vent dna polymerase  (New England Biolabs)


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    Structured Review

    New England Biolabs vent dna polymerase
    <t>RT-PCR</t> amplification of spliced B4-B5 exons. The first lane contains a <t>DNA</t> ladder of markers (Stratagene). The second and third lanes, respectively, represent RNA isolated from the yeast null strain (QBY320) that contained plasmids expressing wild-type LeuRS from yeast and M. tuberculosis . The unspliced and spliced B4-B5 exon products are indicated by respective bands at about 1.5 kbp and 250 bp. The fourth and fifth lanes, respectively, show that the intron remains unspliced when genes containing either the W286C mutant or G288SΔC5 deletion of M. tuberculosis LeuRS are used in attempts to complement the yeast null strain.
    Vent Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A prokaryote and human tRNA synthetase provide an essential RNA splicing function in yeast mitochondria"

    Article Title: A prokaryote and human tRNA synthetase provide an essential RNA splicing function in yeast mitochondria

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

    doi:

    RT-PCR amplification of spliced B4-B5 exons. The first lane contains a DNA ladder of markers (Stratagene). The second and third lanes, respectively, represent RNA isolated from the yeast null strain (QBY320) that contained plasmids expressing wild-type LeuRS from yeast and M. tuberculosis . The unspliced and spliced B4-B5 exon products are indicated by respective bands at about 1.5 kbp and 250 bp. The fourth and fifth lanes, respectively, show that the intron remains unspliced when genes containing either the W286C mutant or G288SΔC5 deletion of M. tuberculosis LeuRS are used in attempts to complement the yeast null strain.
    Figure Legend Snippet: RT-PCR amplification of spliced B4-B5 exons. The first lane contains a DNA ladder of markers (Stratagene). The second and third lanes, respectively, represent RNA isolated from the yeast null strain (QBY320) that contained plasmids expressing wild-type LeuRS from yeast and M. tuberculosis . The unspliced and spliced B4-B5 exon products are indicated by respective bands at about 1.5 kbp and 250 bp. The fourth and fifth lanes, respectively, show that the intron remains unspliced when genes containing either the W286C mutant or G288SΔC5 deletion of M. tuberculosis LeuRS are used in attempts to complement the yeast null strain.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Isolation, Expressing, Mutagenesis

    2) 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

    3) 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

    4) Product Images from "Identification and characterisation of a developmentally regulated mammalian gene that utilises -1 programmed ribosomal frameshifting"

    Article Title: Identification and characterisation of a developmentally regulated mammalian gene that utilises -1 programmed ribosomal frameshifting

    Journal: Nucleic Acids Research

    doi:

    Edr is a conserved mammalian gene and maps to the proximal region of mouse chromosome 6. ( A ) Genomic Southern blot analysis: 10 µg genomic DNA prepared from human term placenta (hu) or M.musculus (mo) was digested with Bam HI (B), Xba I (X) or Eco RI (E) restriction endonucleases, prior to electrophoresis and transfer to nitrocellulose. The filter was hybridised with 32 P-labelled Edr partial cDNA (395 bp Eco RI fragment encompassing nucleotides 1358–1753 in Edr cDNA) and washed to 0.1× SSC 0.1% SDS 65°C prior to autoradiography. ( B ) Comparison of the SDP of a Bam HI RFLP at the Edr locus with others mapped in the BxD series of RI mice revealed linkage with D6Mit86 (0/26 recombinants and Met 4/26 recombinants). These linkage data map Edr to the proximal end of mouse chromosome 6.
    Figure Legend Snippet: Edr is a conserved mammalian gene and maps to the proximal region of mouse chromosome 6. ( A ) Genomic Southern blot analysis: 10 µg genomic DNA prepared from human term placenta (hu) or M.musculus (mo) was digested with Bam HI (B), Xba I (X) or Eco RI (E) restriction endonucleases, prior to electrophoresis and transfer to nitrocellulose. The filter was hybridised with 32 P-labelled Edr partial cDNA (395 bp Eco RI fragment encompassing nucleotides 1358–1753 in Edr cDNA) and washed to 0.1× SSC 0.1% SDS 65°C prior to autoradiography. ( B ) Comparison of the SDP of a Bam HI RFLP at the Edr locus with others mapped in the BxD series of RI mice revealed linkage with D6Mit86 (0/26 recombinants and Met 4/26 recombinants). These linkage data map Edr to the proximal end of mouse chromosome 6.

    Techniques Used: Southern Blot, Electrophoresis, Autoradiography, Mouse Assay

    5) Product Images from "Phosphorylation of RNA polymerase II CTD regulates H3 methylation in yeast"

    Article Title: Phosphorylation of RNA polymerase II CTD regulates H3 methylation in yeast

    Journal: Genes & Development

    doi: 10.1101/gad.1055503

    Set2 methylates at the coding regions, as well as promoters, of genes. Chromatin immunoprecipitation assays were used to examine the H3 Lys 36 methylation status on several genes in vivo. Whole-cell extracts prepared from formaldehyde-fixed wild-type or set2 Δ cells were sonicated to shear their chromatin and then immunoprecipitated with either the H3 Lys 36 methylation-specific antibody [α-Me(Lys 36)H3], the H3 Lys 4 methylation-specific antibody [α-Me(Lys 4)H3], the CTD Ser 5 phosphorylation-specific antibody (H14 [α-Ser5P]), or the Ctk1-phosphorylated CTD antibody (α-Ctk1-PCTD). DNA from the enriched precipitates was isolated and used in PCR reactions with promoter or coding-specific primer pairs for the genes indicated. Primer pairs (labeled as A–D) include their sequence location relative to the translation initiation site. Although we show several genes positive for the presence of H3 Lys 36 methylation, other genes or distinct regions of chromatin examined ( HIS3 , rDNA, and telomeres) showed no enrichment for H3 Lys 36 methylation above background levels observed in set2 Δ (data not shown).
    Figure Legend Snippet: Set2 methylates at the coding regions, as well as promoters, of genes. Chromatin immunoprecipitation assays were used to examine the H3 Lys 36 methylation status on several genes in vivo. Whole-cell extracts prepared from formaldehyde-fixed wild-type or set2 Δ cells were sonicated to shear their chromatin and then immunoprecipitated with either the H3 Lys 36 methylation-specific antibody [α-Me(Lys 36)H3], the H3 Lys 4 methylation-specific antibody [α-Me(Lys 4)H3], the CTD Ser 5 phosphorylation-specific antibody (H14 [α-Ser5P]), or the Ctk1-phosphorylated CTD antibody (α-Ctk1-PCTD). DNA from the enriched precipitates was isolated and used in PCR reactions with promoter or coding-specific primer pairs for the genes indicated. Primer pairs (labeled as A–D) include their sequence location relative to the translation initiation site. Although we show several genes positive for the presence of H3 Lys 36 methylation, other genes or distinct regions of chromatin examined ( HIS3 , rDNA, and telomeres) showed no enrichment for H3 Lys 36 methylation above background levels observed in set2 Δ (data not shown).

    Techniques Used: Chromatin Immunoprecipitation, Methylation, In Vivo, Sonication, Immunoprecipitation, Isolation, Polymerase Chain Reaction, Labeling, Sequencing

    6) 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

    7) Product Images from "Comparative Characterization of Rep Proteins from the Helper-Dependent Adeno-Associated Virus Type 2 and the Autonomous Goose Parvovirus"

    Article Title: Comparative Characterization of Rep Proteins from the Helper-Dependent Adeno-Associated Virus Type 2 and the Autonomous Goose Parvovirus

    Journal: Journal of Virology

    doi:

    (A) Diagram of Rep1 and Rep78 showing the extent of sequence identity and similar functional motifs. Y indicates the tyrosine residue that, in Rep78, is believed to form a covalent attachment to viral DNA. Zn finger indicates clusters of cysteine and histidine residues (three in Rep78 and two in Rep1) which could form Zn fingers. (B) Synthetic oligonucleotides used as DNA substrates. The sequences, termed AAV ori and GPV ori , correspond to the putative minimal AAV or GPV origins, respectively. RS1 is an unrelated sequence used as a negative control. The number of base pairs derived from AAV or GPV viral DNA are indicated in parentheses. The duplex oligonucleotides are flanked by single-stranded tails useful for labeling with Klenow polymerase. Underlined nucleotides are those added during the labeling reaction as described in Materials and Methods. Sequences corresponding to the RBS for AAV and the putative RBS for GPV are overlined. The locations of the single-stranded cleavage site (trs) in AAV and the putative trs in GPV are indicated by slashes.
    Figure Legend Snippet: (A) Diagram of Rep1 and Rep78 showing the extent of sequence identity and similar functional motifs. Y indicates the tyrosine residue that, in Rep78, is believed to form a covalent attachment to viral DNA. Zn finger indicates clusters of cysteine and histidine residues (three in Rep78 and two in Rep1) which could form Zn fingers. (B) Synthetic oligonucleotides used as DNA substrates. The sequences, termed AAV ori and GPV ori , correspond to the putative minimal AAV or GPV origins, respectively. RS1 is an unrelated sequence used as a negative control. The number of base pairs derived from AAV or GPV viral DNA are indicated in parentheses. The duplex oligonucleotides are flanked by single-stranded tails useful for labeling with Klenow polymerase. Underlined nucleotides are those added during the labeling reaction as described in Materials and Methods. Sequences corresponding to the RBS for AAV and the putative RBS for GPV are overlined. The locations of the single-stranded cleavage site (trs) in AAV and the putative trs in GPV are indicated by slashes.

    Techniques Used: Sequencing, Functional Assay, Negative Control, Derivative Assay, Labeling

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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:

    13) 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

    14) Product Images from "Functional Analysis of the SIN3-Histone Deacetylase RPD3-RbAp48-Histone H4 Connection in the Xenopus Oocyte"

    Article Title: Functional Analysis of the SIN3-Histone Deacetylase RPD3-RbAp48-Histone H4 Connection in the Xenopus Oocyte

    Journal: Molecular and Cellular Biology

    doi:

    Nuclear distribution, transcriptional repression, and deacetylase activity of RPD3, SIN3, and RbAp48 in Xenopus oocytes. (A) Nuclear and cytoplasmic distribution of SIN3, RbAp48, and RPD3. Uninjected oocytes or oocytes which had been microinjected with SIN3, RPD3, or RbAp48 mRNA (+mRNA) were manually separated into nuclei and cytoplasm. Soluble material of five oocyte nuclei or cytoplasm equivalents was analyzed by Western blotting after SDS-PAGE. Antibodies were against SIN3, RbAp48, and RPD3. Endogenous proteins were all detected on the same Western blot. (B) Deacetylase activity of oocytes in the presence or absence of exogenous RPD3, SIN3, or RbAp48. Oocytes were uninjected (control) or microinjected with mRNA encoding RPD3, SIN3, or RbAp48, resulting in similar levels of expression of exogenous protein. Oocytes were manually dissected into nuclei (N) and cytoplasm (C), and equivalent oocyte homogenates were assayed for deacetylase activity. The error in the deacetylase assays is ±5%. (C) Exogenous RPD3 but not SIN3 or RbAp48 represses transcription from the TRβA promoter in oocytes. Oocytes were microinjected with 0.5, 1, and 1.5 ng of mRNA for RPD3 (lanes 2, 3, and 4), 1 and 1.5 ng of mRNA for SIN3 (lanes 5 and 6), or 1.5 ng of mRNA for RbAp48 (lane 7). Oocytes were maintained for 6 h to allow translation and then microinjected with 0.5 ng of double-stranded DNA for TRβA promoter. After overnight incubation, the transcript levels were analyzed by primer extension (top left). H4 serves as an internal control. The primer extension results were quantitated with a PhosphorImager and are represented graphically (top right). Protein expression was verified by labeling with [ 35 S]methionine (RPD3 or RbAp48) or by Western blotting (SIN3) (bottom left). The distribution of endogenous RPD3 between the nucleus and cytoplasm in the presence (+SIN3) or absence (−SIN3) of exogenous SIN3 was determined by detection of RPD3 by Western blotting (bottom right).
    Figure Legend Snippet: Nuclear distribution, transcriptional repression, and deacetylase activity of RPD3, SIN3, and RbAp48 in Xenopus oocytes. (A) Nuclear and cytoplasmic distribution of SIN3, RbAp48, and RPD3. Uninjected oocytes or oocytes which had been microinjected with SIN3, RPD3, or RbAp48 mRNA (+mRNA) were manually separated into nuclei and cytoplasm. Soluble material of five oocyte nuclei or cytoplasm equivalents was analyzed by Western blotting after SDS-PAGE. Antibodies were against SIN3, RbAp48, and RPD3. Endogenous proteins were all detected on the same Western blot. (B) Deacetylase activity of oocytes in the presence or absence of exogenous RPD3, SIN3, or RbAp48. Oocytes were uninjected (control) or microinjected with mRNA encoding RPD3, SIN3, or RbAp48, resulting in similar levels of expression of exogenous protein. Oocytes were manually dissected into nuclei (N) and cytoplasm (C), and equivalent oocyte homogenates were assayed for deacetylase activity. The error in the deacetylase assays is ±5%. (C) Exogenous RPD3 but not SIN3 or RbAp48 represses transcription from the TRβA promoter in oocytes. Oocytes were microinjected with 0.5, 1, and 1.5 ng of mRNA for RPD3 (lanes 2, 3, and 4), 1 and 1.5 ng of mRNA for SIN3 (lanes 5 and 6), or 1.5 ng of mRNA for RbAp48 (lane 7). Oocytes were maintained for 6 h to allow translation and then microinjected with 0.5 ng of double-stranded DNA for TRβA promoter. After overnight incubation, the transcript levels were analyzed by primer extension (top left). H4 serves as an internal control. The primer extension results were quantitated with a PhosphorImager and are represented graphically (top right). Protein expression was verified by labeling with [ 35 S]methionine (RPD3 or RbAp48) or by Western blotting (SIN3) (bottom left). The distribution of endogenous RPD3 between the nucleus and cytoplasm in the presence (+SIN3) or absence (−SIN3) of exogenous SIN3 was determined by detection of RPD3 by Western blotting (bottom right).

    Techniques Used: Histone Deacetylase Assay, Activity Assay, Western Blot, SDS Page, Expressing, Incubation, Labeling

    15) Product Images from "Phosphorylation of RNA polymerase II CTD regulates H3 methylation in yeast"

    Article Title: Phosphorylation of RNA polymerase II CTD regulates H3 methylation in yeast

    Journal: Genes & Development

    doi: 10.1101/gad.1055503

    Set2 methylates at the coding regions, as well as promoters, of genes. Chromatin immunoprecipitation assays were used to examine the H3 Lys 36 methylation status on several genes in vivo. Whole-cell extracts prepared from formaldehyde-fixed wild-type or set2 Δ cells were sonicated to shear their chromatin and then immunoprecipitated with either the H3 Lys 36 methylation-specific antibody [α-Me(Lys 36)H3], the H3 Lys 4 methylation-specific antibody [α-Me(Lys 4)H3], the CTD Ser 5 phosphorylation-specific antibody (H14 [α-Ser5P]), or the Ctk1-phosphorylated CTD antibody (α-Ctk1-PCTD). DNA from the enriched precipitates was isolated and used in PCR reactions with promoter or coding-specific primer pairs for the genes indicated. Primer pairs (labeled as A–D) include their sequence location relative to the translation initiation site. Although we show several genes positive for the presence of H3 Lys 36 methylation, other genes or distinct regions of chromatin examined ( HIS3 , rDNA, and telomeres) showed no enrichment for H3 Lys 36 methylation above background levels observed in set2 Δ (data not shown).
    Figure Legend Snippet: Set2 methylates at the coding regions, as well as promoters, of genes. Chromatin immunoprecipitation assays were used to examine the H3 Lys 36 methylation status on several genes in vivo. Whole-cell extracts prepared from formaldehyde-fixed wild-type or set2 Δ cells were sonicated to shear their chromatin and then immunoprecipitated with either the H3 Lys 36 methylation-specific antibody [α-Me(Lys 36)H3], the H3 Lys 4 methylation-specific antibody [α-Me(Lys 4)H3], the CTD Ser 5 phosphorylation-specific antibody (H14 [α-Ser5P]), or the Ctk1-phosphorylated CTD antibody (α-Ctk1-PCTD). DNA from the enriched precipitates was isolated and used in PCR reactions with promoter or coding-specific primer pairs for the genes indicated. Primer pairs (labeled as A–D) include their sequence location relative to the translation initiation site. Although we show several genes positive for the presence of H3 Lys 36 methylation, other genes or distinct regions of chromatin examined ( HIS3 , rDNA, and telomeres) showed no enrichment for H3 Lys 36 methylation above background levels observed in set2 Δ (data not shown).

    Techniques Used: Chromatin Immunoprecipitation, Methylation, In Vivo, Sonication, Immunoprecipitation, Isolation, Polymerase Chain Reaction, Labeling, Sequencing

    16) 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

    17) Product Images from "Protein Modulator of Multidrug Efflux Gene Expression in Pseudomonas aeruginosa ▿"

    Article Title: Protein Modulator of Multidrug Efflux Gene Expression in Pseudomonas aeruginosa ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00543-07

    ) were incubated in 10-μl (final volume) mixtures and subjected to nondenaturing polyacrylamide (12%, wt/vol) gel electrophoresis under reducing conditions at 77 V for 3 h at room temperature. Note that PA3719 has a high pI and migrates in the opposite direction when it is not in a complex with MexR. Thus, a control experiment run with PA3719 and operator DNA only shows no PA3719 and free, unshifted DNA (data not shown).
    Figure Legend Snippet: ) were incubated in 10-μl (final volume) mixtures and subjected to nondenaturing polyacrylamide (12%, wt/vol) gel electrophoresis under reducing conditions at 77 V for 3 h at room temperature. Note that PA3719 has a high pI and migrates in the opposite direction when it is not in a complex with MexR. Thus, a control experiment run with PA3719 and operator DNA only shows no PA3719 and free, unshifted DNA (data not shown).

    Techniques Used: Incubation, Nucleic Acid Electrophoresis

    PA3719 and MexR control of in vitro mexA transcription. (A) Impact of PA3719 on MexR repression of mexA transcription. Phage T7 RNA polymerase-based in vitro transcription reactions were initiated from a 836-bp PCR-generated template (250 ng) carrying a T7 promoter and mexA sequences extending from the MexR operator to 585 bp into the mexA gene (lanes 1 to 4). The results for control reactions with a T7 promoter-containing DNA template, pTRI-Xef, provided with the T7 in vitro transcription kit, are shown in lanes 5 to 7. Reactions were carried out at 37°C for 3 h, and RNA was purified and subjected to electrophoresis prior to visualization with ethidium bromide. MexR (1 μM) and PA3719 (150 nM [+ 1 ] or 500 nM [+ 2] ) were included in the reaction mixtures as indicated. Equal volumes of RNA were loaded in each instance. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 1.1 μg; lane 3, 2.0 μg; lane 4, 4.2 μg; lane 5, 1.7 μg; lane 6, 1.6 μg; and lane 7, 1.6 μg. (B) Impact of PA3719 on mexA transcription. In vitro transcription of the mex (lanes 1 to 3) and pTRI-Xef (lanes 4 to 6) templates was carried out as described above with or without PA3719 (500 nM [+ 2 ] or 2 μM [+ 3 ]), as indicated. The following amounts of RNA were produced: lane 1, 4.4 μg; lane 2, 4.5 μg; lane 3, 4.6 μg; lane 4, 4.6 μg; lane 5, 4.6 μg; and lane 6, 4.4 μg. (C) Differential impact of PA3719 on the repressor activities of MexR and MexR I104F . In vitro transcription of the mex (lanes 1 to 5) and pTRI-Xef (lanes 6 to 8) templates was carried out as described above with or without PA3719 (500 nM) and MexR/MexR I104F (1 μM), as indicated. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 0.8 μg; lane 3, 3.8 μg; lane 4, 0.9 μg; lane 5, 1.9 μg; lane 6, 6.6 μg; lane 7, 6.5 μg; and lane 8, 6.4 μg. RNA was quantitated prior to loading, and the values reported reflect the amount loaded and not the total amount produced. Because reaction, recovery, and loading volumes were kept constant throughout, these values accurately reflect the relative transcript levels produced in each instance.
    Figure Legend Snippet: PA3719 and MexR control of in vitro mexA transcription. (A) Impact of PA3719 on MexR repression of mexA transcription. Phage T7 RNA polymerase-based in vitro transcription reactions were initiated from a 836-bp PCR-generated template (250 ng) carrying a T7 promoter and mexA sequences extending from the MexR operator to 585 bp into the mexA gene (lanes 1 to 4). The results for control reactions with a T7 promoter-containing DNA template, pTRI-Xef, provided with the T7 in vitro transcription kit, are shown in lanes 5 to 7. Reactions were carried out at 37°C for 3 h, and RNA was purified and subjected to electrophoresis prior to visualization with ethidium bromide. MexR (1 μM) and PA3719 (150 nM [+ 1 ] or 500 nM [+ 2] ) were included in the reaction mixtures as indicated. Equal volumes of RNA were loaded in each instance. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 1.1 μg; lane 3, 2.0 μg; lane 4, 4.2 μg; lane 5, 1.7 μg; lane 6, 1.6 μg; and lane 7, 1.6 μg. (B) Impact of PA3719 on mexA transcription. In vitro transcription of the mex (lanes 1 to 3) and pTRI-Xef (lanes 4 to 6) templates was carried out as described above with or without PA3719 (500 nM [+ 2 ] or 2 μM [+ 3 ]), as indicated. The following amounts of RNA were produced: lane 1, 4.4 μg; lane 2, 4.5 μg; lane 3, 4.6 μg; lane 4, 4.6 μg; lane 5, 4.6 μg; and lane 6, 4.4 μg. (C) Differential impact of PA3719 on the repressor activities of MexR and MexR I104F . In vitro transcription of the mex (lanes 1 to 5) and pTRI-Xef (lanes 6 to 8) templates was carried out as described above with or without PA3719 (500 nM) and MexR/MexR I104F (1 μM), as indicated. The following amounts of RNA were produced: lane 1, 4.2 μg; lane 2, 0.8 μg; lane 3, 3.8 μg; lane 4, 0.9 μg; lane 5, 1.9 μg; lane 6, 6.6 μg; lane 7, 6.5 μg; and lane 8, 6.4 μg. RNA was quantitated prior to loading, and the values reported reflect the amount loaded and not the total amount produced. Because reaction, recovery, and loading volumes were kept constant throughout, these values accurately reflect the relative transcript levels produced in each instance.

    Techniques Used: In Vitro, Polymerase Chain Reaction, Generated, Purification, Electrophoresis, Produced

    18) 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

    19) Product Images from "Guanosine triphosphate acts as a cofactor to promote assembly of initial P-element transposase-DNA synaptic complexes"

    Article Title: Guanosine triphosphate acts as a cofactor to promote assembly of initial P-element transposase-DNA synaptic complexes

    Journal: Genes & Development

    doi: 10.1101/gad.1317605

    The P-element transposition reaction. ( A ) P-element transposition takes place in two stages, donor cleavage and target joining or integration. Following DNA repair, a new P-element insertion is created. ( B ) SDS-polyacrylamide gel electrophoresis analysis of purifed P-element transposase after elution indicated by arrow (lane 1 ), and the resin after elution (lane 2 ), stained with Coomassie blue. Lane M is protein molecular weight standards, indicated in kilodaltons at left . ( C ) LM-PCR assay for donor DNA cleavage at the P-element ends analyzed by polyacrylamide gel electrophoresis using untagged transposase (H0.1) (lanes 1,2 ) or the 3′ Py-tagged transposase (lanes 3,4 ), either with (+) or without (-) added GTP. Lane M is a 100-bp DNA marker, indicated in base pairs at left .
    Figure Legend Snippet: The P-element transposition reaction. ( A ) P-element transposition takes place in two stages, donor cleavage and target joining or integration. Following DNA repair, a new P-element insertion is created. ( B ) SDS-polyacrylamide gel electrophoresis analysis of purifed P-element transposase after elution indicated by arrow (lane 1 ), and the resin after elution (lane 2 ), stained with Coomassie blue. Lane M is protein molecular weight standards, indicated in kilodaltons at left . ( C ) LM-PCR assay for donor DNA cleavage at the P-element ends analyzed by polyacrylamide gel electrophoresis using untagged transposase (H0.1) (lanes 1,2 ) or the 3′ Py-tagged transposase (lanes 3,4 ), either with (+) or without (-) added GTP. Lane M is a 100-bp DNA marker, indicated in base pairs at left .

    Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Molecular Weight, Polymerase Chain Reaction, Marker

    20) 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

    21) Product Images from "Selective DNA Binding and Association with the CREB Binding Protein Coactivator Contribute to Differential Activation of Alpha/Beta Interferon Genes by Interferon Regulatory Factors 3 and 7"

    Article Title: Selective DNA Binding and Association with the CREB Binding Protein Coactivator Contribute to Differential Activation of Alpha/Beta Interferon Genes by Interferon Regulatory Factors 3 and 7

    Journal: Molecular and Cellular Biology

    doi:

    Amino acids of IRF-7 involved in different DNA binding specificities. (A) Sequence alignment of the DBDs of IRF-7, IRF-3, IRF-2, and IRF-1. The sequential numbering of IRF-7 is shown at the top. Identical residues in IRF-1, IRF-2, and IRF-3 but not IRF-7 are shown in boldface type. (B) Binding of mutated forms of IRF-3 or IRF3 and IRF7 chimeric recombinant proteins to the PRDI-like- and TG sites from the IFNA1 and IFNA2 promoter s. An EMSA was performed with the indicated amounts of recombinant GST fusion proteins and 32 P-labeled probes corresponding to the following PRDI-like sites: IFNA1, 5′-GGAAAGCAAAAACAGAAATGGAAAGTGG-3′; and IFNA2, 5′-GAAAGCAAAAAGAGAAGTAGAAAGTAA-3′.
    Figure Legend Snippet: Amino acids of IRF-7 involved in different DNA binding specificities. (A) Sequence alignment of the DBDs of IRF-7, IRF-3, IRF-2, and IRF-1. The sequential numbering of IRF-7 is shown at the top. Identical residues in IRF-1, IRF-2, and IRF-3 but not IRF-7 are shown in boldface type. (B) Binding of mutated forms of IRF-3 or IRF3 and IRF7 chimeric recombinant proteins to the PRDI-like- and TG sites from the IFNA1 and IFNA2 promoter s. An EMSA was performed with the indicated amounts of recombinant GST fusion proteins and 32 P-labeled probes corresponding to the following PRDI-like sites: IFNA1, 5′-GGAAAGCAAAAACAGAAATGGAAAGTGG-3′; and IFNA2, 5′-GAAAGCAAAAAGAGAAGTAGAAAGTAA-3′.

    Techniques Used: Binding Assay, Sequencing, Recombinant, Labeling

    DNA binding activity of IRF-7/3 chimeric proteins. (A) An EMSA was performed with whole-cell extracts (20 μg) derived from 293 cells transfected with various Flag-tagged IRF-7 or IRF-7/3 expression plasmids. At 24 h posttransfection, cells were infected with Sendai virus for 6 h (+) or left uninfected (−), as indicated. The 32 P-labeled probe corresponded to the PRDI-PRDIII (5′-GAAAACTGAAAGGGAGAAGTGAAAGTG-3′) motif of the IFNB promoter. (B) Twenty micrograms of whole-cell extracts from panel A was analyzed by immunoblotting (IB) with anti-Flag antibody. F7(2D), F 7 D477/479.
    Figure Legend Snippet: DNA binding activity of IRF-7/3 chimeric proteins. (A) An EMSA was performed with whole-cell extracts (20 μg) derived from 293 cells transfected with various Flag-tagged IRF-7 or IRF-7/3 expression plasmids. At 24 h posttransfection, cells were infected with Sendai virus for 6 h (+) or left uninfected (−), as indicated. The 32 P-labeled probe corresponded to the PRDI-PRDIII (5′-GAAAACTGAAAGGGAGAAGTGAAAGTG-3′) motif of the IFNB promoter. (B) Twenty micrograms of whole-cell extracts from panel A was analyzed by immunoblotting (IB) with anti-Flag antibody. F7(2D), F 7 D477/479.

    Techniques Used: Binding Assay, Activity Assay, Derivative Assay, Transfection, Expressing, Infection, Labeling

    Gel shift analysis of selected IRF-3 and IRF-7 binding sites. Oligonucleotides selected with recombinant IRF-3–GST (lanes 1 to 5) or IRF-7–GST (lanes 6 to 10) at each round were amplified by PCR using 32 P-labeled primers and subsequently used as probes in a gel shift analysis. Fifty nanograms of IRF-3–GST (A) or IRF-7–GST (B) was used in each binding reaction. The number of selection cycles is shown above each lane. Arrows indicate protein-DNA complexes.
    Figure Legend Snippet: Gel shift analysis of selected IRF-3 and IRF-7 binding sites. Oligonucleotides selected with recombinant IRF-3–GST (lanes 1 to 5) or IRF-7–GST (lanes 6 to 10) at each round were amplified by PCR using 32 P-labeled primers and subsequently used as probes in a gel shift analysis. Fifty nanograms of IRF-3–GST (A) or IRF-7–GST (B) was used in each binding reaction. The number of selection cycles is shown above each lane. Arrows indicate protein-DNA complexes.

    Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay, Recombinant, Amplification, Polymerase Chain Reaction, Labeling, Selection

    22) 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:

    23) Product Images from "Lytic gene expression in the temperate bacteriophage GIL01 is activated by a phage-encoded LexA homologue"

    Article Title: Lytic gene expression in the temperate bacteriophage GIL01 is activated by a phage-encoded LexA homologue

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky646

    LexA and gp6 can simultaneously bind to the P3 promoter region. ( A ) The binding of purified LexA and gp6 protein to a P 32 end-labelled P3 promoter fragment was assayed using EMSA. The concentration of LexA used was 0.2, 0.4 and 0.8 μM in lanes 6–8 and 11–13, respectively. The concentration of gp6 used was 0.3, 0.6, 1.35 μM in lanes 2–4 and 1.35 μM in lanes 10–13. The location of free DNA and the gp6, LexA and LexA–gp6–DNA complexes is marked. Above the EMSA is a schematic representation of the P3 promoter fragment used for this EMSA analysis. The promoter elements are marked with black boxes, orange boxes show the position of LexA dinBox 2 and dinBox 3, and the green pentagon represents the gp6-binding site. The GIL01 genome coordinates of this fragment are also given. ( B ) A DNase I footprint experiment investigating the binding of gp6, in the presence or absence of LexA, to a P 32 end-labelled P3 promoter fragment (GIL01 genome coordinates 4801–5000). The gel was calibrated using Maxam–Gilbert G+A sequencing reactions of the labelled fragment (designated as GA), and selected positions are indicated. The concentration of gp6 used in lanes 2–4 and 6–8 was 0.3, 0.6 and 1.35 μM and the concentration of LexA used in lanes 5–8 was 0.8 μM. The location of the gp6 and LexA-binding sites and the P3 promoter elements are shown.
    Figure Legend Snippet: LexA and gp6 can simultaneously bind to the P3 promoter region. ( A ) The binding of purified LexA and gp6 protein to a P 32 end-labelled P3 promoter fragment was assayed using EMSA. The concentration of LexA used was 0.2, 0.4 and 0.8 μM in lanes 6–8 and 11–13, respectively. The concentration of gp6 used was 0.3, 0.6, 1.35 μM in lanes 2–4 and 1.35 μM in lanes 10–13. The location of free DNA and the gp6, LexA and LexA–gp6–DNA complexes is marked. Above the EMSA is a schematic representation of the P3 promoter fragment used for this EMSA analysis. The promoter elements are marked with black boxes, orange boxes show the position of LexA dinBox 2 and dinBox 3, and the green pentagon represents the gp6-binding site. The GIL01 genome coordinates of this fragment are also given. ( B ) A DNase I footprint experiment investigating the binding of gp6, in the presence or absence of LexA, to a P 32 end-labelled P3 promoter fragment (GIL01 genome coordinates 4801–5000). The gel was calibrated using Maxam–Gilbert G+A sequencing reactions of the labelled fragment (designated as GA), and selected positions are indicated. The concentration of gp6 used in lanes 2–4 and 6–8 was 0.3, 0.6 and 1.35 μM and the concentration of LexA used in lanes 5–8 was 0.8 μM. The location of the gp6 and LexA-binding sites and the P3 promoter elements are shown.

    Techniques Used: Binding Assay, Purification, Concentration Assay, Sequencing

    Real-time analysis of LexA and gp6 binding to the P3 promoter. The figure shows SPR sensorgrams of ( A ) LexA, ( B ) gp6 and ( C ) LexA and gp6 binding to an immobilized P3 promoter fragment. Sensorgrams are also shown of gp6 binding a shorter 36 bp DNA fragment, carrying the ( D ) wild-type and ( E ) a mutated gp6 target site. Proteins were injected over the immobilized DNA (∼30 RU) for 120 s at 100 μl/min. The DNA fragments used in these experiments are schematically represented above the graphs. The promoter elements are marked with black boxes, orange boxes show the position of LexA dinBox 2 and dinBox 3, and the green pentagon represents the gp6-binding site. The GIL01 genome coordinates of fragments are also given. In panels (D) and (E) the gp6-binding site, as determined by DNase I footprint analysis, is shown boxed, and in (E), base substitutions are shown in red. In each case, representative sensorgrams are shown and the experiments were performed in duplicate.
    Figure Legend Snippet: Real-time analysis of LexA and gp6 binding to the P3 promoter. The figure shows SPR sensorgrams of ( A ) LexA, ( B ) gp6 and ( C ) LexA and gp6 binding to an immobilized P3 promoter fragment. Sensorgrams are also shown of gp6 binding a shorter 36 bp DNA fragment, carrying the ( D ) wild-type and ( E ) a mutated gp6 target site. Proteins were injected over the immobilized DNA (∼30 RU) for 120 s at 100 μl/min. The DNA fragments used in these experiments are schematically represented above the graphs. The promoter elements are marked with black boxes, orange boxes show the position of LexA dinBox 2 and dinBox 3, and the green pentagon represents the gp6-binding site. The GIL01 genome coordinates of fragments are also given. In panels (D) and (E) the gp6-binding site, as determined by DNase I footprint analysis, is shown boxed, and in (E), base substitutions are shown in red. In each case, representative sensorgrams are shown and the experiments were performed in duplicate.

    Techniques Used: Binding Assay, SPR Assay, Injection

    Regulation of the lytic/lysogenic switch in the Bacillus thuringiensis temperate phage GIL01. The figure shows the genetic map of GIL01, highlighting key genes and regulatory sites. ( A ) Maintenance of the GIL01 lysogenic state. Host LexA protein, in conjunction with the product of ORF7, gp7, represses the expression of phage functions directed from the P1, P2 and P3 promoters to maintain lysogeny. ( B ) The lytic cycle. Upon persistent DNA damage, LexA undergoes auto-cleavage and its cellular concentration drops below a threshold level, which results in derepression of P1 and P2 , and high-level expression of the replication and regulatory genes to initiate the lytic cycle. Substantial intracellular accumulation of gp6 protein activates transcription from P3 , (located between ORF8 and ORF9), resulting in the expression of the downstream phage structural and lysis genes and eventual host cell lysis and death.
    Figure Legend Snippet: Regulation of the lytic/lysogenic switch in the Bacillus thuringiensis temperate phage GIL01. The figure shows the genetic map of GIL01, highlighting key genes and regulatory sites. ( A ) Maintenance of the GIL01 lysogenic state. Host LexA protein, in conjunction with the product of ORF7, gp7, represses the expression of phage functions directed from the P1, P2 and P3 promoters to maintain lysogeny. ( B ) The lytic cycle. Upon persistent DNA damage, LexA undergoes auto-cleavage and its cellular concentration drops below a threshold level, which results in derepression of P1 and P2 , and high-level expression of the replication and regulatory genes to initiate the lytic cycle. Substantial intracellular accumulation of gp6 protein activates transcription from P3 , (located between ORF8 and ORF9), resulting in the expression of the downstream phage structural and lysis genes and eventual host cell lysis and death.

    Techniques Used: Expressing, Concentration Assay, Lysis

    24) 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

    25) Product Images from "Increasing cleavage specificity and activity of restriction endonuclease KpnI"

    Article Title: Increasing cleavage specificity and activity of restriction endonuclease KpnI

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt734

    Binding kinetics to the canonical site. DNA-binding kinetics of WT KpnI and the mutants was assayed by SPR spectroscopy. Biotinylated oligonucleotide harboring the canonical (-GGTACC-) site was immobilized onto a streptavidin chip. The proteins were passed over the DNA with restricted flow rates to measure the association and dissociation rates, as described in ‘Materials and Methods’ section. ( A ) Sensorgrams of WT, D16N, D148E and D16N/D148E. Graphical representation of the association ( B ) and dissociation ( C ) rates of the proteins is shown.
    Figure Legend Snippet: Binding kinetics to the canonical site. DNA-binding kinetics of WT KpnI and the mutants was assayed by SPR spectroscopy. Biotinylated oligonucleotide harboring the canonical (-GGTACC-) site was immobilized onto a streptavidin chip. The proteins were passed over the DNA with restricted flow rates to measure the association and dissociation rates, as described in ‘Materials and Methods’ section. ( A ) Sensorgrams of WT, D16N, D148E and D16N/D148E. Graphical representation of the association ( B ) and dissociation ( C ) rates of the proteins is shown.

    Techniques Used: Binding Assay, SPR Assay, Spectroscopy, Chromatin Immunoprecipitation, Flow Cytometry

    Molecular basis for the high fidelity of variant D148E. ( A ) Mg 2+ -dependent DNA cleavage of mutant D148E. Different concentrations of metal ions (0–10 mM) were incubated with 1 unit of WT or mutant D148E in a buffer containing 10 mM Tris–HCl (pH 7.4), on ice for 5 min. DNA cleavage reaction was initiated by the addition of (i) pUC18 DNA (14 nM) or (ii) end labeled canonical oligonucleotide (10 nM) and incubated at 37°C for 1 h. (iii) Graphical representation of the Mg 2+ activation profile of WT and D148E variant on the oligonucleotide substrate. Mutant D148E requires a Mg 2+ concentration of 200 μM for the 90% digestion of the substrate, compared with 50 μM required by WT, showing that the decrease in binding affinity toward Mg 2+ ( Figure 2 A) directly affects the cleavage activity of KpnI. ( B ) Canonical versus non-canonical discrimination by mutant D148E. EMSA was carried to determine the binding affinity of mutant D148E to end-labeled canonical (GGTACC) and non-canonical (GaTACC) substrates over a range of enzyme concentrations between 0 and 256 nM. The intensity of the shifted DNA band is expressed as percentage bound. ( C ) Mutant D148E does not cleave star substrate at binding concentrations. End-labeled oligonucleotide duplex containing the 5′-GaTACC-3′ sequence (10 nM) was incubated with WT (2.5–10 nM) or mutant D148E (100–500 nM) at 37°C for 30 min in the presence of 2 mM Mg 2+ . The cleavage products were analyzed on 12% urea-PAGE. Lane C contains the substrate DNA without the enzyme. S and P represent the substrate and product, respectively.
    Figure Legend Snippet: Molecular basis for the high fidelity of variant D148E. ( A ) Mg 2+ -dependent DNA cleavage of mutant D148E. Different concentrations of metal ions (0–10 mM) were incubated with 1 unit of WT or mutant D148E in a buffer containing 10 mM Tris–HCl (pH 7.4), on ice for 5 min. DNA cleavage reaction was initiated by the addition of (i) pUC18 DNA (14 nM) or (ii) end labeled canonical oligonucleotide (10 nM) and incubated at 37°C for 1 h. (iii) Graphical representation of the Mg 2+ activation profile of WT and D148E variant on the oligonucleotide substrate. Mutant D148E requires a Mg 2+ concentration of 200 μM for the 90% digestion of the substrate, compared with 50 μM required by WT, showing that the decrease in binding affinity toward Mg 2+ ( Figure 2 A) directly affects the cleavage activity of KpnI. ( B ) Canonical versus non-canonical discrimination by mutant D148E. EMSA was carried to determine the binding affinity of mutant D148E to end-labeled canonical (GGTACC) and non-canonical (GaTACC) substrates over a range of enzyme concentrations between 0 and 256 nM. The intensity of the shifted DNA band is expressed as percentage bound. ( C ) Mutant D148E does not cleave star substrate at binding concentrations. End-labeled oligonucleotide duplex containing the 5′-GaTACC-3′ sequence (10 nM) was incubated with WT (2.5–10 nM) or mutant D148E (100–500 nM) at 37°C for 30 min in the presence of 2 mM Mg 2+ . The cleavage products were analyzed on 12% urea-PAGE. Lane C contains the substrate DNA without the enzyme. S and P represent the substrate and product, respectively.

    Techniques Used: Variant Assay, Mutagenesis, Incubation, Labeling, Activation Assay, Concentration Assay, Binding Assay, Activity Assay, Sequencing, Polyacrylamide Gel Electrophoresis

    Mutant D148E exhibits high fidelity DNA cleavage. ( A ) HNH motif of KpnI. The KpnI monomer model is shown as a ribbon with helices as spirals and strands as arrows. The Mg 2+ and Zn 2+ ions are shown as spheres. The side chains of active site residues, D148, H149 and Q175, are shown as sticks and labeled. ( B ) Effect of D148E mutation of KpnI on DNA cleavage specificity. Plasmid DNA pUC18 (14 nM) was incubated with various concentrations (25, 50 and 100 nM) of WT or mutant enzyme for 1 h at 37°C in the presence of 5 mM Mg 2+ and analyzed on 1% agarose gel. C indicates DNA cleavage reaction in the absence of enzyme. Nc, L and Sc indicate the positions of nicked circular, linear and supercoiled forms of the plasmid, respectively. The asterisk denotes the promiscuous DNA cleavage products.
    Figure Legend Snippet: Mutant D148E exhibits high fidelity DNA cleavage. ( A ) HNH motif of KpnI. The KpnI monomer model is shown as a ribbon with helices as spirals and strands as arrows. The Mg 2+ and Zn 2+ ions are shown as spheres. The side chains of active site residues, D148, H149 and Q175, are shown as sticks and labeled. ( B ) Effect of D148E mutation of KpnI on DNA cleavage specificity. Plasmid DNA pUC18 (14 nM) was incubated with various concentrations (25, 50 and 100 nM) of WT or mutant enzyme for 1 h at 37°C in the presence of 5 mM Mg 2+ and analyzed on 1% agarose gel. C indicates DNA cleavage reaction in the absence of enzyme. Nc, L and Sc indicate the positions of nicked circular, linear and supercoiled forms of the plasmid, respectively. The asterisk denotes the promiscuous DNA cleavage products.

    Techniques Used: Mutagenesis, Labeling, Plasmid Preparation, Incubation, Agarose Gel Electrophoresis

    Ca 2+ ion binds to KpnI D148E but does not support DNA cleavage. ( A ) Fluorescence emission spectra of WT and mutant D148E in the presence of Ca 2+ and Mg 2+ with increasing amounts of CaCl 2 (Blue) or MgCl 2 (Red) (0–10 mM). Representative spectra and the calculated K d values are shown. ( B ) Metal ion competition assay. Reactions were carried out by assaying Mg 2+ -mediated DNA cleavage activity in the presence of titrating Ca 2+ ions. Reactions contained 2.5 nM D148E and competition was performed by pre-incubation of the enzyme in a buffer containing 10 mM Tris–HCl (pH 7.4), on ice with the indicated metal ions. The reactions were initiated by addition of pUC18 DNA (14 nM) and incubated at 37°C for 1 h. Enhanced solvent accessibility of His149 in the presence of Ca 2+ . KpnI WT or mutant D148E enzymes were pre-incubated with 5 mM of ( C ) CaCl 2 or ( D ) MgCl 2 and treated with increasing concentrations (25–100 μM) of DEPC. Residual activity was assayed by cleavage of pUC18 DNA (14 nM) in the presence of 2 mM MgCl 2 .
    Figure Legend Snippet: Ca 2+ ion binds to KpnI D148E but does not support DNA cleavage. ( A ) Fluorescence emission spectra of WT and mutant D148E in the presence of Ca 2+ and Mg 2+ with increasing amounts of CaCl 2 (Blue) or MgCl 2 (Red) (0–10 mM). Representative spectra and the calculated K d values are shown. ( B ) Metal ion competition assay. Reactions were carried out by assaying Mg 2+ -mediated DNA cleavage activity in the presence of titrating Ca 2+ ions. Reactions contained 2.5 nM D148E and competition was performed by pre-incubation of the enzyme in a buffer containing 10 mM Tris–HCl (pH 7.4), on ice with the indicated metal ions. The reactions were initiated by addition of pUC18 DNA (14 nM) and incubated at 37°C for 1 h. Enhanced solvent accessibility of His149 in the presence of Ca 2+ . KpnI WT or mutant D148E enzymes were pre-incubated with 5 mM of ( C ) CaCl 2 or ( D ) MgCl 2 and treated with increasing concentrations (25–100 μM) of DEPC. Residual activity was assayed by cleavage of pUC18 DNA (14 nM) in the presence of 2 mM MgCl 2 .

    Techniques Used: Fluorescence, Mutagenesis, Competitive Binding Assay, Activity Assay, Incubation

    26) 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

    27) Product Images from "Bacteriophage-Based Genetic System for Selection of Nonsplicing Inteins"

    Article Title: Bacteriophage-Based Genetic System for Selection of Nonsplicing Inteins

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.70.5.3158-3162.2004

    Genetic selection system for protein splicing. Lysis of E. coli cells by the T4 gp43 − phage requires complementation of the phage DNA polymerase defect. A plasmid-borne RB69 DNA polymerase containing an intein can complement the T4 gp43 − phage defect only if protein splicing occurs. See the text for details.
    Figure Legend Snippet: Genetic selection system for protein splicing. Lysis of E. coli cells by the T4 gp43 − phage requires complementation of the phage DNA polymerase defect. A plasmid-borne RB69 DNA polymerase containing an intein can complement the T4 gp43 − phage defect only if protein splicing occurs. See the text for details.

    Techniques Used: Selection, Lysis, Plasmid Preparation

    Construction of pTli Pol-2/I A and pTli Pol-2/I IN . (A) The sequence surrounding the RB69 family B DNA polymerase region I (underlined) in the native gene (i) and the mutated gene (ii) are shown with the engineered restriction enzymes sites also underlined. Schematic diagram of the RB69 DNA polymerase (white boxes, exteins) and Pol-2 Tli intein (gray box) precursor containing either an active intein (B) pTli Pol-2/I A ) or an inactive intein (C) (pTli Pol-2/I IN ). Splicing is required to generate a functional RB69 DNA polymerase. Intein amino acid sequences are indicated above the precursor, and DNA polymerase sequences (exteins) are indicated below. pTli Pol-2/I IN has a Ser1-to-Ala mutation that results in C-terminal cleavage in the absence of splicing.
    Figure Legend Snippet: Construction of pTli Pol-2/I A and pTli Pol-2/I IN . (A) The sequence surrounding the RB69 family B DNA polymerase region I (underlined) in the native gene (i) and the mutated gene (ii) are shown with the engineered restriction enzymes sites also underlined. Schematic diagram of the RB69 DNA polymerase (white boxes, exteins) and Pol-2 Tli intein (gray box) precursor containing either an active intein (B) pTli Pol-2/I A ) or an inactive intein (C) (pTli Pol-2/I IN ). Splicing is required to generate a functional RB69 DNA polymerase. Intein amino acid sequences are indicated above the precursor, and DNA polymerase sequences (exteins) are indicated below. pTli Pol-2/I IN has a Ser1-to-Ala mutation that results in C-terminal cleavage in the absence of splicing.

    Techniques Used: Sequencing, Functional Assay, Mutagenesis

    Complementation of the DNA polymerase defect in T4 gp43 − phage. Cells expressing active intein fusions (pTli Pol-2/I A ) (A) or inactive intein fusions (pTli Pol-2/I IN ) (B) were challenged with no phage (plates I), T4 gp43 − phage (plates II), or wild-type T4 phage (plates III) at the indicated temperatures.
    Figure Legend Snippet: Complementation of the DNA polymerase defect in T4 gp43 − phage. Cells expressing active intein fusions (pTli Pol-2/I A ) (A) or inactive intein fusions (pTli Pol-2/I IN ) (B) were challenged with no phage (plates I), T4 gp43 − phage (plates II), or wild-type T4 phage (plates III) at the indicated temperatures.

    Techniques Used: Expressing

    28) 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

    29) 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

    30) 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:

    31) Product Images from "PCR-based generation of shRNA libraries from cDNAs"

    Article Title: PCR-based generation of shRNA libraries from cDNAs

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-6-28

    Generation of shRNA constructs using YIU. (A) Intermediate products produced during the YIU procedure. Lanes 2 and 3. The Y oligonucleotide, designed with a single 3' T overhang and therefore unable to self-ligate. Lane 2, without ligase. Lane 3, with ligase. Note that the forked shape of the Y oligonucleotide causes abnormal mobility on PAGE. The ligated and MmeI-digested YI molecules are indicated by an arrowhead in lane 4; the digested YI molecules shift in migration when ligated to the U oligonucleotide (arrowhead, lane 5). Note that the YIU molecules are a minor fraction of the product molecules because the Y and U oligonucleotides were added at a large molar excess over cDNAs and that the ligation was efficient, as almost all of the U oligonucleotides have been converted to dimers. (B) PCR amplification of YIU ligation products. The template used for PCR in lane 2 was the PAGE-purified DNA band corresponding to the one labeled with arrowhead in lane 5 of Panel A. The template for lane 3 was the whole YIU ligation mixture shown in lane 5 of Panel A. The calculated size of YIU double-stranded DNA is 160 bp. (C) Conversion of the X molecule into double-stranded DNA by a
    Figure Legend Snippet: Generation of shRNA constructs using YIU. (A) Intermediate products produced during the YIU procedure. Lanes 2 and 3. The Y oligonucleotide, designed with a single 3' T overhang and therefore unable to self-ligate. Lane 2, without ligase. Lane 3, with ligase. Note that the forked shape of the Y oligonucleotide causes abnormal mobility on PAGE. The ligated and MmeI-digested YI molecules are indicated by an arrowhead in lane 4; the digested YI molecules shift in migration when ligated to the U oligonucleotide (arrowhead, lane 5). Note that the YIU molecules are a minor fraction of the product molecules because the Y and U oligonucleotides were added at a large molar excess over cDNAs and that the ligation was efficient, as almost all of the U oligonucleotides have been converted to dimers. (B) PCR amplification of YIU ligation products. The template used for PCR in lane 2 was the PAGE-purified DNA band corresponding to the one labeled with arrowhead in lane 5 of Panel A. The template for lane 3 was the whole YIU ligation mixture shown in lane 5 of Panel A. The calculated size of YIU double-stranded DNA is 160 bp. (C) Conversion of the X molecule into double-stranded DNA by a "PCR+1" protocol. PCR amplification of YIU with 20 cycles results in an extra band (the X molecule) seen in the 200–300 bp region on agarose gels (lane 2). However, when the PCR products are diluted with fresh PCR reaction mix (1 × buffer, dNTPs, primers and enzyme) and subjected to one additional PCR cycle, the high molecular weight band is lost (lane 3). (D) Scheme of the generation of X molecules. Vent polymerase, through its strand-displacement activity, can open hairpin structures to generate fully double-stranded products. As the PCR progresses, however, YIU double-stranded DNA molecules accumulate and the concentration of primers decreases. When the templates are heat denatured and cooled, two heterogeneous molecules (shown in red or purple) can form an X-shaped (cruciform) heterodimer via the common complementary regions (shown in green). (E) Restriction digestion of double-stranded DNA of YIU. The YIU double-stranded DNA generated by PCR (the "uncut" lane) is digested with AflII or MlyI individually, or in combination to generate suitable inserts for cloning.

    Techniques Used: shRNA, Construct, Produced, Polyacrylamide Gel Electrophoresis, Migration, Ligation, Polymerase Chain Reaction, Amplification, Purification, Labeling, Molecular Weight, Activity Assay, Concentration Assay, Generated, Clone Assay

    A . Total RNA isolated from HEK293T cells transfected with either a
    Figure Legend Snippet: A . Total RNA isolated from HEK293T cells transfected with either a "classic" or "YIU" construct targeting luciferase was analyzed by Northern blot hybridization to a radioactively labeled luciferase 19-nt sense-strand oligonucleotide and to a 19-nt oligonucleotide complementary to 5S RNA. 19- and 27-nt single-stranded DNA oligonucleotides were used as size standards; because DNA rather than RNA oligonucleotides were used, the indicated sizes are only approximate. The expected approximate sizes of the hairpin transcripts are 49 nt ("classic") and 62 nt ("YIU"), whereas both processed forms are expected to be ~21 nt. B . and C . Constructs expressing luciferase mRNA fused with a fragment of CCND1 mRNA were cotransfected with shRNA expression constructs targeting either luciferase or CCND1 , as described in Table 1. The negative control shRNA expression construct was a "classic" construct targeting BCL2 . Samples were analyzed in duplicate, and values were normalized using Renilla luciferase expression. Error bars represent the standard deviation. The shRNA constructs targeting CCND1 was cotransfected with a luciferase- CCND1 fusion construct containing the target sequence, with the exception of #3, whose target sequence is absent from both fusion constructs. Construct #3 thus serves as an additional negative control. The small degree of suppression seen with #3 and the apparent small stimulation by some shRNA cassettes may be either due to experimental error or to off-target effects of #3 and of the control shRNA construct. The data shown are representative of three individual experiments, which all showed similar results.

    Techniques Used: Isolation, Transfection, Construct, Luciferase, Northern Blot, Hybridization, Labeling, Expressing, shRNA, Negative Control, Standard Deviation, Sequencing

    32) 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

    33) Product Images from "Isolation of Helicobacter pylori Genes That Modulate Urease Activity"

    Article Title: Isolation of Helicobacter pylori Genes That Modulate Urease Activity

    Journal: Journal of Bacteriology

    doi:

    Schematic maps of pUDF104 and pBS- flbA ). The size of each complete gene and the size of the total insert are shown. The flbA gene plus flanking DNA was subcloned into the Bam HI (B) and Cla I (C) sites in pBluescript by PCR as described in the Materials and Methods section to yield pBS- flbA . Truncated genes are denoted by jagged edges. H, Hin dIII; X, Xba I; N, Nhe I. Thick lines, pBluescript vector sequences. Arrows show directions of transcription.
    Figure Legend Snippet: Schematic maps of pUDF104 and pBS- flbA ). The size of each complete gene and the size of the total insert are shown. The flbA gene plus flanking DNA was subcloned into the Bam HI (B) and Cla I (C) sites in pBluescript by PCR as described in the Materials and Methods section to yield pBS- flbA . Truncated genes are denoted by jagged edges. H, Hin dIII; X, Xba I; N, Nhe I. Thick lines, pBluescript vector sequences. Arrows show directions of transcription.

    Techniques Used: Polymerase Chain Reaction, Plasmid Preparation

    34) 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

    35) 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

    36) Product Images from "RNA-binding strategies common to cold-shock domain- and RNA recognition motif-containing proteins"

    Article Title: RNA-binding strategies common to cold-shock domain- and RNA recognition motif-containing proteins

    Journal: Nucleic Acids Research

    doi:

    RNA sequences selected from a pool of randomised 25mers by RRM3 NUC and CSD FRG . ( A ) Sequence of the oligodeoxynucleotide used to generate the pool of random RNA transcripts. ( B and C ) Alignments of the sequences of the DNA fragments resulting from the retrotranscription of the aptamer RNAs selected by either RRM3 NUC (B) or CSD FRG (C). In each case the corresponding SELEX RNA consensus sequence is indicated below the alignment.
    Figure Legend Snippet: RNA sequences selected from a pool of randomised 25mers by RRM3 NUC and CSD FRG . ( A ) Sequence of the oligodeoxynucleotide used to generate the pool of random RNA transcripts. ( B and C ) Alignments of the sequences of the DNA fragments resulting from the retrotranscription of the aptamer RNAs selected by either RRM3 NUC (B) or CSD FRG (C). In each case the corresponding SELEX RNA consensus sequence is indicated below the alignment.

    Techniques Used: Sequencing

    37) 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

    38) 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

    39) Product Images from "Matrix Attachment Region-Dependent Function of the Immunoglobulin ? Enhancer Involves Histone Acetylation at a Distance without Changes in Enhancer Occupancy"

    Article Title: Matrix Attachment Region-Dependent Function of the Immunoglobulin ? Enhancer Involves Histone Acetylation at a Distance without Changes in Enhancer Occupancy

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.1.196-208.2001

    Acetylation of H3 and H4 histones in the VDJ region of wild-type and ΔMAR μ transgenes. Chromatin extracts from formaldehyde-cross-linked wild-type and ΔMAR μ transgenic pre-B cells were immunoprecipitated with α-AcH4, α-AcH3, or α-B220 (negative control) antibodies. The DNAs of the bound fractions were isolated, and the transgenic VDJ sequences in the immunoprecipitated DNA were quantified by PCR. The amplification of the promoter region of the endogenous mb-1 gene was done as an internal control. The levels of enrichment in the immunoprecipitations were estimated by comparison with the amplification products of DNA isolated from the bulk chromatin extracts (input DNA). Eight serial fourfold dilutions of template DNA were done to allow a quantitative determination in the PCR assays. The amounts of template DNA (in nanograms) were 20 (lane 1), 5 (lane 2), 1.25 (lane 3), 0.31 (lane 4), 0.078 (lane 5), 0.019 (lane 6), 0.009 (lane 7), and 0.002 (lane 8). The VDJ region of the wild-type μ transgenes was immunoprecipitated with the α-AcH4 and α-AcH3 antibodies more efficiently than the VDJ region of the ΔMAR μ transgenes, indicating a preferential acetylation of H4 and H3 in the VDJ region of MAR-containing transgenes. These differences are not observed in the endogenous mb-1 promoter control.
    Figure Legend Snippet: Acetylation of H3 and H4 histones in the VDJ region of wild-type and ΔMAR μ transgenes. Chromatin extracts from formaldehyde-cross-linked wild-type and ΔMAR μ transgenic pre-B cells were immunoprecipitated with α-AcH4, α-AcH3, or α-B220 (negative control) antibodies. The DNAs of the bound fractions were isolated, and the transgenic VDJ sequences in the immunoprecipitated DNA were quantified by PCR. The amplification of the promoter region of the endogenous mb-1 gene was done as an internal control. The levels of enrichment in the immunoprecipitations were estimated by comparison with the amplification products of DNA isolated from the bulk chromatin extracts (input DNA). Eight serial fourfold dilutions of template DNA were done to allow a quantitative determination in the PCR assays. The amounts of template DNA (in nanograms) were 20 (lane 1), 5 (lane 2), 1.25 (lane 3), 0.31 (lane 4), 0.078 (lane 5), 0.019 (lane 6), 0.009 (lane 7), and 0.002 (lane 8). The VDJ region of the wild-type μ transgenes was immunoprecipitated with the α-AcH4 and α-AcH3 antibodies more efficiently than the VDJ region of the ΔMAR μ transgenes, indicating a preferential acetylation of H4 and H3 in the VDJ region of MAR-containing transgenes. These differences are not observed in the endogenous mb-1 promoter control.

    Techniques Used: Transgenic Assay, Immunoprecipitation, Negative Control, Isolation, Polymerase Chain Reaction, Amplification

    40) Product Images from "Reconstituting ParA/ParB-mediated transport of DNA cargo"

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo

    Journal: Methods in cell biology

    doi: 10.1016/bs.mcb.2015.01.021

    Creating a supercoiled and fluorescent-labeled sopC-plasmid. The plasmid pBR322:: sopC is fluorescently labeled to visualize its movement over the DNA-carpeted flow cell. We have developed an efficient labeling protocol that does not require intercalating dyes and produces a negatively supercoiled plasmid. The restriction enzyme Nt.BspQ1 nicks the pBR322 backbone at a site located approximately 180° from sopC . DNA polymerase I is used with dNTPs and Alexa647-labeled dCTP to label the DNA. Ethidium Bromide promotes negative supercoiling before a final ligation reaction that covalently closes the nick. The final product is a negatively supercoiled and fluorescently labeled plasmid bearing the sopC centromere site. This protocol can be used to incorporate a variety of dyes without significant perturbation to plasmid topology.
    Figure Legend Snippet: Creating a supercoiled and fluorescent-labeled sopC-plasmid. The plasmid pBR322:: sopC is fluorescently labeled to visualize its movement over the DNA-carpeted flow cell. We have developed an efficient labeling protocol that does not require intercalating dyes and produces a negatively supercoiled plasmid. The restriction enzyme Nt.BspQ1 nicks the pBR322 backbone at a site located approximately 180° from sopC . DNA polymerase I is used with dNTPs and Alexa647-labeled dCTP to label the DNA. Ethidium Bromide promotes negative supercoiling before a final ligation reaction that covalently closes the nick. The final product is a negatively supercoiled and fluorescently labeled plasmid bearing the sopC centromere site. This protocol can be used to incorporate a variety of dyes without significant perturbation to plasmid topology.

    Techniques Used: Labeling, Plasmid Preparation, Flow Cytometry, Ligation

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    New England Biolabs vent dna polymerase
    <t>RT-PCR</t> amplification of spliced B4-B5 exons. The first lane contains a <t>DNA</t> ladder of markers (Stratagene). The second and third lanes, respectively, represent RNA isolated from the yeast null strain (QBY320) that contained plasmids expressing wild-type LeuRS from yeast and M. tuberculosis . The unspliced and spliced B4-B5 exon products are indicated by respective bands at about 1.5 kbp and 250 bp. The fourth and fifth lanes, respectively, show that the intron remains unspliced when genes containing either the W286C mutant or G288SΔC5 deletion of M. tuberculosis LeuRS are used in attempts to complement the yeast null strain.
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    RT-PCR amplification of spliced B4-B5 exons. The first lane contains a DNA ladder of markers (Stratagene). The second and third lanes, respectively, represent RNA isolated from the yeast null strain (QBY320) that contained plasmids expressing wild-type LeuRS from yeast and M. tuberculosis . The unspliced and spliced B4-B5 exon products are indicated by respective bands at about 1.5 kbp and 250 bp. The fourth and fifth lanes, respectively, show that the intron remains unspliced when genes containing either the W286C mutant or G288SΔC5 deletion of M. tuberculosis LeuRS are used in attempts to complement the yeast null strain.

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

    Article Title: A prokaryote and human tRNA synthetase provide an essential RNA splicing function in yeast mitochondria

    doi:

    Figure Lengend Snippet: RT-PCR amplification of spliced B4-B5 exons. The first lane contains a DNA ladder of markers (Stratagene). The second and third lanes, respectively, represent RNA isolated from the yeast null strain (QBY320) that contained plasmids expressing wild-type LeuRS from yeast and M. tuberculosis . The unspliced and spliced B4-B5 exon products are indicated by respective bands at about 1.5 kbp and 250 bp. The fourth and fifth lanes, respectively, show that the intron remains unspliced when genes containing either the W286C mutant or G288SΔC5 deletion of M. tuberculosis LeuRS are used in attempts to complement the yeast null strain.

    Article Snippet: The pC3 -Mtb plasmid was engineered by PCR amplification of the M. tuberculosis LeuRS gene from plasmid pLEU10 using Vent DNA polymerase (New England Biolabs) and 5′ and 3′ primers that introduced, respectively, Xba I and Bgl II sites.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Isolation, Expressing, Mutagenesis

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gki794

    Figure Lengend 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.

    Article Snippet: The PCR was carried out with 2 U of Vent DNA polymerase (New England Biolabs, Pickering, ON, Canada) and the sense 5′-CTCACGCGTCTGCAG-3′ and antisense 5′-GGCTGATTATGCTCTAGATCG-3′ primers.

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gki794

    Figure Lengend 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.

    Article Snippet: The PCR was carried out with 2 U of Vent DNA polymerase (New England Biolabs, Pickering, ON, Canada) and the sense 5′-CTCACGCGTCTGCAG-3′ and antisense 5′-GGCTGATTATGCTCTAGATCG-3′ primers.

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

    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.

    Journal: PLoS Genetics

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

    doi: 10.1371/journal.pgen.1005422

    Figure Lengend 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.

    Article Snippet: Oligonucleotides containing hLtrA sequence were synthesized by HHMI/Keck Oligonucleotide Synthesis Facility (Yale) and PCR reactions were carried out by using Vent DNA polymerase (New England Biolabs), high annealing temperatures (58–60°C), and manual hot start–i .e ., adding Vent DNA polymerase after sample temperature reached 94°C).

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

    Edr is a conserved mammalian gene and maps to the proximal region of mouse chromosome 6. ( A ) Genomic Southern blot analysis: 10 µg genomic DNA prepared from human term placenta (hu) or M.musculus (mo) was digested with Bam HI (B), Xba I (X) or Eco RI (E) restriction endonucleases, prior to electrophoresis and transfer to nitrocellulose. The filter was hybridised with 32 P-labelled Edr partial cDNA (395 bp Eco RI fragment encompassing nucleotides 1358–1753 in Edr cDNA) and washed to 0.1× SSC 0.1% SDS 65°C prior to autoradiography. ( B ) Comparison of the SDP of a Bam HI RFLP at the Edr locus with others mapped in the BxD series of RI mice revealed linkage with D6Mit86 (0/26 recombinants and Met 4/26 recombinants). These linkage data map Edr to the proximal end of mouse chromosome 6.

    Journal: Nucleic Acids Research

    Article Title: Identification and characterisation of a developmentally regulated mammalian gene that utilises -1 programmed ribosomal frameshifting

    doi:

    Figure Lengend Snippet: Edr is a conserved mammalian gene and maps to the proximal region of mouse chromosome 6. ( A ) Genomic Southern blot analysis: 10 µg genomic DNA prepared from human term placenta (hu) or M.musculus (mo) was digested with Bam HI (B), Xba I (X) or Eco RI (E) restriction endonucleases, prior to electrophoresis and transfer to nitrocellulose. The filter was hybridised with 32 P-labelled Edr partial cDNA (395 bp Eco RI fragment encompassing nucleotides 1358–1753 in Edr cDNA) and washed to 0.1× SSC 0.1% SDS 65°C prior to autoradiography. ( B ) Comparison of the SDP of a Bam HI RFLP at the Edr locus with others mapped in the BxD series of RI mice revealed linkage with D6Mit86 (0/26 recombinants and Met 4/26 recombinants). These linkage data map Edr to the proximal end of mouse chromosome 6.

    Article Snippet: PCR-mediated site-directed mutagenesis of Edr was carried out by the same strategy as described previously using Vent DNA polymerase (New England Biolabs).

    Techniques: Southern Blot, Electrophoresis, Autoradiography, Mouse Assay