTS1 regions amplified by PCR is shown. Primer sets for PCR (see Materials and Methods) are represented by filled-in arrows and were used to amplify 234-bp and 473-bp fragments of MPS and RTS1 , respectively. (B) Chromatin immunoprecipitation analysis. Related SP976 (untagged swi1 ) and BSP4 ( swi1-myc ) strains were used to determine the localization of Swi1p-myc to the MPS and RTS1 regions. (C) Chromatin immunoprecipitation analysis of related strains SP982 (untagged swi3 ) and BSP16 ( swi3-myc ) was used to determine the localization of Swi3p-myc to the MPS and RTS1 regions. DNA recovered from each chromatin precipitate was analyzed by hot PCR with the indicated primer sets (see panel A) and deoxynucleoside triphosphates, including [α- 32 p]dCTP. Inp (input) denotes DNA extracted from the total cell extract that was subjected to immunoprecipitation experiments. It was loaded after manyfold dilution; −, mock precipitation without antibody; +, precipitation with anti-c-myc antibody. The lys11 and trp5 primer sets (see Materials and Methods) were used to amplify fragments of 345 bp for lys11 and 370 bp for trp5 for nonspecific DNA binding controls in both panels B and C.

    Techniques Used: Amplification, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunoprecipitation, Binding Assay

    24) Product Images from "Identification of a Transcriptional Activator (ChnR) and a 6-Oxohexanoate Dehydrogenase (ChnE) in the Cyclohexanol Catabolic Pathway in Acinetobacter sp. Strain NCIMB 9871 and Localization of the Genes That Encode Them †"

    Article Title: Identification of a Transcriptional Activator (ChnR) and a 6-Oxohexanoate Dehydrogenase (ChnE) in the Cyclohexanol Catabolic Pathway in Acinetobacter sp. Strain NCIMB 9871 and Localization of the Genes That Encode Them †

    Journal: Applied and Environmental Microbiology

    doi:

    Restriction and gene map of the DNA insert in plasmid pCM100 and its derivatives. Abbreviations: B, Bam HI; Sa, Sal I; A, Acc I; Sp, Sph I; N, Nhe I; E, Eco RI. The arrows indicate the locations and directions of transcription of the CHMO-encoding gene ( chnB ), the 6-oxohexanoate dehydrogenase-encoding gene ( orf1 or chnE ), and a regulatory chnR gene. The ability (+) or inability (−) of each plasmid to express ChnB activity is indicated.
    Figure Legend Snippet: Restriction and gene map of the DNA insert in plasmid pCM100 and its derivatives. Abbreviations: B, Bam HI; Sa, Sal I; A, Acc I; Sp, Sph I; N, Nhe I; E, Eco RI. The arrows indicate the locations and directions of transcription of the CHMO-encoding gene ( chnB ), the 6-oxohexanoate dehydrogenase-encoding gene ( orf1 or chnE ), and a regulatory chnR gene. The ability (+) or inability (−) of each plasmid to express ChnB activity is indicated.

    Techniques Used: Plasmid Preparation, Activity Assay

    25) Product Images from "Loss of Albino3 Leads to the Specific Depletion of the Light-Harvesting System"

    Article Title: Loss of Albino3 Leads to the Specific Depletion of the Light-Harvesting System

    Journal: The Plant Cell

    doi: 10.1105/tpc.003442

    DNA Gel Blot Analysis of ac29 Mutants. DNA was isolated from the diploid parent ( nic7 ac29-2 mt − pf14/ ++ mt + +) and the diploid acy9 , acy10 , acy20 , acy25 , and acy32 mutant strains and from the haploid ac29-3 mutant containing the mt + chromosome from acy32 . The DNAs of the blots were digested with BglII (A) or SacI ( [B] and [C] ) and probed with the acB probe ( [A] and [B] ) or the acC probe (C) .
    Figure Legend Snippet: DNA Gel Blot Analysis of ac29 Mutants. DNA was isolated from the diploid parent ( nic7 ac29-2 mt − pf14/ ++ mt + +) and the diploid acy9 , acy10 , acy20 , acy25 , and acy32 mutant strains and from the haploid ac29-3 mutant containing the mt + chromosome from acy32 . The DNAs of the blots were digested with BglII (A) or SacI ( [B] and [C] ) and probed with the acB probe ( [A] and [B] ) or the acC probe (C) .

    Techniques Used: Western Blot, Isolation, Mutagenesis

    26) Product Images from "Spiked Genes: A Method to Introduce Random Point Nucleotide Mutations Evenly throughout an Entire Gene Using a Complete Set of Spiked Oligonucleotides for the Assembly"

    Article Title: Spiked Genes: A Method to Introduce Random Point Nucleotide Mutations Evenly throughout an Entire Gene Using a Complete Set of Spiked Oligonucleotides for the Assembly

    Journal: ACS Omega

    doi: 10.1021/acsomega.7b00508

    Strategy for the assembly of the synthetic gene library. Each continuous arrow represents a spiked oligonucleotide, wherein each position was doped with 0.25% of each of the other three bases. Primer sequences are listed in Table S1 . (A) Scheme of the single-step PCR assembly reaction to synthesize the emKate gene library. (B) PCR reactions, using three different starting concentrations of the internal primers (4, 8, and 16 nM) and outermost primers at 400 nM, analyzed by agarose gel electrophoresis and the GeneRuler 100 bp Plus DNA ladder as the molecular marker.
    Figure Legend Snippet: Strategy for the assembly of the synthetic gene library. Each continuous arrow represents a spiked oligonucleotide, wherein each position was doped with 0.25% of each of the other three bases. Primer sequences are listed in Table S1 . (A) Scheme of the single-step PCR assembly reaction to synthesize the emKate gene library. (B) PCR reactions, using three different starting concentrations of the internal primers (4, 8, and 16 nM) and outermost primers at 400 nM, analyzed by agarose gel electrophoresis and the GeneRuler 100 bp Plus DNA ladder as the molecular marker.

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker

    27) Product Images from "A New F131V Mutation in Chlamydomonas Phytoene Desaturase Locates a Cluster of Norflurazon Resistance Mutations near the FAD-Binding Site in 3D Protein Models"

    Article Title: A New F131V Mutation in Chlamydomonas Phytoene Desaturase Locates a Cluster of Norflurazon Resistance Mutations near the FAD-Binding Site in 3D Protein Models

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0099894

    Nuclear transformation vector and analysis of pNFR1 transformants. ( A ) pNFR1 and pWTPDS1 transformation vectors. The two vectors are distinguished by a single base substitution conferring norflurazon resistance in pNFR1. Shown are coding regions (black boxes), pBluescript vector backbone (thin grey line), extent of cDNA probe used for DNA hybridization and restriction enzyme sites. ( B ) Map of the PDS1 gene (white box) located on chromosome 12 (Chr12). Restriction sites and overlap region of the cDNA probe are shown. ( C ) Blots of digested DNA from pNFR1 transformants and untransformed cw92 strain hybridized with a PDS1 cDNA probe. Restriction enzyme used, band sizes and MW standards are indicated. ( D ) Protein blot of fractionated total cell protein from pNFR1 cw92 transformants, the cc621 WT strain and the non-transformed cw92 strain incubated with an affinity-purified polyclonal antibody raised against a PDS peptide. The PDS band is arrowed.
    Figure Legend Snippet: Nuclear transformation vector and analysis of pNFR1 transformants. ( A ) pNFR1 and pWTPDS1 transformation vectors. The two vectors are distinguished by a single base substitution conferring norflurazon resistance in pNFR1. Shown are coding regions (black boxes), pBluescript vector backbone (thin grey line), extent of cDNA probe used for DNA hybridization and restriction enzyme sites. ( B ) Map of the PDS1 gene (white box) located on chromosome 12 (Chr12). Restriction sites and overlap region of the cDNA probe are shown. ( C ) Blots of digested DNA from pNFR1 transformants and untransformed cw92 strain hybridized with a PDS1 cDNA probe. Restriction enzyme used, band sizes and MW standards are indicated. ( D ) Protein blot of fractionated total cell protein from pNFR1 cw92 transformants, the cc621 WT strain and the non-transformed cw92 strain incubated with an affinity-purified polyclonal antibody raised against a PDS peptide. The PDS band is arrowed.

    Techniques Used: Transformation Assay, Plasmid Preparation, DNA Hybridization, Incubation, Affinity Purification

    28) Product Images from "The Dynamic Organization of the Perinucleolar Compartment in the Cell Nucleus"

    Article Title: The Dynamic Organization of the Perinucleolar Compartment in the Cell Nucleus

    Journal: The Journal of Cell Biology

    doi:

    The PNC dissociates at prophase and reforms at late telophase. The horizontal rows show different stages of mitosis. The left column shows the immunolabeling of the PNC with antibody SH54, the center column the immunolabeling of fibrillarin with human anti-fibrillarin antibody, and the right column DNA staining by Dapi. The dissociation of the PNC at prophase ( D ) appears to be a gradual event. A concentrated PTB labeling is still spatially linked with the partially dissociated nucleolus ( D and E , arrows ). Both PTB ( G ) and fibrillarin ( H ) are diffusely distributed in metaphase cells. The earliest detectable PNCs ( J , arrows ) in the daughter cell nuclei are associated with nucleolar regions ( K , arrows ). Bar, 10 μm.
    Figure Legend Snippet: The PNC dissociates at prophase and reforms at late telophase. The horizontal rows show different stages of mitosis. The left column shows the immunolabeling of the PNC with antibody SH54, the center column the immunolabeling of fibrillarin with human anti-fibrillarin antibody, and the right column DNA staining by Dapi. The dissociation of the PNC at prophase ( D ) appears to be a gradual event. A concentrated PTB labeling is still spatially linked with the partially dissociated nucleolus ( D and E , arrows ). Both PTB ( G ) and fibrillarin ( H ) are diffusely distributed in metaphase cells. The earliest detectable PNCs ( J , arrows ) in the daughter cell nuclei are associated with nucleolar regions ( K , arrows ). Bar, 10 μm.

    Techniques Used: Immunolabeling, Staining, Labeling

    29) Product Images from "PpoR is a conserved unpaired LuxR solo of Pseudomonas putida which binds N-acyl homoserine lactones"

    Article Title: PpoR is a conserved unpaired LuxR solo of Pseudomonas putida which binds N-acyl homoserine lactones

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-9-125

    Alignment showing similarity of deduced sequence of PpoR to its orthologs . Multiple sequence alignment was performed using the ClustalW2 program (Thompson et al. 1994). The protein sequences used for the alignment are as follows; P. putida KT2440 (AAN70220.1), P . putida F1 (ABQ80629.1), P. putida RD8MR3 (this study; accession number FM992078 ), P . putida GB-1 (ABZ00528.1), P. putida WCS358 (this study; accession number FM992077 ) and P . putida W619 (ACA71296.1). The amino acids that are conserved in QS LuxR family proteins are indicated in bold [ 3 ]. In the alignment, all identical amino acids (*), similar amino acids (:) and completely different amino acids (.) at a particular position are indicated. Also indicated are the regions of the protein sequence of PpoR of P. putida KT2440 that constitutes the AHL binding domain (bold line from 17 to 162 amino acids; PFAM 03472) and the DNA binding domain (dashed line from 176 to 213 amino acids; PFAM 00196).
    Figure Legend Snippet: Alignment showing similarity of deduced sequence of PpoR to its orthologs . Multiple sequence alignment was performed using the ClustalW2 program (Thompson et al. 1994). The protein sequences used for the alignment are as follows; P. putida KT2440 (AAN70220.1), P . putida F1 (ABQ80629.1), P. putida RD8MR3 (this study; accession number FM992078 ), P . putida GB-1 (ABZ00528.1), P. putida WCS358 (this study; accession number FM992077 ) and P . putida W619 (ACA71296.1). The amino acids that are conserved in QS LuxR family proteins are indicated in bold [ 3 ]. In the alignment, all identical amino acids (*), similar amino acids (:) and completely different amino acids (.) at a particular position are indicated. Also indicated are the regions of the protein sequence of PpoR of P. putida KT2440 that constitutes the AHL binding domain (bold line from 17 to 162 amino acids; PFAM 03472) and the DNA binding domain (dashed line from 176 to 213 amino acids; PFAM 00196).

    Techniques Used: Sequencing, Binding Assay

    30) Product Images from "Alternative Excision Repair of Ultraviolet B- and C-Induced DNA Damage in Dormant and Developing Spores of Bacillus subtilis"

    Article Title: Alternative Excision Repair of Ultraviolet B- and C-Induced DNA Damage in Dormant and Developing Spores of Bacillus subtilis

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01340-12

    (A to C) Levels of β-galactosidase from B. subtilis wild-type (A) and Δσ G (B) strains containing a ywjD-lacZ fusion and RT-PCR analysis of ywjD transcription (C). (A and B) B. subtilis strains PERM557 ( ywjD-lacZ ) (A) and PERM755 ( sigGΔ1 ywjD-lacZ ). (C) RNA samples (∼1 μg) isolated from a B. subtilis 168 DSM culture at the times indicated were processed for RT-PCR analysis as described in Materials and Methods. The arrowhead shows the size of the expected RT-PCR products. Lanes: M, DNA markers, 1-kb Plus ladder; Veg, logarithmic growth; T 0 , the time when the slopes of the logarithmic and stationary phases of growth intersected; T 1 to T 9 , times in hours after T 0 .
    Figure Legend Snippet: (A to C) Levels of β-galactosidase from B. subtilis wild-type (A) and Δσ G (B) strains containing a ywjD-lacZ fusion and RT-PCR analysis of ywjD transcription (C). (A and B) B. subtilis strains PERM557 ( ywjD-lacZ ) (A) and PERM755 ( sigGΔ1 ywjD-lacZ ). (C) RNA samples (∼1 μg) isolated from a B. subtilis 168 DSM culture at the times indicated were processed for RT-PCR analysis as described in Materials and Methods. The arrowhead shows the size of the expected RT-PCR products. Lanes: M, DNA markers, 1-kb Plus ladder; Veg, logarithmic growth; T 0 , the time when the slopes of the logarithmic and stationary phases of growth intersected; T 1 to T 9 , times in hours after T 0 .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation

    31) Product Images from "The Crystal Structure of the R280K Mutant of Human p53 Explains the Loss of DNA Binding"

    Article Title: The Crystal Structure of the R280K Mutant of Human p53 Explains the Loss of DNA Binding

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19041184

    Comparison of p53R280K DBD (backbone in purple) with ( a ) human DNA-free (2OCJ, blue) and ( b ) DNA-bound (2AC0, gray) wt p53 DBD structures. Regions with the highest structural variations are encircled by gray dashed lines. In ( a ), the S7/S8 turn is the region with the highest structural variation, while in ( b ), the S7/S8 turn, L1 loop, and L2 loop are the regions with the most significant structural differences. The L2 loop is located at the back in this figure. ( c ) Backbone superposition of p53R280K DBD (purple) with the p53 DBD in complex with DNA (2AC0, gray). The double-stranded DNA is represented in cyan blue. The side chains at position 280 are shown in both structures, lysine from mutant p53 and arginine from wt p53. The hydrogen contacts between the R280 and guanine, and a hypothetical contact between K280 and guanine, are represented by gray dashed lines; carbon atoms are colored light blue, nitrogens are navy blue and oxygens are red. Atomic distances are indicated.
    Figure Legend Snippet: Comparison of p53R280K DBD (backbone in purple) with ( a ) human DNA-free (2OCJ, blue) and ( b ) DNA-bound (2AC0, gray) wt p53 DBD structures. Regions with the highest structural variations are encircled by gray dashed lines. In ( a ), the S7/S8 turn is the region with the highest structural variation, while in ( b ), the S7/S8 turn, L1 loop, and L2 loop are the regions with the most significant structural differences. The L2 loop is located at the back in this figure. ( c ) Backbone superposition of p53R280K DBD (purple) with the p53 DBD in complex with DNA (2AC0, gray). The double-stranded DNA is represented in cyan blue. The side chains at position 280 are shown in both structures, lysine from mutant p53 and arginine from wt p53. The hydrogen contacts between the R280 and guanine, and a hypothetical contact between K280 and guanine, are represented by gray dashed lines; carbon atoms are colored light blue, nitrogens are navy blue and oxygens are red. Atomic distances are indicated.

    Techniques Used: Mutagenesis

    Crystal structure of p53R280K DBD. ( a ) Ribbon diagram of p53R280K DBD; strands are shown in dark blue, helices in purple, linked by gray coils. The zinc(II) ion is represented by a gray sphere near L3 loop and H1 helix and its coordinating amino acid residues (C176, H179, C238, C242) are shown as yellow sticks. ( b ) Backbone superposition of the four molecules of p53R280K DBD in the asymmetric unit of the crystals. Molecule A, purple; molecule B, yellow; molecule C, blue; molecule D, orange. Encircled by a gray dashed line is the region with the highest structural variation, the S7/S8 turn. ( c ) Electron density map calculated around the C-terminus residues and the zinc(II) ion, in green (2mF o -DF c map at 1σ level and 2 Å resolution). The p53R280K DBD polypeptide chain is represented in blue ribbon. Clear electron density for the lysine residue at position 280 is seen in all molecules of the asymmetric unit in the p53R280K DBD domain structure. The DNA fragment and the arginine residue of the wt form (PDB code 2AC0) are superposed to illustrate the orientation of the protein in relation to the DNA and are depicted in color-coded wire-frame and labeled in italic. The two direct hydrogen contacts with guanine, that are disrupted in the p53R280K structure, are depicted as pink thin lines. Residues from p53R280K are labeled in bold, while residues in the wt complex are labeled in italics.
    Figure Legend Snippet: Crystal structure of p53R280K DBD. ( a ) Ribbon diagram of p53R280K DBD; strands are shown in dark blue, helices in purple, linked by gray coils. The zinc(II) ion is represented by a gray sphere near L3 loop and H1 helix and its coordinating amino acid residues (C176, H179, C238, C242) are shown as yellow sticks. ( b ) Backbone superposition of the four molecules of p53R280K DBD in the asymmetric unit of the crystals. Molecule A, purple; molecule B, yellow; molecule C, blue; molecule D, orange. Encircled by a gray dashed line is the region with the highest structural variation, the S7/S8 turn. ( c ) Electron density map calculated around the C-terminus residues and the zinc(II) ion, in green (2mF o -DF c map at 1σ level and 2 Å resolution). The p53R280K DBD polypeptide chain is represented in blue ribbon. Clear electron density for the lysine residue at position 280 is seen in all molecules of the asymmetric unit in the p53R280K DBD domain structure. The DNA fragment and the arginine residue of the wt form (PDB code 2AC0) are superposed to illustrate the orientation of the protein in relation to the DNA and are depicted in color-coded wire-frame and labeled in italic. The two direct hydrogen contacts with guanine, that are disrupted in the p53R280K structure, are depicted as pink thin lines. Residues from p53R280K are labeled in bold, while residues in the wt complex are labeled in italics.

    Techniques Used: Labeling

    32) Product Images from "STING Recognition of Cytoplasmic DNA Instigates Cellular Defense"

    Article Title: STING Recognition of Cytoplasmic DNA Instigates Cellular Defense

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2013.01.039

    (A-C) Sting +/+ or Sting -/- MEFs were treated with poly(I:C), dsDNA90, HSV DNA 120mer, CMV DNA 120mer or ADV DNA 120mer for 3 hours. Total RNA was purified and examined by real time PCR for gene expression of IFNβ ( A ), CCL5( B ) or TNFα( C ). Error bars indicate s.d. (D-E) 293T cells were transfected with indicated plasmids. Cell lysates were precipitated with biotin-dsDNA90, biotin-ADV DNA 120mer, biotin-CMV DNA 120mer ( D ) or biotin-HSV DNA 120mer ( E ) agarose beads and analyzed by immunoblotting using anti-HA antibody. (F-H) In vitro translation products were incubated with biotin-dsDNA90 agarose beads and analyzed by immunoblotting using anti-HA antibody. (I) In vitro translation products were incubated with biotin-ssDNA90 agarose beads and analyzed by immunoblotting using anti-HA antibody.
    Figure Legend Snippet: (A-C) Sting +/+ or Sting -/- MEFs were treated with poly(I:C), dsDNA90, HSV DNA 120mer, CMV DNA 120mer or ADV DNA 120mer for 3 hours. Total RNA was purified and examined by real time PCR for gene expression of IFNβ ( A ), CCL5( B ) or TNFα( C ). Error bars indicate s.d. (D-E) 293T cells were transfected with indicated plasmids. Cell lysates were precipitated with biotin-dsDNA90, biotin-ADV DNA 120mer, biotin-CMV DNA 120mer ( D ) or biotin-HSV DNA 120mer ( E ) agarose beads and analyzed by immunoblotting using anti-HA antibody. (F-H) In vitro translation products were incubated with biotin-dsDNA90 agarose beads and analyzed by immunoblotting using anti-HA antibody. (I) In vitro translation products were incubated with biotin-ssDNA90 agarose beads and analyzed by immunoblotting using anti-HA antibody.

    Techniques Used: Purification, Real-time Polymerase Chain Reaction, Expressing, Transfection, In Vitro, Incubation

    33) Product Images from "Interaction of Staufen1 with the 5? end of mRNA facilitates translation of these RNAs"

    Article Title: Interaction of Staufen1 with the 5? end of mRNA facilitates translation of these RNAs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki794

    Binding of Stau1 55 to the 5′ end increases translation of structure-repressed transcripts. (A) Schematic representation of 5′-structure-repressed transcripts. RNAs coding for the R luc reporter protein are shown with one copy of the SBS or two copies of the MS2-binding site (MS2bs) at the 5′ end. (B) HEK293T cells were co-transfected with plasmids expressing either R luc or SBS- R luc transcripts and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Resulting luciferase activity was quantified 24 h post-transfection. In the absence of Stau1 55 -HA 3 , a 100-fold repression of translation of the SBS- R luc RNA was observed as compared with translation of R luc RNA. Results are expressed as luciferase activity versus concentration of the Stau1 55 -HA 3 coding plasmid. To facilitate comparison, the luciferase activity in the absence of Stau1 55 -HA 3 was defined as 1. P ≤ 0.01, n = 3. Black bars, SBS- R luc RNA; hatched bars, R luc RNA. (C) HEK293T cells were co-transfected with plasmids expressing the SBS- R luc transcript and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Twenty-four hours post-transfection, RNA was isolated, reverse transcribed and PCR amplified. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (−RT). RNA coding for GAPDH was RT–PCR and used to normalize the results. (D) HEK293T cells were co-transfected with plasmids expressing the MS2bs- R luc transcript and different concentrations of plasmids coding for either MS2-Stau1 55 -HA 3 , MS2-HA or Stau1 55 -HA 3 . Resulting luciferase activity was quantified 24 h post-transfection. In the absence of MS2-Stau1 55 -HA 3 , a 100-fold repression of translation of the MS2bs- R luc RNA was observed as compared with translation of R luc RNA. To facilitate comparison, the luciferase activity in the absence of expressor plasmids was defined as 1, n = 3.
    Figure Legend Snippet: Binding of Stau1 55 to the 5′ end increases translation of structure-repressed transcripts. (A) Schematic representation of 5′-structure-repressed transcripts. RNAs coding for the R luc reporter protein are shown with one copy of the SBS or two copies of the MS2-binding site (MS2bs) at the 5′ end. (B) HEK293T cells were co-transfected with plasmids expressing either R luc or SBS- R luc transcripts and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Resulting luciferase activity was quantified 24 h post-transfection. In the absence of Stau1 55 -HA 3 , a 100-fold repression of translation of the SBS- R luc RNA was observed as compared with translation of R luc RNA. Results are expressed as luciferase activity versus concentration of the Stau1 55 -HA 3 coding plasmid. To facilitate comparison, the luciferase activity in the absence of Stau1 55 -HA 3 was defined as 1. P ≤ 0.01, n = 3. Black bars, SBS- R luc RNA; hatched bars, R luc RNA. (C) HEK293T cells were co-transfected with plasmids expressing the SBS- R luc transcript and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Twenty-four hours post-transfection, RNA was isolated, reverse transcribed and PCR amplified. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (−RT). RNA coding for GAPDH was RT–PCR and used to normalize the results. (D) HEK293T cells were co-transfected with plasmids expressing the MS2bs- R luc transcript and different concentrations of plasmids coding for either MS2-Stau1 55 -HA 3 , MS2-HA or Stau1 55 -HA 3 . Resulting luciferase activity was quantified 24 h post-transfection. In the absence of MS2-Stau1 55 -HA 3 , a 100-fold repression of translation of the MS2bs- R luc RNA was observed as compared with translation of R luc RNA. To facilitate comparison, the luciferase activity in the absence of expressor plasmids was defined as 1, n = 3.

    Techniques Used: Binding Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Concentration Assay, Isolation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    Stau1 55 mediated translational up-regulation does not involved RNA modification. (A) TAR-CAT RNA was incubated in RRL in the presence of 400 nM of bacterially expressed and purified Stau1 55 Δ2-his 6 or BSA for increasing periods of time. TAR-CAT RNA was then reverse transcribed and PCR amplified for 14 cycles to stay in the non-saturated part of the amplification curve. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (right panel). (B) HEK293T cells were co-transfected with plasmids expressing either R luc or TAR- R luc transcripts and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Twenty-four hours post-transfection, RNA was isolated, reverse transcribed and PCR amplified. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (−RT). RNA coding for GAPDH was RT–PCR and used to normalize the results. (C) Bacterially expressed and column-purified Stau1 55 Δ2-his 6 (Stau) and La-his 6 (La) (left panel) were incubated with [ 32 P]labelled double-stranded RNA in the presence of different combinations of ribonucleotides (right panel). RNA was resolved on agarose gel and revealed by autoradiography. While La-his 6 displayed an helicase activity, Stau1 55 Δ2-his 6 was inactive in this assay.
    Figure Legend Snippet: Stau1 55 mediated translational up-regulation does not involved RNA modification. (A) TAR-CAT RNA was incubated in RRL in the presence of 400 nM of bacterially expressed and purified Stau1 55 Δ2-his 6 or BSA for increasing periods of time. TAR-CAT RNA was then reverse transcribed and PCR amplified for 14 cycles to stay in the non-saturated part of the amplification curve. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (right panel). (B) HEK293T cells were co-transfected with plasmids expressing either R luc or TAR- R luc transcripts and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Twenty-four hours post-transfection, RNA was isolated, reverse transcribed and PCR amplified. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (−RT). RNA coding for GAPDH was RT–PCR and used to normalize the results. (C) Bacterially expressed and column-purified Stau1 55 Δ2-his 6 (Stau) and La-his 6 (La) (left panel) were incubated with [ 32 P]labelled double-stranded RNA in the presence of different combinations of ribonucleotides (right panel). RNA was resolved on agarose gel and revealed by autoradiography. While La-his 6 displayed an helicase activity, Stau1 55 Δ2-his 6 was inactive in this assay.

    Techniques Used: Modification, Incubation, Purification, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Transfection, Expressing, Plasmid Preparation, Isolation, Reverse Transcription Polymerase Chain Reaction, Autoradiography, Activity Assay

    34) Product Images from "Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution"

    Article Title: Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni116

    Analysis of the NExT DNA shuffling technology. ( a ) 1% agarose gel showing the uracil-PCR products of CAT_Nd10 clones obtained with different amounts of uridine in the reactions. For the PCR program, an extended elongation time of 2 min was chosen based on a test series showing that the yield was significantly improved compared to shorter times (data not shown). %U was calculated by c(dUTP)/[c(dUTP) + c(dTTP)] × 100. ( b ) Polyacrylamide urea gel stained with ethidium bromide showing UDG/piperidine digests of CAT_Nd10 PCR products obtained with various dUTP:dTTP ratios (1:0, 0:1, 1:1, 1:2, 1:3, 1:4, 1:5) to determine an optimal ratio. Digests between 1 and 3 h yielded equivalent results, indicating a selective and consistent reaction. From left to right: lane 1, oligonucleotides with 58, 48 and 36 bases as size marker; lane 2, 100% dUTP PCR digested; lane 3, 0% dUTP digested; lane 4, 0% dUTP undigested; lane 5, 50% dUTP digested; lane 6, 33.3% dUTP digested; lane 7, 25% dUTP digested; lane 8, 20% dUTP digested; lane 9, 16.7% dUTP digested; lane 10, 100 bp DNA ladder. Note that residual amounts of piperidine contribute to slightly distorted lanes. ( c ) 1% agarose gel of CAT_Nd10_Cd9 gene fragment libraries from DNA containing 33.3% U showing the reassembly process with Vent DNA polymerase and the amplification of reassembled genes with Taq polymerase. Lane 1, fragments without reassembly PCR; lane 2, fragments after 16 cycles of reassembly; lane 3, fragments after 26 cycles of reassembly; lane 4, fragments after 36 cycles of reassembly; lane 5, 100 bp DNA ladder; lane 6, amplification PCR of fragments without reassembly; lane 7, amplification PCR of fragments subjected to 16 reassembly cycles; lane 8, amplification PCR of fragments subjected to 26 reassembly cycles; lane 9, amplification PCR of fragments subjected to 36 reassembly cycles. ( d ) Polyacrylamide urea gel with UDG/T4 endonuclease V digests of CAT wild-type PCR products containing various dUTP:dTTP ratios to analyze enzymatic fragmentation. Lanes 1–3, oligonucleotides with 68, 48 and 36 bases; lanes 4–10, digests of PCR products obtained with 100%, 0%, 50%, 33.3%, 25%, 20% and 16.7% dUTP; lanes 11–12, PCR products without digest obtained with 100% and 0% dUTP; lane 13, pBR322/HpaII DNA marker. Note that the migration behavior of DNA without uracil incorporation is influenced by the digestion with UDG/piperidine or UDG/T4 endonuclease V. A small fraction of the cleavage might be attributed to this treatment.
    Figure Legend Snippet: Analysis of the NExT DNA shuffling technology. ( a ) 1% agarose gel showing the uracil-PCR products of CAT_Nd10 clones obtained with different amounts of uridine in the reactions. For the PCR program, an extended elongation time of 2 min was chosen based on a test series showing that the yield was significantly improved compared to shorter times (data not shown). %U was calculated by c(dUTP)/[c(dUTP) + c(dTTP)] × 100. ( b ) Polyacrylamide urea gel stained with ethidium bromide showing UDG/piperidine digests of CAT_Nd10 PCR products obtained with various dUTP:dTTP ratios (1:0, 0:1, 1:1, 1:2, 1:3, 1:4, 1:5) to determine an optimal ratio. Digests between 1 and 3 h yielded equivalent results, indicating a selective and consistent reaction. From left to right: lane 1, oligonucleotides with 58, 48 and 36 bases as size marker; lane 2, 100% dUTP PCR digested; lane 3, 0% dUTP digested; lane 4, 0% dUTP undigested; lane 5, 50% dUTP digested; lane 6, 33.3% dUTP digested; lane 7, 25% dUTP digested; lane 8, 20% dUTP digested; lane 9, 16.7% dUTP digested; lane 10, 100 bp DNA ladder. Note that residual amounts of piperidine contribute to slightly distorted lanes. ( c ) 1% agarose gel of CAT_Nd10_Cd9 gene fragment libraries from DNA containing 33.3% U showing the reassembly process with Vent DNA polymerase and the amplification of reassembled genes with Taq polymerase. Lane 1, fragments without reassembly PCR; lane 2, fragments after 16 cycles of reassembly; lane 3, fragments after 26 cycles of reassembly; lane 4, fragments after 36 cycles of reassembly; lane 5, 100 bp DNA ladder; lane 6, amplification PCR of fragments without reassembly; lane 7, amplification PCR of fragments subjected to 16 reassembly cycles; lane 8, amplification PCR of fragments subjected to 26 reassembly cycles; lane 9, amplification PCR of fragments subjected to 36 reassembly cycles. ( d ) Polyacrylamide urea gel with UDG/T4 endonuclease V digests of CAT wild-type PCR products containing various dUTP:dTTP ratios to analyze enzymatic fragmentation. Lanes 1–3, oligonucleotides with 68, 48 and 36 bases; lanes 4–10, digests of PCR products obtained with 100%, 0%, 50%, 33.3%, 25%, 20% and 16.7% dUTP; lanes 11–12, PCR products without digest obtained with 100% and 0% dUTP; lane 13, pBR322/HpaII DNA marker. Note that the migration behavior of DNA without uracil incorporation is influenced by the digestion with UDG/piperidine or UDG/T4 endonuclease V. A small fraction of the cleavage might be attributed to this treatment.

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Clone Assay, Staining, Marker, Amplification, Migration

    35) Product Images from "Molecular and Functional Analyses of the Fast Skeletal Myosin Light Chain2 Gene of the Korean Oily Bitterling, Acheilognathus koreensis"

    Article Title: Molecular and Functional Analyses of the Fast Skeletal Myosin Light Chain2 Gene of the Korean Oily Bitterling, Acheilognathus koreensis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms140816672

    Analysis of DsRed reporter activity of the Akmlc2f promoter in the zebrafish embryo. The fluorescent protein reporter of the Akmlc2f promoter, pDsRed2-1/Akmlc2f P2657 was digested with the restriction endonuclease Apa LI and then the DNA solution at concentrations of 25 ng/mL was injected into the one-cell stage embryos using an air-pressure microinjector (WPI). Digital images of embryo (6 days post hatching) were captured using a macro zoom fluorescence microscope (Olympus, Tokyo, Japan). Scale bar = 200 μm
    Figure Legend Snippet: Analysis of DsRed reporter activity of the Akmlc2f promoter in the zebrafish embryo. The fluorescent protein reporter of the Akmlc2f promoter, pDsRed2-1/Akmlc2f P2657 was digested with the restriction endonuclease Apa LI and then the DNA solution at concentrations of 25 ng/mL was injected into the one-cell stage embryos using an air-pressure microinjector (WPI). Digital images of embryo (6 days post hatching) were captured using a macro zoom fluorescence microscope (Olympus, Tokyo, Japan). Scale bar = 200 μm

    Techniques Used: Activity Assay, Injection, Fluorescence, Microscopy

    36) Product Images from "The Transposon-Like Correia Elements Encode Numerous Strong Promoters and Provide a Potential New Mechanism for Phase Variation in the Meningococcus"

    Article Title: The Transposon-Like Correia Elements Encode Numerous Strong Promoters and Provide a Potential New Mechanism for Phase Variation in the Meningococcus

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1001277

    Transcriptional analysis of three loci containing CEs. (A) Schematic diagram of the region between the NMA0073 and NMA0074 ORFs showing the location of primers I, II and III (sequences given in Table S2 ) used in the accompanying PCR analysis. The solid black arrows denote genes. The hatched box represents a strong predicted transcriptional terminator, with the arrow(s) indicating its polarity: in this case, the terminator is predicted to function in both directions. The CE inverted repeats are indicated by grey arrowheads and the direction of transcription from the α-right Y128T repeat is indicated by a bent arrow. Primer I was used for reverse transcription (RT). The subsequent PCR step was performed with the indicated pairs of primers and analyzed on an ethidium bromide-stained 3% Metaphor agarose TAE gel (right panels). Results from the RT-PCR are shown in the middle panel and those from the genomic DNA PCR are shown on the far right. The latter provides molecular weight standards for the RT-PCR products and a control for the efficiency of the various primer pairs. (B) The region between NMA0057 and NMA0059 is shown. Annotations are as in (A). The inverted repeats of an ATR element are depicted as black arrowheads. (C) The region between NMA0530 and NMA0531 is shown. The short NMA0530A ORF is not likely to code for a protein and has been omitted from the schematic. Annotations are as in (A).
    Figure Legend Snippet: Transcriptional analysis of three loci containing CEs. (A) Schematic diagram of the region between the NMA0073 and NMA0074 ORFs showing the location of primers I, II and III (sequences given in Table S2 ) used in the accompanying PCR analysis. The solid black arrows denote genes. The hatched box represents a strong predicted transcriptional terminator, with the arrow(s) indicating its polarity: in this case, the terminator is predicted to function in both directions. The CE inverted repeats are indicated by grey arrowheads and the direction of transcription from the α-right Y128T repeat is indicated by a bent arrow. Primer I was used for reverse transcription (RT). The subsequent PCR step was performed with the indicated pairs of primers and analyzed on an ethidium bromide-stained 3% Metaphor agarose TAE gel (right panels). Results from the RT-PCR are shown in the middle panel and those from the genomic DNA PCR are shown on the far right. The latter provides molecular weight standards for the RT-PCR products and a control for the efficiency of the various primer pairs. (B) The region between NMA0057 and NMA0059 is shown. Annotations are as in (A). The inverted repeats of an ATR element are depicted as black arrowheads. (C) The region between NMA0530 and NMA0531 is shown. The short NMA0530A ORF is not likely to code for a protein and has been omitted from the schematic. Annotations are as in (A).

    Techniques Used: Polymerase Chain Reaction, Staining, Reverse Transcription Polymerase Chain Reaction, Molecular Weight

    37) Product Images from "Development of a Genetic System for the Chemolithoautotrophic Bacterium Thiobacillus denitrificans ▿"

    Article Title: Development of a Genetic System for the Chemolithoautotrophic Bacterium Thiobacillus denitrificans ▿

    Journal:

    doi: 10.1128/AEM.02928-06

    (A) Electropherogram of PCR products from wild-type (WT) T. denitrificans , the hynL mutant, and the complemented (Compl.) hynL mutant, as well as digested plasmid DNA from the complemented mutant. Lane 1, HyperLadder III, Bioline; lane 2, wild-type DNA,
    Figure Legend Snippet: (A) Electropherogram of PCR products from wild-type (WT) T. denitrificans , the hynL mutant, and the complemented (Compl.) hynL mutant, as well as digested plasmid DNA from the complemented mutant. Lane 1, HyperLadder III, Bioline; lane 2, wild-type DNA,

    Techniques Used: Polymerase Chain Reaction, Mutagenesis, Plasmid Preparation

    38) Product Images from "Alternative Excision Repair of Ultraviolet B- and C-Induced DNA Damage in Dormant and Developing Spores of Bacillus subtilis"

    Article Title: Alternative Excision Repair of Ultraviolet B- and C-Induced DNA Damage in Dormant and Developing Spores of Bacillus subtilis

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01340-12

    (A to C) Levels of β-galactosidase from B. subtilis wild-type (A) and Δσ G (B) strains containing a ywjD-lacZ fusion and RT-PCR analysis of ywjD transcription (C). (A and B) B. subtilis strains PERM557 ( ywjD-lacZ ) (A) and PERM755 ( sigGΔ1 ywjD-lacZ ). (C) RNA samples (∼1 μg) isolated from a B. subtilis 168 DSM culture at the times indicated were processed for RT-PCR analysis as described in Materials and Methods. The arrowhead shows the size of the expected RT-PCR products. Lanes: M, DNA markers, 1-kb Plus ladder; Veg, logarithmic growth; T 0 , the time when the slopes of the logarithmic and stationary phases of growth intersected; T 1 to T 9 , times in hours after T 0 .
    Figure Legend Snippet: (A to C) Levels of β-galactosidase from B. subtilis wild-type (A) and Δσ G (B) strains containing a ywjD-lacZ fusion and RT-PCR analysis of ywjD transcription (C). (A and B) B. subtilis strains PERM557 ( ywjD-lacZ ) (A) and PERM755 ( sigGΔ1 ywjD-lacZ ). (C) RNA samples (∼1 μg) isolated from a B. subtilis 168 DSM culture at the times indicated were processed for RT-PCR analysis as described in Materials and Methods. The arrowhead shows the size of the expected RT-PCR products. Lanes: M, DNA markers, 1-kb Plus ladder; Veg, logarithmic growth; T 0 , the time when the slopes of the logarithmic and stationary phases of growth intersected; T 1 to T 9 , times in hours after T 0 .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation

    39) Product Images from "Expression of the Multidrug Resistance Transporter NorA from Staphylococcus aureus Is Modified by a Two-Component Regulatory System"

    Article Title: Expression of the Multidrug Resistance Transporter NorA from Staphylococcus aureus Is Modified by a Two-Component Regulatory System

    Journal: Journal of Bacteriology

    doi:

    Gel mobility shift analysis of the interaction of protein extracts from the wild-type strain ISP794 with different fragments of the norA promoter and the effect of unlabeled DNA. The radiolabeled fragment (arrow) was incubated with increasing amounts of protein extracts. The labeled fragments used in these experiments are L4-R1 (315 bp) (A), L2-R2 (153 bp) (B), L2-R3 (87 bp) (C), L1-R2 (60 bp) (D), and L5-R2 (39 bp) (E). The protein(s) binds to the tested fragment and retards its mobility (a different gel was used for each fragment). An unlabeled fragment of 350 bp amplified by PCR from a Klebsiella oxytoca promoter and an unlabeled fragment of the tested fragment serve as specificity control (NSPE DNA and SPE DNA, respectively). Protein and DNA concentrations and ratios of unlabeled fragments to labeled fragments used in this assay are indicated in the tables above the figures.
    Figure Legend Snippet: Gel mobility shift analysis of the interaction of protein extracts from the wild-type strain ISP794 with different fragments of the norA promoter and the effect of unlabeled DNA. The radiolabeled fragment (arrow) was incubated with increasing amounts of protein extracts. The labeled fragments used in these experiments are L4-R1 (315 bp) (A), L2-R2 (153 bp) (B), L2-R3 (87 bp) (C), L1-R2 (60 bp) (D), and L5-R2 (39 bp) (E). The protein(s) binds to the tested fragment and retards its mobility (a different gel was used for each fragment). An unlabeled fragment of 350 bp amplified by PCR from a Klebsiella oxytoca promoter and an unlabeled fragment of the tested fragment serve as specificity control (NSPE DNA and SPE DNA, respectively). Protein and DNA concentrations and ratios of unlabeled fragments to labeled fragments used in this assay are indicated in the tables above the figures.

    Techniques Used: Mobility Shift, Incubation, Labeling, Amplification, Polymerase Chain Reaction

    Isolation of the protein from the wild-type strain ISP794 binding to different fragments of the norA promoter. Different fragments of DNA were immobilized on magnetic beads. Proteins binding to these fragments were then used for different analyses. (A) SDS-PAGE analysis of protein released from DNA affinity magnetic beads. Lane 1, standard proteins (in kilodaltons); lane 2, fragment L2-R2; lane 3, fragment L1-R2; lane 4, fragment L2-R3. The 18-kDa protein is indicated by an arrow on the left. (B) Gel mobility shift analysis of fragment L2-R2 with affinity-purified extracts from strain ISP794. Lane 1, control DNA without protein; lane 2, purified protein; lane 3, 0.5 μg of protein from crude extracts of ISP794. Free DNA is indicated by an arrow.
    Figure Legend Snippet: Isolation of the protein from the wild-type strain ISP794 binding to different fragments of the norA promoter. Different fragments of DNA were immobilized on magnetic beads. Proteins binding to these fragments were then used for different analyses. (A) SDS-PAGE analysis of protein released from DNA affinity magnetic beads. Lane 1, standard proteins (in kilodaltons); lane 2, fragment L2-R2; lane 3, fragment L1-R2; lane 4, fragment L2-R3. The 18-kDa protein is indicated by an arrow on the left. (B) Gel mobility shift analysis of fragment L2-R2 with affinity-purified extracts from strain ISP794. Lane 1, control DNA without protein; lane 2, purified protein; lane 3, 0.5 μg of protein from crude extracts of ISP794. Free DNA is indicated by an arrow.

    Techniques Used: Isolation, Binding Assay, Magnetic Beads, SDS Page, Mobility Shift, Affinity Purification, Purification

    Isolation of the protein from the mutant BF15 binding to the fragment L2-R2. (A) SDS-PAGE analysis of protein released from affinity-purified extracts from different strains. Lane 1, standard proteins (in kilodaltons); lane 2, purified protein from ISP794; lane 3, purified protein from BF15. The 18-kDa protein is indicated by an arrow on the left. (B) Gel mobility shift analysis of fragment L2-R2 with affinity-purified protein extracts from BF15 and fragment L2-R2. Lane 1, control DNA without protein; lane 2, purified protein; lane 3, 0.5 μg of protein from crude extracts of BF15. Free DNA is indicated by an arrow.
    Figure Legend Snippet: Isolation of the protein from the mutant BF15 binding to the fragment L2-R2. (A) SDS-PAGE analysis of protein released from affinity-purified extracts from different strains. Lane 1, standard proteins (in kilodaltons); lane 2, purified protein from ISP794; lane 3, purified protein from BF15. The 18-kDa protein is indicated by an arrow on the left. (B) Gel mobility shift analysis of fragment L2-R2 with affinity-purified protein extracts from BF15 and fragment L2-R2. Lane 1, control DNA without protein; lane 2, purified protein; lane 3, 0.5 μg of protein from crude extracts of BF15. Free DNA is indicated by an arrow.

    Techniques Used: Isolation, Mutagenesis, Binding Assay, SDS Page, Affinity Purification, Purification, Mobility Shift

    40) Product Images from "The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3"

    Article Title: The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3

    Journal: Genes & Development

    doi: 10.1101/gad.566910

    DAXX favors deposition of H3.3 in vitro. ( A ) Purification and reconstitution of recombinant DAXX/histone complex. Histones H3.1–H4 (lane 1 ) or H3.3–H4 (lane 2 ) and full-length DAXX were expressed in bacteria and mixed at equimolar ratio (lanes 3 , 4 ). ( B ) DAXX facilitates the deposition of (H3.3–H4) 2 tetramers on DNA. Negatively supercoiled DNA corresponding to topoisomer −1 was incubated with increasing amounts of (H3.3–H4) 2 tetramers (at the indicated histone/DNA ratio, rw) either in the presence (lanes 6 – 8 ) or the absence (lanes 3 – 5 ) of equimolar (to the tetramers) amounts of GST-DAXX. The reaction products were then analyzed on native 4.5% polyacrylamide gel. (Lane 1 ) Topoisomer −1 DNA. (Lane 2 ) (H3.3–H4) 2 tetrasomes reconstituted on topoisomer −1 by salt dialysis. Positions of the open circular DNA (OC), the naked topoisomer −1 DNA, and the (H3.3–H4) 2 tetrasome are indicated. ( C ) DAXX deposits more efficiently (H3.3–H4) 2 than (H3.1–H4) 2 tetramers. Topoisomer −1 was incubated with increasing amounts (at the indicated histone/DNA ratio, rw) of (H3.1–H4) 2 (lanes 3 – 5 ) or (H3.3–H4) 2 (lanes 6 – 8 ) tetramers in the presence of equimolar (to the tetramers) amounts of GST-DAXX. The reaction products were then analyzed on native 4.5% polyacrylamide gel. (Lane 1 ) Topoisomer −1 DNA. (Lane 2 ) (H3.3–H4) 2 tetrasomes reconstituted on topoisomer −1 by salt dialysis. Positions of the open circular DNA (OC), the naked topoisomer −1 DNA, and the (H3.3–H4) 2 tetrasome are indicated.
    Figure Legend Snippet: DAXX favors deposition of H3.3 in vitro. ( A ) Purification and reconstitution of recombinant DAXX/histone complex. Histones H3.1–H4 (lane 1 ) or H3.3–H4 (lane 2 ) and full-length DAXX were expressed in bacteria and mixed at equimolar ratio (lanes 3 , 4 ). ( B ) DAXX facilitates the deposition of (H3.3–H4) 2 tetramers on DNA. Negatively supercoiled DNA corresponding to topoisomer −1 was incubated with increasing amounts of (H3.3–H4) 2 tetramers (at the indicated histone/DNA ratio, rw) either in the presence (lanes 6 – 8 ) or the absence (lanes 3 – 5 ) of equimolar (to the tetramers) amounts of GST-DAXX. The reaction products were then analyzed on native 4.5% polyacrylamide gel. (Lane 1 ) Topoisomer −1 DNA. (Lane 2 ) (H3.3–H4) 2 tetrasomes reconstituted on topoisomer −1 by salt dialysis. Positions of the open circular DNA (OC), the naked topoisomer −1 DNA, and the (H3.3–H4) 2 tetrasome are indicated. ( C ) DAXX deposits more efficiently (H3.3–H4) 2 than (H3.1–H4) 2 tetramers. Topoisomer −1 was incubated with increasing amounts (at the indicated histone/DNA ratio, rw) of (H3.1–H4) 2 (lanes 3 – 5 ) or (H3.3–H4) 2 (lanes 6 – 8 ) tetramers in the presence of equimolar (to the tetramers) amounts of GST-DAXX. The reaction products were then analyzed on native 4.5% polyacrylamide gel. (Lane 1 ) Topoisomer −1 DNA. (Lane 2 ) (H3.3–H4) 2 tetrasomes reconstituted on topoisomer −1 by salt dialysis. Positions of the open circular DNA (OC), the naked topoisomer −1 DNA, and the (H3.3–H4) 2 tetrasome are indicated.

    Techniques Used: In Vitro, Purification, Recombinant, Incubation

    DAXX targets H3.3 to PML-NBs. Resting DAXX −/− MEF cells were transiently transfected with GFP-tagged H3.3 expression vector (+GFP-H3.3) in combination ( a–h ) or not ( i–p ) with HA-DAXX expression vector (+HA-DAXX). Forty hours later, cells were supplemented with serum and were paraformaldehyde-fixed after an additional 8 h. Distribution of HA-DAXX ( b , j ) or endogenous PML ( f , n ) in GFP-H3.3-positive cells was investigated by immunofluorescence staining using anti-HA or anti-PML antibody, respectively. ( a , e , i , m ) DNA was stained with DAPI. ( d , h , l , p ) Merged images correspond to the overlay of red (HA-DAXX or PML) and green fluorescence (GFP-H3.3).
    Figure Legend Snippet: DAXX targets H3.3 to PML-NBs. Resting DAXX −/− MEF cells were transiently transfected with GFP-tagged H3.3 expression vector (+GFP-H3.3) in combination ( a–h ) or not ( i–p ) with HA-DAXX expression vector (+HA-DAXX). Forty hours later, cells were supplemented with serum and were paraformaldehyde-fixed after an additional 8 h. Distribution of HA-DAXX ( b , j ) or endogenous PML ( f , n ) in GFP-H3.3-positive cells was investigated by immunofluorescence staining using anti-HA or anti-PML antibody, respectively. ( a , e , i , m ) DNA was stained with DAPI. ( d , h , l , p ) Merged images correspond to the overlay of red (HA-DAXX or PML) and green fluorescence (GFP-H3.3).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Immunofluorescence, Staining, Fluorescence

    DAXX-dependent deposition of H3.3 on pericentric heterochromatin. ( A ) DAXX and ATRX are present on pericentric DNA repeats in wild-type MEFs. Presence of DAXX ( left panel) and ATRX ( right panel) on pericentric DNA repeats was investigated by ChIP assays using specific antibodies. (−Ab) Control sample in which primary antibody was omitted. Results are expressed as percentage of chromatin input used for immunoprecipitation. ( B ) The level of transcripts from pericentric DNA repeats is reduced in DAXX-deficient cells. Relative mRNA level for pericentric DNA repeats in wild-type and DAXX −/− MEFs was determined by quantitative RT–PCR. Results are represented as relative expression level of pericentric DNA repeats versus GAPDH . Mean ± standard deviation of four independent experiments. ( C ) Depletion of H3.3A and H3.3B resulted in a decrease in transcription from pericentric DNA repeats. MEFs were transfected with control siRNA (siCTRL) or a mixture of H3.3A and H3.3B siRNA (siH3.3). Relative mRNA levels for pericentric DNA repeats, H3.3A , and H3.3B were determined by quantitative RT–PCR. Results were normalized to GAPDH and were set at 1 in cells transfected with control siRNA. Mean ± standard deviation of three independent experiments. ( D ) DAXX is required for deposition of H3.3 onto pericentric DNA repeats outside of S phase. DAXX −/− MEFs were deprived of serum for 48 h before being cotransfected with empty vector (CTRL) or else epitope-tagged H3.1 or H3.3 expression vector in combination with DAXX expression vector where indicated. Forty hours later, cells were reinduced for 8 h with 20% FCS in the presence of aphidicolin and were subjected to ChIP assays. Results are expressed as percentage of chromatin input immunoprecipitated. Mean ± standard deviation of three independent experiments.
    Figure Legend Snippet: DAXX-dependent deposition of H3.3 on pericentric heterochromatin. ( A ) DAXX and ATRX are present on pericentric DNA repeats in wild-type MEFs. Presence of DAXX ( left panel) and ATRX ( right panel) on pericentric DNA repeats was investigated by ChIP assays using specific antibodies. (−Ab) Control sample in which primary antibody was omitted. Results are expressed as percentage of chromatin input used for immunoprecipitation. ( B ) The level of transcripts from pericentric DNA repeats is reduced in DAXX-deficient cells. Relative mRNA level for pericentric DNA repeats in wild-type and DAXX −/− MEFs was determined by quantitative RT–PCR. Results are represented as relative expression level of pericentric DNA repeats versus GAPDH . Mean ± standard deviation of four independent experiments. ( C ) Depletion of H3.3A and H3.3B resulted in a decrease in transcription from pericentric DNA repeats. MEFs were transfected with control siRNA (siCTRL) or a mixture of H3.3A and H3.3B siRNA (siH3.3). Relative mRNA levels for pericentric DNA repeats, H3.3A , and H3.3B were determined by quantitative RT–PCR. Results were normalized to GAPDH and were set at 1 in cells transfected with control siRNA. Mean ± standard deviation of three independent experiments. ( D ) DAXX is required for deposition of H3.3 onto pericentric DNA repeats outside of S phase. DAXX −/− MEFs were deprived of serum for 48 h before being cotransfected with empty vector (CTRL) or else epitope-tagged H3.1 or H3.3 expression vector in combination with DAXX expression vector where indicated. Forty hours later, cells were reinduced for 8 h with 20% FCS in the presence of aphidicolin and were subjected to ChIP assays. Results are expressed as percentage of chromatin input immunoprecipitated. Mean ± standard deviation of three independent experiments.

    Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Quantitative RT-PCR, Expressing, Standard Deviation, Transfection, Plasmid Preparation

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    Article Title: Identification by In Vivo Genomic Footprinting of a Transcriptional Switch Containing NF-?B and Sp1 That Regulates the I?B? Promoter
    Article Snippet: .. For each sample, 2 μg of DNA were submitted to ligation-mediated PCR (LM-PCR) using Vent DNA polymerase (New England Biolabs, Mississauga, Ontario, Canada) as described elsewhere ( , ). .. PCR amplification was for 2 min for the first cycle and was progressively increased to 10 min in the last cycle.

    Article Title: Purification and In Vitro Characterization of the Serratia marcescens NucC Protein, a Zinc-Binding Transcription Factor Homologous to P2 Ogr
    Article Snippet: .. A 310-bp fragment containing the nucC gene was amplified from pSE380TacNucC by PCR with Vent DNA polymerase (New England Biolabs) and primers SM1 (5′-CGGAATTCT ATG ATGCACTGTCCACT-3′) and SM2 (5′-CACGTTGCATTTGCGAG-3′). ..

    Article Title: The Plasmid-Encoded Signal Peptidase SipP Can Functionally Replace the Major Signal Peptidases SipS and SipT of Bacillus subtilis
    Article Snippet: .. PCR was carried out with Vent DNA polymerase (New England Biolabs) as described by van Dijl et al. ( ). .. To construct an Orf1p–β-lactamase fusion protein, the 5′ sequences of ORF1, specifying the 50 amino-terminal residues of Orf1p, were amplified by PCR with primers orf1-1 (5′-TAT GGA TCC TAT TGA ATT TTG CTA GGA GGG-3′) and orf1-2 (5′-GAT CGT CGA CTC ATA ACT TTT TGT TGA AGA TTG GG-3′), using plasmid pTA1015 as the template.

    Article Title: The Glycan Moieties and the N-Terminal Polypeptide Backbone of a Fimbria-Associated Adhesin, Fap1, Play Distinct Roles in the Biofilm Development of Streptococcus parasanguinis ▿
    Article Snippet: .. PCR was carried out in a Techne Genius PCR apparatus using Vent DNA polymerase (New England Biolabs). .. The PCR fragment was purified using QIAGEN PCR purification column.

    Article Title: Catabolite Repression of the Citrate Fermentation Genes in Klebsiella pneumoniae: Evidence for Involvement of the Cyclic AMP Receptor Protein
    Article Snippet: .. Chromosomal DNA of K. pneumoniae ATCC 13882 was used as template for the PCR with Vent DNA polymerase (New England Biolabs). ..

    Amplification:

    Article Title: Purification and In Vitro Characterization of the Serratia marcescens NucC Protein, a Zinc-Binding Transcription Factor Homologous to P2 Ogr
    Article Snippet: .. A 310-bp fragment containing the nucC gene was amplified from pSE380TacNucC by PCR with Vent DNA polymerase (New England Biolabs) and primers SM1 (5′-CGGAATTCT ATG ATGCACTGTCCACT-3′) and SM2 (5′-CACGTTGCATTTGCGAG-3′). ..

    Article Title: Structure of the sporulation-specific transcription factor Ndt80 bound to DNA
    Article Snippet: .. The NDT80 open reading frame was amplified with Vent DNA polymerase (New England Biolabs) and ligated between the Bam HI and Sal I sites in the expression vector pMAL-C2 (New England Biolabs). .. MBP–Ndt80 fusion protein was expressed and purified by maltose affinity chromatography as described previously ( ).

    Article Title: Tissue distribution of products of the mouse decay-accelerating factor (DAF) genes. Exploitation of a Daf1 knock-out mouse and site-specific monoclonal antibodies
    Article Snippet: .. The coding regions for mouse DAF CCP1-4, CCP2-3, CCP3-4, CCP2-4, CCP1-2,4 and CCP1,3-4 were amplified from mouse Daf1 cDNA with proofreading Vent DNA polymerase (New England Biolabs, Beverly, MA). .. PCR products were cloned into the Eco RI and Xba I sites of the expression vector pPICZαA.

    Expressing:

    Article Title: Structure of the sporulation-specific transcription factor Ndt80 bound to DNA
    Article Snippet: .. The NDT80 open reading frame was amplified with Vent DNA polymerase (New England Biolabs) and ligated between the Bam HI and Sal I sites in the expression vector pMAL-C2 (New England Biolabs). .. MBP–Ndt80 fusion protein was expressed and purified by maltose affinity chromatography as described previously ( ).

    Plasmid Preparation:

    Article Title: Structure of the sporulation-specific transcription factor Ndt80 bound to DNA
    Article Snippet: .. The NDT80 open reading frame was amplified with Vent DNA polymerase (New England Biolabs) and ligated between the Bam HI and Sal I sites in the expression vector pMAL-C2 (New England Biolabs). .. MBP–Ndt80 fusion protein was expressed and purified by maltose affinity chromatography as described previously ( ).

    Ligation:

    Article Title: Identification by In Vivo Genomic Footprinting of a Transcriptional Switch Containing NF-?B and Sp1 That Regulates the I?B? Promoter
    Article Snippet: .. For each sample, 2 μg of DNA were submitted to ligation-mediated PCR (LM-PCR) using Vent DNA polymerase (New England Biolabs, Mississauga, Ontario, Canada) as described elsewhere ( , ). .. PCR amplification was for 2 min for the first cycle and was progressively increased to 10 min in the last cycle.

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    New England Biolabs vent dna polymerase
    Targeting strategy for inactivation of the mouse Daf genes. The tandem <t>Daf1</t> and Daf2 genes are shown diagrammatically (not drawn to scale). The black-filled boxes represent exons and the open boxes represent selection marker genes as marked. As the figure shows, the pDAFup construct integrates to the Daf1 gene first to introduce a loxP site and a TK gene within exon 3, then the pDAFdown construct may integrate into its homologous region in either the Daf1 or Daf2 gene together with another loxP site and TK gene. Upon recombination induced by Cre recombinase, the <t>DNA</t> fragment flanked by the two loxP sites is deleted together with the two TK genes to generate either Daf1 or Daf2 knock-out ES cells.
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    Targeting strategy for inactivation of the mouse Daf genes. The tandem Daf1 and Daf2 genes are shown diagrammatically (not drawn to scale). The black-filled boxes represent exons and the open boxes represent selection marker genes as marked. As the figure shows, the pDAFup construct integrates to the Daf1 gene first to introduce a loxP site and a TK gene within exon 3, then the pDAFdown construct may integrate into its homologous region in either the Daf1 or Daf2 gene together with another loxP site and TK gene. Upon recombination induced by Cre recombinase, the DNA fragment flanked by the two loxP sites is deleted together with the two TK genes to generate either Daf1 or Daf2 knock-out ES cells.

    Journal: Immunology

    Article Title: Tissue distribution of products of the mouse decay-accelerating factor (DAF) genes. Exploitation of a Daf1 knock-out mouse and site-specific monoclonal antibodies

    doi: 10.1046/j.0019-2805.2001.01287.x

    Figure Lengend Snippet: Targeting strategy for inactivation of the mouse Daf genes. The tandem Daf1 and Daf2 genes are shown diagrammatically (not drawn to scale). The black-filled boxes represent exons and the open boxes represent selection marker genes as marked. As the figure shows, the pDAFup construct integrates to the Daf1 gene first to introduce a loxP site and a TK gene within exon 3, then the pDAFdown construct may integrate into its homologous region in either the Daf1 or Daf2 gene together with another loxP site and TK gene. Upon recombination induced by Cre recombinase, the DNA fragment flanked by the two loxP sites is deleted together with the two TK genes to generate either Daf1 or Daf2 knock-out ES cells.

    Article Snippet: The coding regions for mouse DAF CCP1-4, CCP2-3, CCP3-4, CCP2-4, CCP1-2,4 and CCP1,3-4 were amplified from mouse Daf1 cDNA with proofreading Vent DNA polymerase (New England Biolabs, Beverly, MA).

    Techniques: Selection, Marker, Construct, Introduce, Knock-Out

    PCR analysis of recombined ES cells. (a1) PCR with Neo and Hph-specific primers P3 and P4 yielded a ∼500-bp fragment verifying that the two markers were brought together by Cre/ loxP recombination. M, 1 kb ladder; C, PCR with the wild-type DNA as template; K/O, recombined DNA as template. (a2) PCR with primers P5 , P6 ( Daf1 -specific) and P7 ( Daf2 -specific) showing that the pDAFdown construct integrated into exon 5 of the Daf1 gene and that the Daf1 gene thus was selectively inactivated. (a3) RT-PCR with primers P8 and P10 of the Daf1 mRNA product in the Daf1 −/− mice. A truncated product corresponding to sequence for CCP1,4 is seen. (b) A diagram of the mouse Daf1 and Daf2 genes is shown. B, Bam HI; E, Eco RI; and S, Sac I. The position of the 1·5-kb Sac I probe is indicated and the hybridized Eco RI and Bam HI fragments are shown by the brackets. (c) Southern blot analyses of Eco RI- and Bam HI-digested genomic DNA from parental and K/O mice. DNA from wild-type, heterozygous ( Daf1 +/− ) and homozygous ( Daf1 −/− ) knock-out mice were hybridized with the Sac I fragment of the Daf1 gene (panel B). The pattern corresponded to the expected deletion from Daf1 exon 3 to exon 5. The high M r band corresponds to the homologous Eco RI fragment in the Daf2 gene.

    Journal: Immunology

    Article Title: Tissue distribution of products of the mouse decay-accelerating factor (DAF) genes. Exploitation of a Daf1 knock-out mouse and site-specific monoclonal antibodies

    doi: 10.1046/j.0019-2805.2001.01287.x

    Figure Lengend Snippet: PCR analysis of recombined ES cells. (a1) PCR with Neo and Hph-specific primers P3 and P4 yielded a ∼500-bp fragment verifying that the two markers were brought together by Cre/ loxP recombination. M, 1 kb ladder; C, PCR with the wild-type DNA as template; K/O, recombined DNA as template. (a2) PCR with primers P5 , P6 ( Daf1 -specific) and P7 ( Daf2 -specific) showing that the pDAFdown construct integrated into exon 5 of the Daf1 gene and that the Daf1 gene thus was selectively inactivated. (a3) RT-PCR with primers P8 and P10 of the Daf1 mRNA product in the Daf1 −/− mice. A truncated product corresponding to sequence for CCP1,4 is seen. (b) A diagram of the mouse Daf1 and Daf2 genes is shown. B, Bam HI; E, Eco RI; and S, Sac I. The position of the 1·5-kb Sac I probe is indicated and the hybridized Eco RI and Bam HI fragments are shown by the brackets. (c) Southern blot analyses of Eco RI- and Bam HI-digested genomic DNA from parental and K/O mice. DNA from wild-type, heterozygous ( Daf1 +/− ) and homozygous ( Daf1 −/− ) knock-out mice were hybridized with the Sac I fragment of the Daf1 gene (panel B). The pattern corresponded to the expected deletion from Daf1 exon 3 to exon 5. The high M r band corresponds to the homologous Eco RI fragment in the Daf2 gene.

    Article Snippet: The coding regions for mouse DAF CCP1-4, CCP2-3, CCP3-4, CCP2-4, CCP1-2,4 and CCP1,3-4 were amplified from mouse Daf1 cDNA with proofreading Vent DNA polymerase (New England Biolabs, Beverly, MA).

    Techniques: Polymerase Chain Reaction, Construct, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Sequencing, Southern Blot, Knock-Out

    Cotranscription of ORF1 and sipP . (A) Schematic presentation of the ORF1- sipP module of pTA1015. The positions of primers used for RT-PCR are indicated. Primers O1 (5′-GAT GGC GCT ACT CTG GG-3′) and O2 (5′-ACT ATC TAC AAT CGG GAC TCC-3′) were used to detect ORF1-specific transcripts; primers P1 (5′-TAG AAA TGA AGA ATG ACC-3′) and P2 (5′-TCG CAT ATT ACT AAA TGG-3′) were used to detect sipP -specific transcripts; primers O1 and P2 were used to detect transcripts of ORF1 and sipP ). (B) DNA fragments amplified by RT-PCR with the primer sets O1-O2 (ORF1), P1-P2 ( sipP ), and O1-P2 (ORF1- sipP ) were separated on a 2% agarose gel containing ethidium bromide (1 μg/ml) and visualized by UV illumination (lanes labeled +RT). As a negative control, reactions were also performed in the absence of reverse transcriptase (lanes labeled −RT). The positions of amplified DNA fragments corresponding to ORF1 (180 bp), sipP (270 bp), and ORF1- sipP (650 bp) are indicated.

    Journal: Journal of Bacteriology

    Article Title: The Plasmid-Encoded Signal Peptidase SipP Can Functionally Replace the Major Signal Peptidases SipS and SipT of Bacillus subtilis

    doi:

    Figure Lengend Snippet: Cotranscription of ORF1 and sipP . (A) Schematic presentation of the ORF1- sipP module of pTA1015. The positions of primers used for RT-PCR are indicated. Primers O1 (5′-GAT GGC GCT ACT CTG GG-3′) and O2 (5′-ACT ATC TAC AAT CGG GAC TCC-3′) were used to detect ORF1-specific transcripts; primers P1 (5′-TAG AAA TGA AGA ATG ACC-3′) and P2 (5′-TCG CAT ATT ACT AAA TGG-3′) were used to detect sipP -specific transcripts; primers O1 and P2 were used to detect transcripts of ORF1 and sipP ). (B) DNA fragments amplified by RT-PCR with the primer sets O1-O2 (ORF1), P1-P2 ( sipP ), and O1-P2 (ORF1- sipP ) were separated on a 2% agarose gel containing ethidium bromide (1 μg/ml) and visualized by UV illumination (lanes labeled +RT). As a negative control, reactions were also performed in the absence of reverse transcriptase (lanes labeled −RT). The positions of amplified DNA fragments corresponding to ORF1 (180 bp), sipP (270 bp), and ORF1- sipP (650 bp) are indicated.

    Article Snippet: PCR was carried out with Vent DNA polymerase (New England Biolabs) as described by van Dijl et al. ( ).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Activated Clotting Time Assay, CTG Assay, Amplification, Agarose Gel Electrophoresis, Labeling, Negative Control

    Temporally controlled transcription of ORF1 and sipP . (A) Schematic presentation of the ORF1- sipP regions of pTAB-OL and pTAB-PL, containing transcriptional ORF1- lacZ and sipP-lacZ gene fusions, respectively. The ORF1- and sipP-lacZ ), an integration plasmid for B. subtilis containing a promoterless spoVG-lacZ gene fusion (see Materials and Methods for details). In pTAB-OL, the transcription of lacZ is directed by the promoter(s) of ORF1. In pTAB-PL, the transcription of lacZ is directed by the promoter(s) of ORF1 and/or sipP . DNA fragments amplified by PCR are indicated with black bars. Only restriction sites relevant for the constructions are shown (Ba, Bam HI; Ec, Eco RI; Sc, Sac I). ‘ rep , 5′ truncated rep gene; ORF1’, 3′ truncated ORF1 gene; sipP ’, 3′ truncated sipP gene; ‘ORF1, 5′ truncated ORF1 gene; ori pBR322, replication functions of pBR322; SL, potential stem-loop structures. (B) Time course of the transcription of sip-lacZ gene fusions were determined in cells growing in TY medium at 37°C. β-Galactosidase activities (in units per OD 600 ) were determined for B. subtilis 8G5(pTAB-PL) (■) ( sipP-lacZ ), B. subtilis 8G5::pGDE22 (○) ( sipS-lacZ ), and B. subtilis ) ( sipV-lacZ ). Time zero indicates the transition point between the exponential and post-exponential growth phases. (C) The time course of transcription of the ORF1- lacZ gene fusion was determined as for panel B, using B. subtilis 8G5(pTAB-OL) (□).

    Journal: Journal of Bacteriology

    Article Title: The Plasmid-Encoded Signal Peptidase SipP Can Functionally Replace the Major Signal Peptidases SipS and SipT of Bacillus subtilis

    doi:

    Figure Lengend Snippet: Temporally controlled transcription of ORF1 and sipP . (A) Schematic presentation of the ORF1- sipP regions of pTAB-OL and pTAB-PL, containing transcriptional ORF1- lacZ and sipP-lacZ gene fusions, respectively. The ORF1- and sipP-lacZ ), an integration plasmid for B. subtilis containing a promoterless spoVG-lacZ gene fusion (see Materials and Methods for details). In pTAB-OL, the transcription of lacZ is directed by the promoter(s) of ORF1. In pTAB-PL, the transcription of lacZ is directed by the promoter(s) of ORF1 and/or sipP . DNA fragments amplified by PCR are indicated with black bars. Only restriction sites relevant for the constructions are shown (Ba, Bam HI; Ec, Eco RI; Sc, Sac I). ‘ rep , 5′ truncated rep gene; ORF1’, 3′ truncated ORF1 gene; sipP ’, 3′ truncated sipP gene; ‘ORF1, 5′ truncated ORF1 gene; ori pBR322, replication functions of pBR322; SL, potential stem-loop structures. (B) Time course of the transcription of sip-lacZ gene fusions were determined in cells growing in TY medium at 37°C. β-Galactosidase activities (in units per OD 600 ) were determined for B. subtilis 8G5(pTAB-PL) (■) ( sipP-lacZ ), B. subtilis 8G5::pGDE22 (○) ( sipS-lacZ ), and B. subtilis ) ( sipV-lacZ ). Time zero indicates the transition point between the exponential and post-exponential growth phases. (C) The time course of transcription of the ORF1- lacZ gene fusion was determined as for panel B, using B. subtilis 8G5(pTAB-OL) (□).

    Article Snippet: PCR was carried out with Vent DNA polymerase (New England Biolabs) as described by van Dijl et al. ( ).

    Techniques: Plasmid Preparation, Amplification, Polymerase Chain Reaction

    Organization of the citrate-specific fermentation genes in K. pneumoniae . The 13-kb DNA cluster encompassing 11 genes involved in citrate fermentation is shown in panel A. The lower part shows an enlargement of the citC - citS intergenic region. The arrows indicate transcription start sites. The positions of the CitB binding sites as deduced from DNase I footprints and of putative CRP binding sites are indicated. The sequence of the citC - citS ), the −10 regions, the CitB binding sites, the putative CRP binding sites, and the hypersensitive sites observed in DNase I footprints (asterisks).

    Journal: Journal of Bacteriology

    Article Title: Catabolite Repression of the Citrate Fermentation Genes in Klebsiella pneumoniae: Evidence for Involvement of the Cyclic AMP Receptor Protein

    doi: 10.1128/JB.183.18.5248-5256.2001

    Figure Lengend Snippet: Organization of the citrate-specific fermentation genes in K. pneumoniae . The 13-kb DNA cluster encompassing 11 genes involved in citrate fermentation is shown in panel A. The lower part shows an enlargement of the citC - citS intergenic region. The arrows indicate transcription start sites. The positions of the CitB binding sites as deduced from DNase I footprints and of putative CRP binding sites are indicated. The sequence of the citC - citS ), the −10 regions, the CitB binding sites, the putative CRP binding sites, and the hypersensitive sites observed in DNase I footprints (asterisks).

    Article Snippet: Chromosomal DNA of K. pneumoniae ATCC 13882 was used as template for the PCR with Vent DNA polymerase (New England Biolabs).

    Techniques: Binding Assay, Sequencing

    Diagram of gly, nss, galT1, galT2 , and fap1 gene loci. (A) Restriction map of the fap1 -positive recombinant phage DNA. A 3.9-kb fragment was PCR amplified from the recombinant phage DNA using a λEMBL3 left-arm-specific sequence (F1) and a fap1 5′-end-specific sequence (800bp2) as primers. The left and right arms represent λEMBL3 cloning vectors. (B) Organization of the fap1 upstream region. Genes that share homology with glycosyltransferase ( gly ), nucleotide-sugar synthetase ( nss ), and galactosyltransferase genes ( galT1 and galT2 ) are located upstream of the fap1 locus. The fap1 gene is 618 bp from the stop codon of galT2 . The HaeII genomic DNA fragment was amplified by inverse PCR using nss 5′-1 and nss 5′-2 primers.

    Journal: Infection and Immunity

    Article Title: The Glycan Moieties and the N-Terminal Polypeptide Backbone of a Fimbria-Associated Adhesin, Fap1, Play Distinct Roles in the Biofilm Development of Streptococcus parasanguinis ▿

    doi: 10.1128/IAI.01544-06

    Figure Lengend Snippet: Diagram of gly, nss, galT1, galT2 , and fap1 gene loci. (A) Restriction map of the fap1 -positive recombinant phage DNA. A 3.9-kb fragment was PCR amplified from the recombinant phage DNA using a λEMBL3 left-arm-specific sequence (F1) and a fap1 5′-end-specific sequence (800bp2) as primers. The left and right arms represent λEMBL3 cloning vectors. (B) Organization of the fap1 upstream region. Genes that share homology with glycosyltransferase ( gly ), nucleotide-sugar synthetase ( nss ), and galactosyltransferase genes ( galT1 and galT2 ) are located upstream of the fap1 locus. The fap1 gene is 618 bp from the stop codon of galT2 . The HaeII genomic DNA fragment was amplified by inverse PCR using nss 5′-1 and nss 5′-2 primers.

    Article Snippet: PCR was carried out in a Techne Genius PCR apparatus using Vent DNA polymerase (New England Biolabs).

    Techniques: Recombinant, Polymerase Chain Reaction, Amplification, Sequencing, Clone Assay, Inverse PCR