Journal: PLoS ONE

Article Title: Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins

doi: 10.1371/journal.pone.0202138

Figure Lengend Snippet: Sites of structural changes induced by the hyper-negative supercoiling detected by Nuclease SI. (A) Experimental scheme. The red-filled circle designates 32 P. The different steps of the experiment are indicated: first (1), the digestion by the Nuclease SI; second (2), the digestion by (BamHI + BglII) or (BahmHI + HindIII); third (3), electrophoresis on a sequencing gel. (B) The enzymatic probe used to map the fine structure of the T -2 and T -6 topoisomers is Nuclease SI. Nuclease SI is at 2 mU microL -1 and DNA at 0.5 nM. After the Nuclease SI reaction, the samples are treated to remove the proteins. The DNAs are precipitated and submitted to the BamHI+HindIII double digestion to only visualize DNA fragments from one of the two radiolabeled strands. The reaction products are analyzed on two different sequencing gels (8% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 5) or the guanines and adenines (G+A lanes; lanes 2 and 6). (C) Same as 3B except that the samples are submitted to the BglII+BamHI double digestion to only visualize DNA fragments from the complementary radiolabeled strands. The reaction products are analyzed on two different sequencing gels (7% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 7) or the guanines and adenines (G+A lanes; lanes 2 and 6).

Article Snippet: Materials Escherichia coli topoisomerase I (Ec TopoI), T4 polynucleotide kinase (PNK), calf intestinal phosphatase, T4 DNA ligase, DNAse I, BamHI, BglII and HindIII were from New England Biolabs.

Techniques: Electrophoresis, Sequencing

Journal: eLife

Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

doi: 10.7554/eLife.19669

Figure Lengend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from spo11 strains with inserts at HIS4 (top) and at URA3 (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.015

Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

Techniques:

Journal: eLife

Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

doi: 10.7554/eLife.19669

Figure Lengend Snippet: Spo11-initiated events at the two insert loci. ( A ) Spo11-catalyzed DSBs are more frequent at HIS4 that at URA3 . Left—Southern blots of Eco RI digests of DNA from vde∆ strains, probed with pBR322 sequences, showing Spo11-DSBs in the Parent 2 insert (see Figure 1 ) in resection/repair-deficient sae2∆ mutant strains. Right—location of DSBs and probe and DSB frequencies (average of 7 and 8 hr samples from a single experiment; error bars represent range). Spo11-DSBs in the Parent 1 inserts at HIS4 and URA3 were at different locations within the insert, but displayed similar ratios between the two loci (data not shown). ( B ) Southern blots of Hin dIII digests of DNA from vde∆ strains, to detect total Spo11-initiated crossovers. ( C ) Southern blots of Hin dIII-VDE double digests of the same samples, to determine the background contribution of Spo11-initiated COs in subsequent experiments measuring VDE-initiated COs, which will be VDE-resistant due to conversion of the VRS site to VRS103 . Probes were as shown in Figure 1 . ( D ) Quantification of data in panels B (total COs; filled circles) and C (VDE-resistant COs; open circles). Data are from a single experiment. DOI: http://dx.doi.org/10.7554/eLife.19669.004

Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

Techniques: Mutagenesis

Journal: eLife

Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

doi: 10.7554/eLife.19669

Figure Lengend Snippet: 70–80% of VDE-DSBs are repaired. ( A ) Fraction of inserts remaining, calculated using Hin dIII digests (see Figure 1 ). For the arg4-VRS103 insert, the ratio (Parent 2 + CO2)/ (0.5 x LC) was calculated at 9 hr, and was then normalized to the 0 hr value. For the arg4-VRS insert, a similar calculation was made: (Parent 1 + NCO + CO1)/(0.5 x LC) ( B ) Relative recovery of interhomolog recombination products, calculated using Hin dIII-VDE double digests (see Figure 1 ). The sum of CO (average of CO1 and CO2) and NCO frequencies was divided by the frequency of total DSBs, as calculated in Figure 2A . Data are the average of two independent experiments; error bars represent range. DOI: http://dx.doi.org/10.7554/eLife.19669.006

Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

Techniques:

Journal: eLife

Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

doi: 10.7554/eLife.19669

Figure Lengend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Probes and gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.009

Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

Techniques:

Journal: eLife

Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

doi: 10.7554/eLife.19669

Figure Lengend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. In the gel with Hin DIII digests of samples from a pch2∆ mm4-mn yen1∆ slx1∆ strain with inserts at URA3 , the 9 hr sample was originally loaded between the 4 and 5 hr samples; this image was cut and spliced as indicated by vertical lines for presentation purposes. DOI: http://dx.doi.org/10.7554/eLife.19669.012

Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

Techniques:

Journal: bioRxiv

Article Title: DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats

doi: 10.1101/2020.06.20.162743

Figure Lengend Snippet: Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.

Article Snippet: Ligation and transformation Purified DNA library (250 ng) was cut with the Hind III and Bam HI restriction enzymes (NEB) for 1h at 37 °C in a reaction mixture containing 1X of the NEB cut smart buffer.

Techniques: High Throughput Screening Assay, Primer Extension Assay, DNA Synthesis, Microarray, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Produced, Acrylamide Gel Assay, Polyacrylamide Gel Electrophoresis, Electrophoresis, Next-Generation Sequencing, Sequencing, Fluorescence, Imaging

Journal: bioRxiv

Article Title: Network analysis of promoter interactions reveals the hierarchical differences in genome organisation between human pluripotent states

doi: 10.1101/2019.12.13.875286

Figure Lengend Snippet: Dynamics of enhancer activity and interactivity between human pluripotent states. (A) Plot showing the number of ROSE-called SEs in naïve and primed PSCs. As illustrated in the diagram, two values are given for shared SEs because a SE in one cell type may overlap with two individually-called SEs in the other cell type. (B) Diagram showing the number of genes that are contacted by SEs in the two pluripotent cell types. Shared genes (orange) are genes that are contacted by SE elements in both naïve and primed PSCs. Naïve-specific genes (blue) and primed-specific genes (red) are contacted by SEs in either naïve or primed PSCs, respectively. (C) Plots showing the log2 FPKM expression of genes that interact with SEs in each cell type (naïve, n=648; primed, n=286; shared, n=174). P-values are derived from a Mann Whitney U test. (D) Plot showing the distribution of ROSE-called enhancers in naïve and primed PSCs. (E) Diagram showing the number of genes that are contacted by enhancers in the two pluripotent cell types. Genes that are also in contact with a SE have been removed from this list of enhancer-interacting genes. (F) Plots showing the log2 FPKM expression of genes that interact with enhancer elements in each cell type (naïve, n=2077; primed, n=1210; shared, n=3158). P-values are derived from a Mann Whitney U test. (G) Genome browser view of the DPPA5 promoter interactomes in naïve (upper) and primed (lower) PSCs. Significant interactions are shown as blue arcs that connect the baited HindIII fragment containing the DPPA5 promoter (shaded in red) with promoter-interacting regions (shaded in green). ChIP-seq (H3K4me1, H3K4me3 and H3K27ac) and RNA-seq tracks are shown. Chromatin states include active chromatin, light green; H3K4me1-only chromatin, dark green; bivalent chromatin, purple; background, grey. ROSE tracks show the location of enhancers (green) and super-enhancers (red), and OSN tracks show the position of shared (orange) and naïve-specific (blue) regions of OSN occupancy. (H) Network graph showing the locations and cell-type-origin of enhancer and SE elements. Colours depict naïve-specific (blue), primed-specific (red) and shared (orange) enhancer and SE elements. Node size represents SE (large nodes) and enhancers (small nodes). Lines represent interactions and are coloured according to the colour of the node of origin. (I-J) Plots show the (I) percent DNA methylation or (J) histone modification levels in naïve and primed PSCs at shared (n=18,735) and cell type-specific active enhancers (naïve, n=26,955; primed, n=34,805). Regions that are in the background chromatin state in both cell types are shown to indicate genome-wide levels (n=467,772). See also Figure S6 .

Article Snippet: Using biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (Life Technologies; all at a final concentration of 30 μM), the HindIII restriction sites were then filled in with Klenow (NEB) for 75 minutes at 37°C, followed by ligation for 4 hours at 16°C (50 units T4 DNA ligase (Life Technologies) per 7 million cells starting material) in a total volume of 5.5 mL ligation buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, 100 μg/mL BSA) per 7 million cells starting material.

Techniques: Activity Assay, Expressing, Derivative Assay, MANN-WHITNEY, Chromatin Immunoprecipitation, RNA Sequencing Assay, DNA Methylation Assay, Modification, Genome Wide

Journal: bioRxiv

Article Title: Network analysis of promoter interactions reveals the hierarchical differences in genome organisation between human pluripotent states

doi: 10.1101/2019.12.13.875286

Figure Lengend Snippet: Multi-scale exploration of promoter-interaction data using force-directed network graphs. (A) Representation of PCHi-C data as arc diagrams (upper) and as corresponding network graphs (lower). Interacting HindIII genomic fragments are depicted as nodes that are connected by edges (significant interactions). A combined network graph is created by merging naïve and primed human PSC datasets whilst retaining cell type-specific information. Blue, naïve-specific nodes and edges; red, primed-specific nodes and edges; grey, shared nodes and edges. (B) Canvas produces a force-directed layout of the combined, whole-network graph. Nodes that interact more frequently are pulled closer together, and less interacting nodes are pushed further apart. (C) Differential gene expression (p-adj

Article Snippet: Using biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (Life Technologies; all at a final concentration of 30 μM), the HindIII restriction sites were then filled in with Klenow (NEB) for 75 minutes at 37°C, followed by ligation for 4 hours at 16°C (50 units T4 DNA ligase (Life Technologies) per 7 million cells starting material) in a total volume of 5.5 mL ligation buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, 100 μg/mL BSA) per 7 million cells starting material.

Techniques: Expressing

Journal: bioRxiv

Article Title: Network analysis of promoter interactions reveals the hierarchical differences in genome organisation between human pluripotent states

doi: 10.1101/2019.12.13.875286

Figure Lengend Snippet: Long-range promoter interactions in primed PSCs drive genome conformation changes between pluripotent states. (A) Plot shows the number of interactions (edges) and the number of interacting HindIII fragments (nodes) for each sub-network in naïve and primed PSCs. Each small circle represents a different sub-network. The lower-left quadrant contains larger sub-networks in primed PSCs, and the upper-right quadrant contains larger sub-networks in naïve PSCs. The protocadherin ( PCDH ), histone H1 ( HISTH1 ) and ‘most changing’ (containing diverse genes) sub-networks are highlighted in red. (B) Multidimensional scaling representation (MDS) of the ‘most changing’ sub-network plotted using the linear genomic distance between nodes as edge weights. The measured stress score of 0.139 indicates there is a reasonable fit between the linear genomic distances and the spacing of the nodes as determined by MDS ( Kruskal and Wish, 1978 ). (C) Plot shows the distribution of linear genomic distances between interacting nodes in naïve and primed PSCs, binned into long-, mid-, and short-range distances. (D) Chart shows the total number of chromatin loops identified on chromosome 5 in naïve and primed PSCs. (E) Hi-C interaction matrix of chromosome 5 at a resolution of 250 kb with Knight-Ruiz (KR) normalisation; upper right, naïve PSCs; lower left, primed PSCs. Areas of contact enrichment were defined separately for naïve and primed PSCs using HiCCUPS and each cell type-specific set of chromatin interactions are highlighted as a black square on their respective heatmap. The two corner numbers indicates the maximum intensity values for the matrix. The tracks below the Hi-C heatmap show the PCHi-C interactions and ChiCAGO scores over the same region. (F) Heatmap shows the aggregate peak analysis (250 kb resolution) of primed-specific chromatin interactions on chromosome 5 for naïve and primed PSCs. Chromatin interactions > 5 Mb from the diagonal were used for the analysis (n=27 loops out of a total of 76). The ‘peak to lower left’ (P2LL) score denotes the enrichment of the central pixel over the pixels in the lower left quadrant. See also Figures S3 and S4 .

Article Snippet: Using biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (Life Technologies; all at a final concentration of 30 μM), the HindIII restriction sites were then filled in with Klenow (NEB) for 75 minutes at 37°C, followed by ligation for 4 hours at 16°C (50 units T4 DNA ligase (Life Technologies) per 7 million cells starting material) in a total volume of 5.5 mL ligation buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, 100 μg/mL BSA) per 7 million cells starting material.

Techniques: Hi-C

Journal: bioRxiv

Article Title: Network analysis of promoter interactions reveals the hierarchical differences in genome organisation between human pluripotent states

doi: 10.1101/2019.12.13.875286

Figure Lengend Snippet: Assigning ChromHMM states to chromatin interacting regions (A) Heatmap shows the number of interacting HindIII fragments that were assigned to each of the ChromHMM-defined chromatin states in both cell types. Note the higher number of bivalently-marked (H3K27me3 and H3K4me1/3) interacting regions in primed compared to naïve PSCs. (B) Chart shows the total number of interacting HindIII fragments for each of the ChromHMM states. (C) Sankey plot reveals the chromatin state composition for the 617 HindIII interacting regions in naïve PSCs that were defined by ChromHMM as being in a ‘mixed’ chromatin state. The left column shows the 617 mixed chromatin state regions and the right column shows the breakdown of individual chromatin states for each of the regions, represented by the different colour bars. Approximately half of the mixed state HindIII fragments contain signatures of active (H3K4me3, green), bivalent (H3K27me3 and H3K4me1/3, orange) and Polycomb (H3K27me3-only, red) chromatin. (D) Heatmap shows the difference in HindIII fragment interaction frequency between cell types as a function of the chromatin state of the interacting regions (rows) and the linear interaction distance (columns, binned distances). Interacting regions that are engaged in long-range promoter interactions, defined as > 1Mb, are highlighted by the dashed box. Nearly all (98%) of the long-range interactions were associated with bivalently-marked promoters and these regions have a higher interaction frequency in primed compared to naïve PSCs.

Article Snippet: Using biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (Life Technologies; all at a final concentration of 30 μM), the HindIII restriction sites were then filled in with Klenow (NEB) for 75 minutes at 37°C, followed by ligation for 4 hours at 16°C (50 units T4 DNA ligase (Life Technologies) per 7 million cells starting material) in a total volume of 5.5 mL ligation buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, 100 μg/mL BSA) per 7 million cells starting material.

Techniques:

Journal: bioRxiv

Article Title: Network analysis of promoter interactions reveals the hierarchical differences in genome organisation between human pluripotent states

doi: 10.1101/2019.12.13.875286

Figure Lengend Snippet: Changes in promoter-interaction frequency and transcriptional levels in four gene clusters (A) Box plots show the number of significant interactions at HindIII fragments located within the HISTH1 , PCDH , KRT and Olfactory gene clusters. (B) Volcano plots show the transcriptional changes between naïve and primed PSCs for genes within the HISTH1 , PCDH , KRT and Olfactory gene clusters. Each dot represents a different gene. Genes coloured in red are differentially expressed between naïve and primed PSCs (log10 fold change > 1.5 or > −1.5 and with an adjusted p-value

Article Snippet: Using biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (Life Technologies; all at a final concentration of 30 μM), the HindIII restriction sites were then filled in with Klenow (NEB) for 75 minutes at 37°C, followed by ligation for 4 hours at 16°C (50 units T4 DNA ligase (Life Technologies) per 7 million cells starting material) in a total volume of 5.5 mL ligation buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, 100 μg/mL BSA) per 7 million cells starting material.

Techniques:

Journal: bioRxiv

Article Title: Network analysis of promoter interactions reveals the hierarchical differences in genome organisation between human pluripotent states

doi: 10.1101/2019.12.13.875286

Figure Lengend Snippet: Long-range promoter interactions create large sub-networks in primed PSCs (A) Scatter plots show the number of interactions (edges) and the number of interacting HindIII fragments (nodes) for each sub-network in naïve and primed PSCs. The lower-left quadrant contains larger sub-networks in primed PSCs, and the upper-right quadrant contains larger sub-networks in naïve PSCs. The HOXA , HOXD , NKX and HISTH1 sub-networks are highlighted. Sub-networks are coloured according to their number of long-range promoter interactions. Note the increased number of long-range promoter interactions within most sub-networks in primed (right) compared to naïve (left) PSCs. (B) Genome browser tracks show the PCHi-C interactions and CHiCAGO scores in naïve and primed PSCs for the HOXA , HOXD , NKX and HISTH1 sub-networks. (C) Dot plots show that the high number of long-range promoter interactions in primed PSCs is independent of the applied CHiCAGO threshold. Each dot represents a PCHi-C interaction, positioned according to the linear genomic distance of the interaction (x-axis) and the assigned CHiCAGO score (y-axis). Black dots show the interactions obtained when applying a CHiCAGO score of > 5 (the threshold used for constructing the network graph) and red dots show the interactions when using a relaxed CHiCAGO score of between 3 and 5. Primed PSCs have more long-range promoter interactions (shaded area; defined as > 1Mb) compared to naïve PSCs when either CHiCAGO threshold score is applied.

Article Snippet: Using biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (Life Technologies; all at a final concentration of 30 μM), the HindIII restriction sites were then filled in with Klenow (NEB) for 75 minutes at 37°C, followed by ligation for 4 hours at 16°C (50 units T4 DNA ligase (Life Technologies) per 7 million cells starting material) in a total volume of 5.5 mL ligation buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, 100 μg/mL BSA) per 7 million cells starting material.

Techniques:

Journal: Iranian Journal of Parasitology

Article Title: Allelic Variations of Plasmodium vivax Apical Membrane Antigen-1 (Pv AMA-1) in Malarious Areas of Southeastern Iran Using PCR-RFLP Technique

doi:

Figure Lengend Snippet: Electrophoretic product of PvAMA-1 gene treated with EcoR I, PvuII, and HindIII, enzymes using RFLP-PCR technique including Columns 1,2 3 treated with EcoRI. Column 4 main bound of Pv AMA-1. Column 5 size marker with 1000bp. Columns 6, 7 and 8 treated with PvuII. Columns 9 10 treated with HindIII

Article Snippet: RFLP In order to determine the presence of different alleles of Pv AMA-1 gene in the region, PCR–RFLP technique was done to digest the gene using three restriction enzymes EcoR-1, Pvu-II and Hind3 (Thermo cat No #ER0271, #ER0631 and ferments cat No #ER0501 respectively) according to the manufacturer’s recommendations.

Techniques: Polymerase Chain Reaction, Marker

Journal: Applied and Environmental Microbiology

Article Title: Molecular and Genetic Analyses of the Putative Proteus O Antigen Gene Locus ▿ O Antigen Gene Locus ▿ †

doi: 10.1128/AEM.02946-09

Figure Lengend Snippet: Dendrogram of Proteus O antigen gene clusters generated by using the BioNumerics software program following restriction digestion by HindIII and EcoRI.

Article Snippet: Eight microliters of purified O antigen PCR product (the sizes are about 14,000 to 20,000 bp) was digested with 7.5 U of HindIII and 7.5 U of EcoRI (Takara).

Techniques: Generated, Software

Journal: Applied and Environmental Microbiology

Article Title: Molecular and Genetic Analyses of the Putative Proteus O Antigen Gene Locus ▿ O Antigen Gene Locus ▿ †

doi: 10.1128/AEM.02946-09

Figure Lengend Snippet: PCR-RFLP analyses of putative O antigen gene clusters following restriction with HindIII and EcoRI. Lane M, DNA marker; lane 1, O69 (G2667); lane 2, O71 (G2669); lane 3, O72a (G2670); lane 4, O73ac (G2673); lane 5, O74 (G2674); lane 6, O75 (G2675).

Article Snippet: Eight microliters of purified O antigen PCR product (the sizes are about 14,000 to 20,000 bp) was digested with 7.5 U of HindIII and 7.5 U of EcoRI (Takara).

Techniques: Polymerase Chain Reaction, Marker

Journal: Journal of Bacteriology

Article Title: PhaQ, a New Class of Poly-?-Hydroxybutyrate (PHB)-Responsive Repressor, Regulates phaQ and phaP (Phasin) Expression in Bacillus megaterium through Interaction with PHB

doi: 10.1128/JB.186.10.3015-3021.2004

Figure Lengend Snippet: DNase I footprinting analysis of PhaQ binding to the phaQ promoter region. (A) A 0.4-kb SmaI-HindIII DNA fragment containing the phaQ promoter region (positions −356 to +39) and labeled with 32 P at its HindIII site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. (B) A 0.36-kb BamHI-EcoRI DNA fragment containing the phaQ promoter region (positions −105 to + 249) and labeled with 32 P at its BamHI site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. The numbers on the left indicate the positions of bases relative to the transcriptional initiation site of phaQ . Solid brackets on the right denote the protected regions.

Article Snippet: The mutated sequences were verified by DNA sequencing. ( To construct a plasmid that overproduces PhaQ in B. megaterium , a 490-bp DNA fragment carrying the Shine-Dalgano sequence plus the phaQ gene and flanked by EcoRI and HindIII sites was amplified by PCR and cloned between the EcoRI and HindIII sites of pHY300PLK (Takara Shuzo Co. Ltd., Kyoto, Japan) to generate plasmid pGS1056.

Techniques: Footprinting, Binding Assay, Labeling, Incubation

Journal: Journal of Bacteriology

Article Title: IS1630 of Mycoplasma fermentans, a Novel IS30-Type Insertion Element That Targets and Duplicates Inverted Repeats of Variable Length and Sequence during Insertion

doi:

Figure Lengend Snippet: Identification of multiple copies of IS 1630 in M. fermentans strains. Genomic DNA from M. fermentans PG18 (lanes 2 and 4) or II-29/1 (lanes 3 and 5) was digested with Eco RI (lanes 2 and 3) or Hin dIII (lanes 4 and 5), transferred to a nylon membrane, and hybridized with DIG-labeled oligonucleotide probe 5 (see Materials and Methods). Lane 1 contains DIG-labeled lambda Hin dIII markers (Boehringer), the sizes of which are indicated in kilobase pairs.

Article Snippet: Bgl II-, Hin dIII-, or Xba I-digested chromosomal DNA from M. fermentans PG18 or Bgl II-digested total DNA from M. fermentans II-29/1 was ligated to Bam HI-, Hin dIII-, or Xba I-digested cloning vector pZero 2 or pZero2.1 under conditions recommended by the vector supplier (Invitrogen).

Techniques: Labeling

Journal: Oncogene

Article Title: Hsa-miR-24-3p Increases Nasopharyngeal Carcinoma Radiosensitivity by Targeting Both the 3’UTR and 5’UTR of Jab1/CSN5

doi: 10.1038/onc.2016.147

Figure Lengend Snippet: MiR-24 directly targets Jab1 mRNA. ( A ) In silico prediction of the binding sites of miR-24 to Jab1 at 3’UTR and 5’UTR by RNAhybrid was shown. Mfe: minimum free energy. 5’UTR: 5’ untranslated region; CDS: coding sequence; 3’UTR: 3’ untranslated region. ( B ) Schematic map showing the luciferase reporter constructs pMIR-Luc-Jab1 3’UTR, pMIR-Luc-Jab1 5’UTR and pMIR-Luc-Jab1 5’UTR upstream and miR-24 binding sites position. Bam H 1, Spe I and Hind III: restriction enzymes; blue bar: the exact predicted binding sites of miR-24 to Jab1. Red underline: deleted sequences for mutation. ( C ) Assay of luciferase activities when co-transfected with pMIR-Luc-Jab1 3’UTR, pMIR-Luc-Jab1 5’UTR or pMIR-Luc-Jab1 5’UTR upstream and miR-24 mimics or miRNA control. The control group was transfected with pMIR-Luc-Jab1 3’UTR, pMIR-Luc-Jab1 5’UTR or pMIR-Luc-Jab1 5’UTR upstream only. Data were normalized to control. *** P

Article Snippet: Inserts were retrieved with Hind III and Spe I and cloned into the same sites of pMIR-REPORT Lucirefase vector (Applied Biosystems) downstream of the firefly open reading frame (ORF) for the Jab1 3’UTR and 5’UTR and in the BamH1 site (pMIR-REPORT) upstream of the luciferase ORF for the Jab1 5’UTR Upstream.

Techniques: In Silico, Binding Assay, Sequencing, Luciferase, Construct, Mutagenesis, Transfection

Journal: Advanced Biomedical Research

Article Title: In Vitro Evaluation of Vegf-Pseudomonas Exotoxin: A Conjugated on Tumor Cells

doi: 10.4103/2277-9175.218691

Figure Lengend Snippet: (a) Schematic representation of recombinant vector containing designed construct and (b) 1% agarose gel electrophoresis of recombinant vector [Lane 1: recombinant vector digested by HindIII and SacI and Lane 2: 1 kb DNA ladder SM0331 (Fermentas, Lithuania)]

Article Snippet: The construct was digested by SacI and HindIII restriction enzymes (Thermo Scientific, Lithuania) and subcloned into pET-28a expression vector (MilliporeMillipore, USA) by T4 DNA ligase (Thermo Scientific, Lithuania).

Techniques: Recombinant, Plasmid Preparation, Construct, Agarose Gel Electrophoresis

Journal: PLoS Genetics

Article Title: Checkpoint Activation of an Unconventional DNA Replication Program in Tetrahymena

doi: 10.1371/journal.pgen.1005405

Figure Lengend Snippet: Two-dimensional gel electrophoresis analysis of rDNA replication intermediates following removal of HU. Cells were synchronized at the G1/S border by starvation and cultured in growth media in the absence or presence of 20 mM HU. DNA was prepared from mock-treated starved cells, mock-treated S phase cells, and HU-treated cells at defined intervals following HU removal. (A) Upper diagram: schematic of the 21 kb rDNA minichromosome and location of relevant restriction sites and probes for Southern blot analysis. Macronuclear rDNA minichromosomes contain two copies of the rRNA coding region and adjacent 5' and 3 'non-transcribed spacer (NTS) regions in an inverted orientation. The 35S rRNA precursor encodes the 17S, 5.8S and 26S rRNAs (grey areas- mature RNA coding regions, black and white stippled areas- processed RNA precursor regions, hatched area- self-splicing 26S rRNA intron). Telomeric DNA repeats (vertical hashes) are present at chromosome termini. The positions of four probes for N/N and N/A 2D gel analysis are shown. Expanded view of the 1.9 kb 5 NTS. Thick arrow- rRNA promoter; grey ovals- position of phase nucleosomes in vegetative rDNA minichromosomes [ 60 ], black boxes- type 1 repeats. Domains 1 and 2 (thin arrows) are 430 bp imperfect tandem repeats with 230 bp nuclease hypersensitive regions Lower diagram: schematic of typical RI patterns detected by N/N 2D gel electrophoresis. Simple Y arc (arrow): passive replication of the probed DNA fragment interval due to initiation elsewhere in the chromosome. Bubble arc (arrowhead): initiation at a central position in the probed DNA fragment. Bubble-to-Y arc: initiation at an asymmetric position in the examined fragment (low MW RIs: bubble arc (arrowhead), high MW RIs: Y arc). Composite: active (complete bubble arc, arrowhead) and passive (complete Y arc, arrow) replication within the probed DNA fragment. X and Double Y: the X spike (arrowhead) is generated from branch migration recombinant intermediates, and the double Y (open arrow) is generated by two converging replication forks. (B) RI patterns detected with the 5’ NTS probe 1 on HindIII digested DNA from mock treated quiescent (starvation) and replicating cell populations (S phase). (C) 5’ NTS analysis on DNA from HU-treated cells 0–120 min after HU removal (arrow: simple Y arc, passive replication; arrowhead: X-spike recombination intermediates). See flow cytometry profiles ( Fig 5A ) and western blots ( Fig 5B ) for cell cycle progression and abundance of Orc1p and Mcm6p, respectively. (D) Two-dimensional N-N gel analysis of the 5.5 kb rRNA coding region ClaI fragment (position 2168–7629, probe 2). (E) Two dimensional neutral-alkaline (N/A) analysis of RIs derived from the rRNA coding region ClaI fragment. Probe 3 spans nucleotides 2169 to 3670, and probe 4 spans nucleotides 5214–6676. Schematic of nascent-strand RIs resolved by N/A 2D gel electrophoresis. The 1n spot corresponds to non-replicating DNA. The vertical smear is derived from nicked, non-replicating DNA, and the horizontal smear represents the parental strand in RIs of different length. The diagonal arc corresponds to nascent-strand replication intermediates that are liberated from the parental strand by alkali denaturation prior to electrophoresis in the second dimension.

Article Snippet: For RI enrichment, 200 μg of genomic DNA were digested with HindIII (rDNA 5’ NTS analysis) or ClaI (rDNA coding region analysis) for 4 h and applied to the 200-μl packed volume benzoylated naphthoylated DEAE (BND)-cellulose (Sigma-Aldrich).

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Cell Culture, Southern Blot, Generated, Migration, Recombinant, Flow Cytometry, Cytometry, Western Blot, Derivative Assay

Journal: Avicenna Journal of Medical Biotechnology

Article Title: Alpha Alumina Nanoparticle Conjugation to Cysteine Peptidase A and B: An Efficient Method for Autophagy Induction

doi:

Figure Lengend Snippet: Digestion analysis of pJET1.2-CPA; A) pJET1.2-CPB; B) PET28a-CPA; C) and PET28a-CPB; D) with HindIII and BamH1. Band 1, 2:pJET1.2 (2964 bp ) and CPA (650 bp ), Band 3, 4: pJET1.2 (2964 bp ) and CPB (950 bp ), Band 5, 6: PET28a (5369 bp ) and CPB (950 bp ), and Band 7,8: PET28a (5369 bp ) and CPA (650 bp ).

Article Snippet: CPB- and CPA-coding DNA was then sub-cloned by insertion between BamHI and HindIII sites in pET28a (expression vector) (Novagen, USA).

Techniques:

Journal: Scientific Reports

Article Title: Differential effects of spinal motor neuron-derived and skeletal muscle-derived Rspo2 on acetylcholine receptor clustering at the neuromuscular junction

doi: 10.1038/s41598-018-31949-7

Figure Lengend Snippet: Transgene constructs and RSPO2 expression in transgenic mice. (A) Schematic presentation of transgene constructs for generation of transgenic mice. The muscle-specific MCK promoter and the SMN-specific VAChT promoter were inserted 5′ to the human RSPO2 gene. Three copies of FLAG cDNA (3 X FLAG) were inserted 3′ to RSPO2 . N, Not I; H, Hin dIII; X, Xba I; and E, Eco RI. (B) Immunofluorescent staining of sections of the tibialis anterior (TA) muscle and spinal cord of wild-type (WT) and transgenic adult mice with anti-FLAG antibody. FLAG-tagged Rspo2 was detected in the tibialis anterior of MCK -RSPO2 mice and in the spinal cord of VAChT -RSPO2 mice. A, anterior; and P, posterior. Scale bar =100 μm. (C) Immunofluorescent staining of the spinal cord of VAChT -RSPO2 transgenic adult mice with anti-FLAG (green) antibody for detecting Rspo2 and anti-ChAT (red) antibody for visualizing SMNs. FLAG-tagged Rspo2 was primarily expressed in ChAT-positive SMNs, but leaky expression was also observed in a small fraction of cells. Scale bar =100 μm. (D) Quantitative RT-PCR of Rspo2 transcripts. Total RNA was isolated from the diaphragms and spinal cords of wild-type (WT), Rspo2 −/− , MCK -RSPO2/Rspo2 −/− , and VAChT- RSPO2/Rspo2 −/− embryos at E18.5. Values are normalized to Gapdh . The normalized Rspo2 expression levels are plotted relative to that in the WT diaphragm to make direct comparisons between the diaphragm and spinal cord possible. Mean and SEM ( n = 3) are indicated.

Article Snippet: To generate CMV-RSPO2 , the coding region of RSPO2 cDNA was amplified from the clone by PCR (see Supplementary Table ) and was inserted into the Hin dIII and Xba l sites of the CMV-driven expression vector, p3XFLAG-CMV-14 (Sigma-Aldrich).

Techniques: Construct, Expressing, Transgenic Assay, Mouse Assay, Staining, Quantitative RT-PCR, Isolation

Journal: Protein Science : A Publication of the Protein Society

Article Title: Genetic selection system for improving recombinant membrane protein expression in E. coli

doi: 10.1002/pro.39

Figure Lengend Snippet: Analysis of plasmid copy number. Strains were retransformed with pSEL1 and pSEL2 encoding rhomboid-Rv1337 and were grown to mid-log phase. Samples were normalized based on OD 600 and were column purified along with HindIII-digested pUC19 plasmid as an internal purification control. The samples were then analyzed on an agarose gel.

Article Snippet: This fragment was then placed between the KpnI and HindIII sites in the MCS of pBAD/HisA (Invitrogen).

Techniques: Plasmid Preparation, Purification, Agarose Gel Electrophoresis

Journal: eLife

Article Title: A universal vector concept for a direct genotyping of transgenic organisms and a systematic creation of homozygous lines

doi: 10.7554/eLife.31677

Figure Lengend Snippet: The pAVOIAF{#1–#2–#3–#4} vector. ( A ) Vector map of pAVOIAF{#1–#2–#3–#4}. The vector is based on the pUC57-Kan vector, from which only the kanamycin resistance cassette and the origin of replication remain. The four-slot cloning site together with the 3’ and 5’ piggyBac terminal repeats is located between the AatII and PciI sites. The light gray band on the inside indicates the transgene. ( B ) Scheme of the four-slot cloning site. Each slot consists of an 18 bp spacer that translates into the amino acids Phe-Arg-Glu-Asp-Asp-Tyr and thus was termed FREDDY spacer. The slots can be accessed individually by unique restriction enzyme site pairs (XmaI/SpeI for #1, HindIII/XbaI for #2, XhoI/NheI for #3 and AflII/AvrII for #4). They are embedded into five PmeI restriction enzyme sites that allow a simple one-enzyme control digestion to determine the size of the sequences that were inserted into the slots. For convenience, the downstream restriction enzyme sites for each slot (SpeI for #1, XbaI for #2, NheI for #3 and AvrII #4) result in identical sticky ends, facilitating cloning procedures that cannot utilize the suggested restriction enzyme site pairs. Extends of the genetic elements are not to scale. ORF, open-reading frame.

Article Snippet: Molecular biology: the pGS[#P’#O(LA)-mEmerald] and pAGOC{#P’#O(LA)-mEmerald} vectors A hybrid sequence, consisting of (i) a HindIII site, (ii) the modular fluorescent protein expression cassette as described above and (iii) a XbaI site, was de novo synthetized and inserted into the unique SfiI site of pMK-RQ (Thermo Fisher Scientific).

Techniques: Plasmid Preparation, Clone Assay

Journal: Nature Communications

Article Title: CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples

doi: 10.1038/s41467-019-12570-2

Figure Lengend Snippet: CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file

Article Snippet: We then used I-DOT One to dispense first 5 ng diluted in 350 nl of gDNA extracted from HeLa cells, followed by 100 nl of 20 U/μl of HindIII (NEB, catalog number R3104) and 50 nl of CutSmart buffer (NEB, catalog number R3104), in 96 of the 384 wells.

Techniques: In Vitro, Sequencing, Formalin-fixed Paraffin-Embedded, Amplification, Polymerase Chain Reaction

Journal: Molecular and Cellular Biology

Article Title: Chorioallantoic Fusion Defects and Embryonic Lethality Resulting from Disruption of Zfp36L1, a Gene Encoding a CCCH Tandem Zinc Finger Protein of the Tristetraprolin Family

doi: 10.1128/MCB.24.14.6445-6455.2004

Figure Lengend Snippet: Southern and Northern analysis confirming Zfp36L1 targeting in ES cells. (A and B) Southern blots of mouse tail DNA from WT and Het. mice. (A) HindIII/XbaI cleavage of DNA and hybridization with the Zfp36L1 5′-flanking probe generated a diagnostic

Article Snippet: The PCR product was cut with BamHI and HindIII, gel purified, and then ligated with BamHI- and HindIII-cut Bluescript KS+ (Stratagene).

Techniques: Northern Blot, Mouse Assay, Hybridization, Generated, Diagnostic Assay

Journal: International journal of medical microbiology : IJMM

Article Title: Random insertion and gene disruption via transposon mutagenesis of Ureaplasma parvum using a mini-transposon plasmid

doi: 10.1016/j.ijmm.2014.09.003

Figure Lengend Snippet: In gel hybridization detection of gentamicin selection gene. Agarose gel separation of total genomic DNA extracted from transposon mutated Ureaplasma strains. Comparing HindIII digested and undigested genomic DNA. A and C show ethidium bromide visualisation

Article Snippet: The genomic DNA and control pMT85 plasmid digestion were performed at 37°C overnight with HindIII (Promega).

Techniques: Hybridization, Selection, Agarose Gel Electrophoresis