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  • 99
    New England Biolabs hindiii
    Generation of floxed mice by the enhanced PITCh system. a Targeting strategy for the generation of flox Col12a1 mice by the enhanced PITCh system. Purple highlights indicate microhomologies between endogenous Col12a1 locus and PITCh-donor. Blue characters indicate CRISPR target sequences. Red characters indicate protospacer adjacent motif (PAM) sequences. Yellow lightnings indicate DSB sites. b Schematic diagram of pronuclear injection of Cas9 protein, Col12a1 -left, -right, and gRNA-s1 crRNAs, tracrRNA, PITCh-donor, and Exo1 mRNA. The red, purple, and blue boxes indicate the insert, Col12a1 microhomologies, and gRNA-s1 target sequences, respectively. c PCR screenings of newborns. d PCR-RFLP (restriction fragment length polymorphism) screenings of floxed newborn mice. e Summary of flox Col12a1 mouse production by the enhanced PITCh system. f Sequences of boundaries between Col12a1 and LoxPs. Blue, green, and red characters indicate microhomologies, <t>HindIII</t> sites, and LoxPs, respectively. g in vitro Cre-recombination assay. Cloned PCR products of flox alleles from three flox Col12a1 mice and genomic PCR of wildtype were incubated with or without Cre-recombinase. LF: left forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, and WT: wildtype
    Hindiii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6537 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hindiii hindiii fragment
    Chromatin conformation in GATA2 locus is significantly changed upon ATRA induction a Diagram showing ChIP-seq, ATAC-seq, and 4C-seq results of GATA2 promoter and upstream regions. (upper panel) <t>HindIII</t> cutting sites, gene annotation, and control-specific ATAC-seq peaks. Transcription factors with enriched motifs in the peak are labeled. (middle panel) Genome browser views of ChIP-seq and ATAC-seq results of control and ATRA-treated HL-60 cells; semitransparent cyan color labels the bait site used in 4C-seq, and semitransparent yellow color labels the putative enhancers recognized in 4C-seq. (lower panel) 4C-seq results of control and ATRA-treated HL-60 cells using the bait mentioned above. Red dots indicate 4C-seq RPMs in the ATRA-treated cells, and blue dots indicate RPMs in the control cells; only fragments with significant interactions with the bait are shown. Log2-foldchange of RPMs is shown in the lowest bar graph. b DNA FISH showing the loss of the chromatin loop between the GATA2 promoter and enhancer. BAC probes containing the promoter (red) and enhancer (green) were more separated in the ATRA-treated cells (right) than in the control cells (left). c Loci of promoter and enhancer interacted more in the control cells than in the ATRA-treated cells. p -Value was determined by Mann–Whitney U -test: ** p -value
    Hindiii Hindiii Fragment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jena Bioscience hindiii
    spa -RFLP patterns obtained from Hin <t>dIII</t> digestion of the <t>PCR</t> products. a Human, b bovine and c food S. aureus isolates. Lane L: a 100 bp DNA ladder. The other lanes: RFLP patterns of S. aureus isolates
    Hindiii, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hindiii
    Electrophoretic product of PvAMA-1 gene treated with EcoR I, PvuII, and <t>HindIII,</t> 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
    Hindiii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6952 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fastdigest hindiii
    Electrophoretic product of PvAMA-1 gene treated with EcoR I, PvuII, and <t>HindIII,</t> 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
    Fastdigest Hindiii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs hindiii hf
    Generation of vPK transgenic mice. FLAG-tagged vPK was cloned, using <t>HinDIII-HF</t> and BamHI-HF, into a plasmid containing a Ub- and hGH-stabilization element. The linearized transgene fragment was microinjected into the embryos of C57BL/6 mice and implanted into pseudopregnant female mice. Each vPK line was developed from breeding a vPK founder to a C57BL/6 mouse. ( A ) Schematic of the linearized construct used to generate the vPK transgenic mice, as well as the cut sites and probe for the Southern blot in D . ( B ) DNA from the tails of 2 WT (wA and wB) and 2 vPK transgenic mice (vA and vB) for lines 1 and 2 was isolated and evaluated by PCR for the vPK transgene. ( C ) Expression of vPK protein in lysates from spleens of the mice in B as determined by SDS-PAGE and Western blot. Lysate from HEK-293 cells that transiently express vPK was used as a positive control for vPK expression. ( D ) Southern blot of WT, vPK1, and vPK2. DNA was isolated from the spleens of WT and vPK lines 1 and 2. D, double digest with HinDIII-HF and BamHI-HF; S, single digestion with AFlII; p-vPK, plasmid Ub.vPK.hGH.
    Hindiii Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 12755 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega hindiii
    In gel hybridization detection of gentamicin selection gene. Agarose gel separation of total genomic DNA extracted from transposon mutated Ureaplasma strains. Comparing <t>HindIII</t> digested and undigested genomic DNA. A and C show ethidium bromide visualisation
    Hindiii, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 2888 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche hindiii
    BoHV-4 gB disruption. A) Overall strategy to delete a 1261 bp sequence from the ORF8 coding for gB, via heat inducible homologous recombination. The 2232 bp Kana-GalK selectable DNA stuffer, flanked by ORF8 homologous regions, was introduced between positions 11640 and 12901 of the BoHV-4-A strain cloned as a BAC. The expected ORF8 locus (A, bottom) has an increased size of the <t>HindIII</t> fragment (5280 instead of 4314 bp), generated by HindIII restriction enzyme digestion. The diagrams here presented are not to scale. B) HindIII Restriction profile and corresponding Southern Blotting of six representative targeted clones, compared to the untargeted control. Southern Blotting was performed with a probe spanning Kana sequence and confirmed the above data. C) Clonal stability of the pBAC-BoHV-4-A-ΔgBKanaGalK in Escherichia coli SW102 cells, passaged for 25 consecutive days and analyzed by HindIII digestion and agarose gel electrophoresis.
    Hindiii, supplied by Roche, used in various techniques. Bioz Stars score: 95/100, based on 832 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Valiant hindiii
    Genetic organization of the antibiotic resistance gene cluster of SGI1 and the new SGI1-H variant of serovar Newport strains 01-2174 and 01-5348. The direction of transcription of the genes is indicated by arrows. Black and gray arrows correspond to SGI1 antibiotic resistance genes and chromosomal genes flanking SGI1, respectively. DR-L and DR-R are the left and right direct repeats, respectively, bracketing SGI1. IRi and IRt are 25-bp imperfect inverted repeats defining the left and right ends of complex class 1 integrons. PCRs used to assess the genetic organization of the antibiotic resistance genes (i.e., PCRs floR , A, B, C, D, E, and F) and the SGI1 junctions to the chromosome (i.e., PCRs LJ and RJ for left and right junctions, respectively) are indicated. Restriction sites: X, XbaI; H, <t>HindIII.</t>
    Hindiii, supplied by Valiant, used in various techniques. Bioz Stars score: 85/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim hindiii
    DNA Gel Blot Hybridization of the Genome-Specific Sequence PSR593 to Genomic DNA from F1 Hybrids and Newly Formed Allopolyploids. (A) Hybridization to genomic DNA from the F1 hybrid between Ae . sharonensis (TH02) and T . monococcum ssp aegilopoides (TMB02), from the S1 generation of the allotetraploid that derived from this hybrid, and from the two parental plants. DNA was digested using EcoRI, EcoRV, DraI, and <t>HindIII.</t> Arrows indicate the bands from the genome of TH02 that disappeared in F1 and in the S1 generation of the allopolyploid. Fragment size is indicated at right in kilobases. (B) Hybridization to genomic DNA from the F1 hybrid between T. turgidum ssp durum (TTR16) and Ae . speltoides (TS01), from the S1 generation of the allohexaploid derived from this hybrid, and from the two parental plants. DNA was digested using HindIII and DraI. Arrows indicate the bands that disappeared in F1 and/or in the S1 generation of the allohexaploid. The upper band of TTR16 is present in F1 of the HindIII digest but is absent in S1. No such difference between F1 and S1 was noted with DraI. This difference between HindIII and DraI probably results from methylation. Fragment size is indicated at right in kilobases.
    Hindiii, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    ACGT Inc hindiii
    Equilibrium between circular and linear conformations of 199-bp DNA fragments with various sticky ends. The probability of finding a fragment in circular conformation, f c , can be estimated from its relative mobility in the polyacrylamide gel since the linear (lane M) and circular (lane 1) forms have different mobility in such a gel. The experiments were performed for sticky ends of 4 AT base pairs created by EcoRI restriction enzyme (lane 2), 2 AT and 2 GC base pairs created by <t>HindIII</t> (lane 3) and 4 GC base pairs created by AgeI (lane 4). The gel electrophoresis was run in TBE buffer containing additional 10 mM of MgCl 2 at 5°C ( A ) and 25°C ( B ).
    Hindiii, supplied by ACGT Inc, used in various techniques. Bioz Stars score: 86/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GenScript hindiii
    Agarose gel electrophoresis showing the presence of HBcAg VLP in the pUC57 vector and (pET28a/VLP). ( A ) The nucleotide sequence coding for HBcAg VLP from the pUC57 vector. Lane 1: uncut pUC 57 vector containing the HBcAg VLP sequence; Lane 2: pUC57 vector containing the HBcAg VLP sequence digested with NheI and <t>HindIII</t> enzymes; Lane 3: 1-kb DNA ladder; Lane 4: uncut pET28a vector; Lane 5: pET28a vector linearized with NheI and HindIII enzymes; ( B ) Confirmation of the presence of the hybrid HBcAg VLP nucleotide sequence in (pET28a/VLP). Lane 1: uncut (pET28a/VLP); Lanes 2–3: pET28a HBcAg VLP digested with NheI and HindIII enzymes; Lane 4: 1-kb DNA ladder; Lane 5: uncut pET28a vector; Lane 6: pET28a vector with NheI and HindIII enzymes.
    Hindiii, supplied by GenScript, used in various techniques. Bioz Stars score: 91/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Toyobo hindiii
    Agarose gel electrophoresis showing the presence of HBcAg VLP in the pUC57 vector and (pET28a/VLP). ( A ) The nucleotide sequence coding for HBcAg VLP from the pUC57 vector. Lane 1: uncut pUC 57 vector containing the HBcAg VLP sequence; Lane 2: pUC57 vector containing the HBcAg VLP sequence digested with NheI and <t>HindIII</t> enzymes; Lane 3: 1-kb DNA ladder; Lane 4: uncut pET28a vector; Lane 5: pET28a vector linearized with NheI and HindIII enzymes; ( B ) Confirmation of the presence of the hybrid HBcAg VLP nucleotide sequence in (pET28a/VLP). Lane 1: uncut (pET28a/VLP); Lanes 2–3: pET28a HBcAg VLP digested with NheI and HindIII enzymes; Lane 4: 1-kb DNA ladder; Lane 5: uncut pET28a vector; Lane 6: pET28a vector with NheI and HindIII enzymes.
    Hindiii, supplied by Toyobo, used in various techniques. Bioz Stars score: 95/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Kaneka Corp hindiii
    pL102 presentation and shortening description. pL102 is composed of a 13.5-kb DNA fragment (▪) inserted in the pUC19 vector (□).The mclC structural gene, the mclI immunity gene, and the mclA and mclB export genes constitute the MccL genetic system. The restriction sites used for the insert shortening were XbaI/SacI on one side and <t>HindIII/PauI</t> on the other side. The HindIII-PauI fragment was deleted. The dotted arrows indicate the start points and the direction of the shortening.
    Hindiii, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 86/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore bglii hindiii
    pL102 presentation and shortening description. pL102 is composed of a 13.5-kb DNA fragment (▪) inserted in the pUC19 vector (□).The mclC structural gene, the mclI immunity gene, and the mclA and mclB export genes constitute the MccL genetic system. The restriction sites used for the insert shortening were XbaI/SacI on one side and <t>HindIII/PauI</t> on the other side. The HindIII-PauI fragment was deleted. The dotted arrows indicate the start points and the direction of the shortening.
    Bglii Hindiii, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa hindiii site
    Cloning of SFD1 . (A) Alignment of the SFD1 sequence with the consensus sequence from COG0240 and Pfam0210.8. Basic Local Alignment Search Tool (BLASTp) analysis of the predicted SFD1 protein sequence revealed homology to DHAP reductases/G3P dehydrogenases in the COG2040 and Pfam0210.8 protein family data sets. The alignment of SFD1 amino acids 89 to 419 to the consensus sequence from COG2040 and Pfam0210.8 is shown. Solid and dashed lines above the SFD1 sequence mark the predicted NAD + and substrate binding domains, respectively. The arrow indicates the Ala 381 that is mutated to yield Thr 381 in sfd1-2 . Amino acids that are identical to SFD1 are shown in red and those that are similar are shown in blue. (B) Complementation of the sfd1-1 and sfd1-2 mutants by SFD1 . Comparison of the morphology of 4-week-old, soil-grown wild-type sfd1-1 , ssi2 npr1 , and sfd1-1 ssi2 npr1 plants and the sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a 5-kb <t>HindIII</t> fragment containing the genomic SFD1 clone. The photographs were taken from the same distance. (C) Restoration of the ssi2 -conferred cell death by SFD1 . Leaves from 4-week-old, soil-grown ssi2 npr1 , sfd1-1 ssi2 npr1 , sfd1-1/SFD1 ssi2 npr1 , and sfd1-2/SFD1 ssi2 npr1 plants were stained with trypan blue. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a 5-kb HindIII fragment containing the genomic SFD1 clone. Trypan blue–stained leaves from the transgenic plants and from the ssi2 npr1 plants show intensely stained dead cells (yellow arrows). All of the photographs were taken at the same magnification. (D) Restoration of the ssi2 -conferred constitutive PR1 expression by SFD1 . PR1 expression was monitored in 4-week-old, soil-grown sfd1-1 ssi2 npr1 ( sfd1-1 ) and sfd1-2 ssi2 npr1 ( sfd1-2 ) plants transformed with pBI121 (Vector) or with pBI121 containing a 5-kb HindIII genomic fragment spanning SFD1 (SFD1). Blots were hybridized with a radiolabeled PR1 probe. Gel loading was monitored by photographing the ethidium bromide (EtBr)–stained gel before transfer to the membrane.
    Hindiii Site, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 237 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher xhol hindiii
    Cloning of SFD1 . (A) Alignment of the SFD1 sequence with the consensus sequence from COG0240 and Pfam0210.8. Basic Local Alignment Search Tool (BLASTp) analysis of the predicted SFD1 protein sequence revealed homology to DHAP reductases/G3P dehydrogenases in the COG2040 and Pfam0210.8 protein family data sets. The alignment of SFD1 amino acids 89 to 419 to the consensus sequence from COG2040 and Pfam0210.8 is shown. Solid and dashed lines above the SFD1 sequence mark the predicted NAD + and substrate binding domains, respectively. The arrow indicates the Ala 381 that is mutated to yield Thr 381 in sfd1-2 . Amino acids that are identical to SFD1 are shown in red and those that are similar are shown in blue. (B) Complementation of the sfd1-1 and sfd1-2 mutants by SFD1 . Comparison of the morphology of 4-week-old, soil-grown wild-type sfd1-1 , ssi2 npr1 , and sfd1-1 ssi2 npr1 plants and the sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a 5-kb <t>HindIII</t> fragment containing the genomic SFD1 clone. The photographs were taken from the same distance. (C) Restoration of the ssi2 -conferred cell death by SFD1 . Leaves from 4-week-old, soil-grown ssi2 npr1 , sfd1-1 ssi2 npr1 , sfd1-1/SFD1 ssi2 npr1 , and sfd1-2/SFD1 ssi2 npr1 plants were stained with trypan blue. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a 5-kb HindIII fragment containing the genomic SFD1 clone. Trypan blue–stained leaves from the transgenic plants and from the ssi2 npr1 plants show intensely stained dead cells (yellow arrows). All of the photographs were taken at the same magnification. (D) Restoration of the ssi2 -conferred constitutive PR1 expression by SFD1 . PR1 expression was monitored in 4-week-old, soil-grown sfd1-1 ssi2 npr1 ( sfd1-1 ) and sfd1-2 ssi2 npr1 ( sfd1-2 ) plants transformed with pBI121 (Vector) or with pBI121 containing a 5-kb HindIII genomic fragment spanning SFD1 (SFD1). Blots were hybridized with a radiolabeled PR1 probe. Gel loading was monitored by photographing the ethidium bromide (EtBr)–stained gel before transfer to the membrane.
    Xhol Hindiii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore hindiii
    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 <t>HindIII</t> 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.
    Hindiii, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher xhoi hindiii
    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 <t>HindIII</t> 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.
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    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 <t>HindIII</t> 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.
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    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 <t>HindIII</t> 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.
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    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 <t>HindIII</t> 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.
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    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 <t>HindIII</t> 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.
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    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 <t>HindIII</t> 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.
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    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 <t>HindIII</t> 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.
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    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 <t>HindIII</t> 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.
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    Image Search Results


    Generation of floxed mice by the enhanced PITCh system. a Targeting strategy for the generation of flox Col12a1 mice by the enhanced PITCh system. Purple highlights indicate microhomologies between endogenous Col12a1 locus and PITCh-donor. Blue characters indicate CRISPR target sequences. Red characters indicate protospacer adjacent motif (PAM) sequences. Yellow lightnings indicate DSB sites. b Schematic diagram of pronuclear injection of Cas9 protein, Col12a1 -left, -right, and gRNA-s1 crRNAs, tracrRNA, PITCh-donor, and Exo1 mRNA. The red, purple, and blue boxes indicate the insert, Col12a1 microhomologies, and gRNA-s1 target sequences, respectively. c PCR screenings of newborns. d PCR-RFLP (restriction fragment length polymorphism) screenings of floxed newborn mice. e Summary of flox Col12a1 mouse production by the enhanced PITCh system. f Sequences of boundaries between Col12a1 and LoxPs. Blue, green, and red characters indicate microhomologies, HindIII sites, and LoxPs, respectively. g in vitro Cre-recombination assay. Cloned PCR products of flox alleles from three flox Col12a1 mice and genomic PCR of wildtype were incubated with or without Cre-recombinase. LF: left forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, and WT: wildtype

    Journal: BMC Genomics

    Article Title: Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

    doi: 10.1186/s12864-016-3331-9

    Figure Lengend Snippet: Generation of floxed mice by the enhanced PITCh system. a Targeting strategy for the generation of flox Col12a1 mice by the enhanced PITCh system. Purple highlights indicate microhomologies between endogenous Col12a1 locus and PITCh-donor. Blue characters indicate CRISPR target sequences. Red characters indicate protospacer adjacent motif (PAM) sequences. Yellow lightnings indicate DSB sites. b Schematic diagram of pronuclear injection of Cas9 protein, Col12a1 -left, -right, and gRNA-s1 crRNAs, tracrRNA, PITCh-donor, and Exo1 mRNA. The red, purple, and blue boxes indicate the insert, Col12a1 microhomologies, and gRNA-s1 target sequences, respectively. c PCR screenings of newborns. d PCR-RFLP (restriction fragment length polymorphism) screenings of floxed newborn mice. e Summary of flox Col12a1 mouse production by the enhanced PITCh system. f Sequences of boundaries between Col12a1 and LoxPs. Blue, green, and red characters indicate microhomologies, HindIII sites, and LoxPs, respectively. g in vitro Cre-recombination assay. Cloned PCR products of flox alleles from three flox Col12a1 mice and genomic PCR of wildtype were incubated with or without Cre-recombinase. LF: left forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, and WT: wildtype

    Article Snippet: For floxCol12a1 screening, PCR products were digested with HindIII (NEB).

    Techniques: Mouse Assay, CRISPR, Injection, Polymerase Chain Reaction, In Vitro, Recombination Assay, Clone Assay, Incubation, Marker

    Visualization of nuclear compartments and chromatin domains in non-treated liver cells ( A ) and the same cells treated according to the 3C protocol up to the ligation step ( B ). The insoluble fraction was collected after HindIII digestion and 1.6% SDS extraction. (a–e) Immunostaining with antibodies against nucleolin (a), Sc35 (b), DNA topoisomerase II (c), H3K9me3 (d) and H3K27me3 (e). (f) Visualization of the chromosome 7 territory (FISH with a library of the chromosome 7–specific probes). In both sections of the Figure, the results of immunostaining are shown in the first row (red) and counterstaining of DNA with DAPI is shown in the second row (blue). The superimposition of the immunostaining and counterstaining of DNA is shown in the third row. Scale bar: 5 µm.

    Journal: Nucleic Acids Research

    Article Title: Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub

    doi: 10.1093/nar/gkt067

    Figure Lengend Snippet: Visualization of nuclear compartments and chromatin domains in non-treated liver cells ( A ) and the same cells treated according to the 3C protocol up to the ligation step ( B ). The insoluble fraction was collected after HindIII digestion and 1.6% SDS extraction. (a–e) Immunostaining with antibodies against nucleolin (a), Sc35 (b), DNA topoisomerase II (c), H3K9me3 (d) and H3K27me3 (e). (f) Visualization of the chromosome 7 territory (FISH with a library of the chromosome 7–specific probes). In both sections of the Figure, the results of immunostaining are shown in the first row (red) and counterstaining of DNA with DAPI is shown in the second row (blue). The superimposition of the immunostaining and counterstaining of DNA is shown in the third row. Scale bar: 5 µm.

    Article Snippet: The nuclei were harvested and suspended in 0.5 ml of 1.2× restriction buffer 2 (New England Biolabs) for subsequent HindIII digestion or 0.25 ml of 1.2× restriction buffer 3 (New England Biolabs) for MboI digestion.

    Techniques: Ligation, Immunostaining, Fluorescence In Situ Hybridization

    Electron microscopic analysis of the insoluble 3C material from liver cells at different steps of the 3C procedure. After formaldehyde cross-linking ( A and A’ ), after isolation of nuclei and extraction with 0.3% SDS followed by 1.8% Triton X-100 ( B and B’ ) and after digestion with HindIII restriction endonuclease followed by extraction with 1.6% SDS ( C and C’ ). Panels below show the enlarged framed region of the above images. Scale bars: 1 µm (A–C) and 250 nm (A’–C’).

    Journal: Nucleic Acids Research

    Article Title: Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub

    doi: 10.1093/nar/gkt067

    Figure Lengend Snippet: Electron microscopic analysis of the insoluble 3C material from liver cells at different steps of the 3C procedure. After formaldehyde cross-linking ( A and A’ ), after isolation of nuclei and extraction with 0.3% SDS followed by 1.8% Triton X-100 ( B and B’ ) and after digestion with HindIII restriction endonuclease followed by extraction with 1.6% SDS ( C and C’ ). Panels below show the enlarged framed region of the above images. Scale bars: 1 µm (A–C) and 250 nm (A’–C’).

    Article Snippet: The nuclei were harvested and suspended in 0.5 ml of 1.2× restriction buffer 2 (New England Biolabs) for subsequent HindIII digestion or 0.25 ml of 1.2× restriction buffer 3 (New England Biolabs) for MboI digestion.

    Techniques: Isolation

    Frequencies of ligation of the fragment harboring the Hbb-b1 promoter with several selected fragments of the β-globin gene domain in soluble and insoluble portions of the 3C material. ( A ) Results of standard 3C analysis performed without fractionating the 3C material. ( B ) Results of 3C analysis performed separately on soluble (super) and insoluble (debris) fractions. ( C ) The same as (B) after normalization of the ligation frequencies to the amount of DNA in the samples. ( D ) The same as (C), soluble fraction only. On the top of each graph, a map of the domain is shown, with β-globin genes, olfactory receptor genes and DNase I hypersensitive sites shown by red arrows, blue arrows and black vertical lines, respectively. Plotted on the horizontal axis are the fragment positions. The scale is in kilobases, and according to GenBank entry NT_039433, the ‘0’ point corresponds to the start of the Hbb-y gene. The black rectangle in the background of each graph shows the anchor fragment, and the gray rectangles indicate test fragments. Plotted on the vertical axis are the ligation frequencies; the highest ligation frequency observed is set to 100 [the frequency of ligation between the anchor fragment and the upstream restriction fragment in the total 3C material from fetal liver cells (A) or in the insoluble portion of the 3C material from fetal liver cells (B and C) or the soluble portion of the 3C material from fetal brain cells (D)]. Red and blue lines show the results for liver and brain cells, respectively; solid lines show the results for the total 3C material (A) or the insoluble portion of the 3C material (B and C); dotted lines show the results for the soluble portion of the 3C material. Ligation frequencies of HindIII and MboI fragments are presented on the left and the right graphs, respectively. The error bars represent SEM for three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub

    doi: 10.1093/nar/gkt067

    Figure Lengend Snippet: Frequencies of ligation of the fragment harboring the Hbb-b1 promoter with several selected fragments of the β-globin gene domain in soluble and insoluble portions of the 3C material. ( A ) Results of standard 3C analysis performed without fractionating the 3C material. ( B ) Results of 3C analysis performed separately on soluble (super) and insoluble (debris) fractions. ( C ) The same as (B) after normalization of the ligation frequencies to the amount of DNA in the samples. ( D ) The same as (C), soluble fraction only. On the top of each graph, a map of the domain is shown, with β-globin genes, olfactory receptor genes and DNase I hypersensitive sites shown by red arrows, blue arrows and black vertical lines, respectively. Plotted on the horizontal axis are the fragment positions. The scale is in kilobases, and according to GenBank entry NT_039433, the ‘0’ point corresponds to the start of the Hbb-y gene. The black rectangle in the background of each graph shows the anchor fragment, and the gray rectangles indicate test fragments. Plotted on the vertical axis are the ligation frequencies; the highest ligation frequency observed is set to 100 [the frequency of ligation between the anchor fragment and the upstream restriction fragment in the total 3C material from fetal liver cells (A) or in the insoluble portion of the 3C material from fetal liver cells (B and C) or the soluble portion of the 3C material from fetal brain cells (D)]. Red and blue lines show the results for liver and brain cells, respectively; solid lines show the results for the total 3C material (A) or the insoluble portion of the 3C material (B and C); dotted lines show the results for the soluble portion of the 3C material. Ligation frequencies of HindIII and MboI fragments are presented on the left and the right graphs, respectively. The error bars represent SEM for three independent experiments.

    Article Snippet: The nuclei were harvested and suspended in 0.5 ml of 1.2× restriction buffer 2 (New England Biolabs) for subsequent HindIII digestion or 0.25 ml of 1.2× restriction buffer 3 (New England Biolabs) for MboI digestion.

    Techniques: Ligation

    Experimental design for SDF and cell clone generation. A) SDF sequence is homologous to the entire wild type eGFP coding sequence. SDF-PCR-WT, 876 bp long was generated by PCR amplification with primer pair 1F/1R ( Table 1 ). SDF-DIG-WT, 752 bp long, was obtained by HindIII and XhoI digestion of pCR-2.1 vector. C/T transition, responsible of fluorescence switching off, is showed. B) Sequencing analysis showing wild type (WT; top panel) and mutated (Mut; bottom panel) pCEP4-eGFP in C1 and D1 cell clones, respectively. Arrows indicate the modified base (C→T). C) FACS density plot of C1 (WT; top) and D1 (Mut; bottom) respectively. D) pCEP4-eGFP copy number determination for each cell clone.

    Journal: PLoS ONE

    Article Title: Small Fragment Homologous Replacement: Evaluation of Factors Influencing Modification Efficiency in an Eukaryotic Assay System

    doi: 10.1371/journal.pone.0030851

    Figure Lengend Snippet: Experimental design for SDF and cell clone generation. A) SDF sequence is homologous to the entire wild type eGFP coding sequence. SDF-PCR-WT, 876 bp long was generated by PCR amplification with primer pair 1F/1R ( Table 1 ). SDF-DIG-WT, 752 bp long, was obtained by HindIII and XhoI digestion of pCR-2.1 vector. C/T transition, responsible of fluorescence switching off, is showed. B) Sequencing analysis showing wild type (WT; top panel) and mutated (Mut; bottom panel) pCEP4-eGFP in C1 and D1 cell clones, respectively. Arrows indicate the modified base (C→T). C) FACS density plot of C1 (WT; top) and D1 (Mut; bottom) respectively. D) pCEP4-eGFP copy number determination for each cell clone.

    Article Snippet: Inc., USA) by XhoI and HindIII restriction (New England Biolabs, Ipswich, MA, USA), and cloned in vector pCR-2.1 (Invitrogen, CA, USA).

    Techniques: Sequencing, Polymerase Chain Reaction, Generated, Amplification, Plasmid Preparation, Fluorescence, Clone Assay, Modification, FACS

    Agarose gel electrophoresis showing the presence of HBcAg VLP in the pUC57 vector and (pET28a/VLP). ( A ) The nucleotide sequence coding for HBcAg VLP from the pUC57 vector. Lane 1: uncut pUC 57 vector containing the HBcAg VLP sequence; Lane 2: pUC57 vector containing the HBcAg VLP sequence digested with NheI and HindIII enzymes; Lane 3: 1-kb DNA ladder; Lane 4: uncut pET28a vector; Lane 5: pET28a vector linearized with NheI and HindIII enzymes; ( B ) Confirmation of the presence of the hybrid HBcAg VLP nucleotide sequence in (pET28a/VLP). Lane 1: uncut (pET28a/VLP); Lanes 2–3: pET28a HBcAg VLP digested with NheI and HindIII enzymes; Lane 4: 1-kb DNA ladder; Lane 5: uncut pET28a vector; Lane 6: pET28a vector with NheI and HindIII enzymes.

    Journal: International Journal of Molecular Sciences

    Article Title: Production and Evaluation of Virus-Like Particles Displaying Immunogenic Epitopes of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

    doi: 10.3390/ijms16048382

    Figure Lengend Snippet: Agarose gel electrophoresis showing the presence of HBcAg VLP in the pUC57 vector and (pET28a/VLP). ( A ) The nucleotide sequence coding for HBcAg VLP from the pUC57 vector. Lane 1: uncut pUC 57 vector containing the HBcAg VLP sequence; Lane 2: pUC57 vector containing the HBcAg VLP sequence digested with NheI and HindIII enzymes; Lane 3: 1-kb DNA ladder; Lane 4: uncut pET28a vector; Lane 5: pET28a vector linearized with NheI and HindIII enzymes; ( B ) Confirmation of the presence of the hybrid HBcAg VLP nucleotide sequence in (pET28a/VLP). Lane 1: uncut (pET28a/VLP); Lanes 2–3: pET28a HBcAg VLP digested with NheI and HindIII enzymes; Lane 4: 1-kb DNA ladder; Lane 5: uncut pET28a vector; Lane 6: pET28a vector with NheI and HindIII enzymes.

    Article Snippet: The 585-bp insert release (Lane 2, A) and the pET28a vector digested with NheI and HindIII enzymes (Lane 5, A) were cloned using T4 DNA ligase enzyme (NEB, Ipswich, MA, USA).

    Techniques: Agarose Gel Electrophoresis, Plasmid Preparation, Sequencing

    Read-count interaction profiles for baited promoters presented in Figures 4 and 5 . Plots show the read counts corresponding to the interactions of baited promoter fragments ( grey line) with other HindIII fragments. Significant interactions detected by CHiCAGO (score ≥12) are shown in red, and sub-threshold interactions (score ≥11 in the cell type shown and score ≥12 in the other cell type) are shown in blue. ( A ) U2AF1 , TBX3 , SUV39H2 and CDKN2B from Figure 4B . ( B ) CARS , PAX9 , CNPY1 , RXRG and AGAP2 from Figure 4—figure supplement 2 . ( C ) RGMB , MAP2 and KCNE3 from Figure 5D–F . DOI: http://dx.doi.org/10.7554/eLife.21926.015

    Journal: eLife

    Article Title: Global reorganisation of cis-regulatory units upon lineage commitment of human embryonic stem cells

    doi: 10.7554/eLife.21926

    Figure Lengend Snippet: Read-count interaction profiles for baited promoters presented in Figures 4 and 5 . Plots show the read counts corresponding to the interactions of baited promoter fragments ( grey line) with other HindIII fragments. Significant interactions detected by CHiCAGO (score ≥12) are shown in red, and sub-threshold interactions (score ≥11 in the cell type shown and score ≥12 in the other cell type) are shown in blue. ( A ) U2AF1 , TBX3 , SUV39H2 and CDKN2B from Figure 4B . ( B ) CARS , PAX9 , CNPY1 , RXRG and AGAP2 from Figure 4—figure supplement 2 . ( C ) RGMB , MAP2 and KCNE3 from Figure 5D–F . DOI: http://dx.doi.org/10.7554/eLife.21926.015

    Article Snippet: Using biotin-14-dATP (Life Technologies (Carlsbad, CA)), dCTP, dGTP and dTTP (all at a final concentration of 30 µM), the HindIII restriction sites were then filled in with Klenow (NEB (Ipswich, MA)) for 75 min at 37°C.

    Techniques:

    Read-count interaction profiles for baited promoters presented in Figures 1 – 3 . Plots show the read counts corresponding to the interactions of baited promoter fragments (grey line) with other HindIII fragments. Significant interactions detected by CHiCAGO (score ≥12) are shown in red, and sub-threshold interactions (score ≥11 in the cell type shown, and score ≥12 in the other cell type) are shown in blue. ( A ) SOX2 , from Figure 1B . ( B ) PAX6 , from Figure 1—figure supplement 2A . ( C ) POU3F2 , from Figure 2A . ( D ) SNAI2 , GLI2 , PRDM1 and POU3F1 , from Figure 3E–H . DOI: http://dx.doi.org/10.7554/eLife.21926.007

    Journal: eLife

    Article Title: Global reorganisation of cis-regulatory units upon lineage commitment of human embryonic stem cells

    doi: 10.7554/eLife.21926

    Figure Lengend Snippet: Read-count interaction profiles for baited promoters presented in Figures 1 – 3 . Plots show the read counts corresponding to the interactions of baited promoter fragments (grey line) with other HindIII fragments. Significant interactions detected by CHiCAGO (score ≥12) are shown in red, and sub-threshold interactions (score ≥11 in the cell type shown, and score ≥12 in the other cell type) are shown in blue. ( A ) SOX2 , from Figure 1B . ( B ) PAX6 , from Figure 1—figure supplement 2A . ( C ) POU3F2 , from Figure 2A . ( D ) SNAI2 , GLI2 , PRDM1 and POU3F1 , from Figure 3E–H . DOI: http://dx.doi.org/10.7554/eLife.21926.007

    Article Snippet: Using biotin-14-dATP (Life Technologies (Carlsbad, CA)), dCTP, dGTP and dTTP (all at a final concentration of 30 µM), the HindIII restriction sites were then filled in with Klenow (NEB (Ipswich, MA)) for 75 min at 37°C.

    Techniques:

    hDNA2 D277A unwinds plasmid- and oligonucleotide-based DNA substrates. ( A ) Representative 1% agarose gel showing hDNA2 D277A helicase activity on a λDNA/HindIII substrate in a time-course experiment with 346 nM hRPA. Heat, heat-denatured DNA substrate. ( B ) Representative 1% agarose gel showing that nuclease- and helicase-deficient hDNA2 D277A K654R (lanes 2–6) and helicase-deficient hDNA2 K654R (lane 8) do not exhibit helicase activity. Lane 7, DNA unwinding by nuclease-deficient DNA2 D277A. Reactions contained 215 nM hRPA. ( C – E ) Representative 10% polyacrylamide gels showing the helicase activity of hDNA2 D277A with ( C ) 5’ overhang, ( D ) 3’ overhang and with ( E ) dsDNA substrates. Reactions contained 7.5 nM RPA. Heat, heat-denatured DNA substrate. ( F ) Representative 1% agarose gels showing DNA unwinding of a 2.7 kbp-long substrate by either hDNA2 D277A (left part, at 37°C) or yDna2 E675A (right part, at 30°C) in a kinetic experiment with 215 nM human RPA or 267 nM yeast RPA respectively. ( G ) Quantitation of experiments such as shown in F. Averages shown, n = 2; error bars, SEM. DOI: http://dx.doi.org/10.7554/eLife.18574.007

    Journal: eLife

    Article Title: Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases

    doi: 10.7554/eLife.18574

    Figure Lengend Snippet: hDNA2 D277A unwinds plasmid- and oligonucleotide-based DNA substrates. ( A ) Representative 1% agarose gel showing hDNA2 D277A helicase activity on a λDNA/HindIII substrate in a time-course experiment with 346 nM hRPA. Heat, heat-denatured DNA substrate. ( B ) Representative 1% agarose gel showing that nuclease- and helicase-deficient hDNA2 D277A K654R (lanes 2–6) and helicase-deficient hDNA2 K654R (lane 8) do not exhibit helicase activity. Lane 7, DNA unwinding by nuclease-deficient DNA2 D277A. Reactions contained 215 nM hRPA. ( C – E ) Representative 10% polyacrylamide gels showing the helicase activity of hDNA2 D277A with ( C ) 5’ overhang, ( D ) 3’ overhang and with ( E ) dsDNA substrates. Reactions contained 7.5 nM RPA. Heat, heat-denatured DNA substrate. ( F ) Representative 1% agarose gels showing DNA unwinding of a 2.7 kbp-long substrate by either hDNA2 D277A (left part, at 37°C) or yDna2 E675A (right part, at 30°C) in a kinetic experiment with 215 nM human RPA or 267 nM yeast RPA respectively. ( G ) Quantitation of experiments such as shown in F. Averages shown, n = 2; error bars, SEM. DOI: http://dx.doi.org/10.7554/eLife.18574.007

    Article Snippet: The PCR products were digested with BamHI and HindIII restriction endonucleases (New England Biolabs, Ipswich, MA) and ligated into a pFastBac1 vector (Invitrogen, Carlsbad, CA) generating pFB-His-hDNA2-FLAG.

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Activity Assay, Recombinase Polymerase Amplification, Quantitation Assay

    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

    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:

    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

    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

    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

    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:

    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

    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:

    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

    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:

    crRNA processing in M. mazei wt and cas6b deletion strains for CRISPR-Cas subtypes I-B and III-C analyzed by an RNA-Seq approach.

    Journal: RNA Biology

    Article Title: Cross-cleavage activity of Cas6b in crRNA processing of two different CRISPR-Cas systems in Methanosarcina mazei Gö1

    doi: 10.1080/15476286.2018.1514234

    Figure Lengend Snippet: crRNA processing in M. mazei wt and cas6b deletion strains for CRISPR-Cas subtypes I-B and III-C analyzed by an RNA-Seq approach.

    Article Snippet: Both fragments were TA-cloned into pCR4-TOPO (Invitrogen) resulting in pRS1078 ( cas6b -IB) and pRS1080 ( cas6b -IIIC) followed by subsequent cloning via Xmn I and Hind III from these plasmids to pMAL-cs (New England BioLabs).

    Techniques: CRISPR, RNA Sequencing Assay

    Chromatin conformation in GATA2 locus is significantly changed upon ATRA induction a Diagram showing ChIP-seq, ATAC-seq, and 4C-seq results of GATA2 promoter and upstream regions. (upper panel) HindIII cutting sites, gene annotation, and control-specific ATAC-seq peaks. Transcription factors with enriched motifs in the peak are labeled. (middle panel) Genome browser views of ChIP-seq and ATAC-seq results of control and ATRA-treated HL-60 cells; semitransparent cyan color labels the bait site used in 4C-seq, and semitransparent yellow color labels the putative enhancers recognized in 4C-seq. (lower panel) 4C-seq results of control and ATRA-treated HL-60 cells using the bait mentioned above. Red dots indicate 4C-seq RPMs in the ATRA-treated cells, and blue dots indicate RPMs in the control cells; only fragments with significant interactions with the bait are shown. Log2-foldchange of RPMs is shown in the lowest bar graph. b DNA FISH showing the loss of the chromatin loop between the GATA2 promoter and enhancer. BAC probes containing the promoter (red) and enhancer (green) were more separated in the ATRA-treated cells (right) than in the control cells (left). c Loci of promoter and enhancer interacted more in the control cells than in the ATRA-treated cells. p -Value was determined by Mann–Whitney U -test: ** p -value

    Journal: Cell Death & Disease

    Article Title: Alterations of specific chromatin conformation affect ATRA-induced leukemia cell differentiation

    doi: 10.1038/s41419-017-0173-6

    Figure Lengend Snippet: Chromatin conformation in GATA2 locus is significantly changed upon ATRA induction a Diagram showing ChIP-seq, ATAC-seq, and 4C-seq results of GATA2 promoter and upstream regions. (upper panel) HindIII cutting sites, gene annotation, and control-specific ATAC-seq peaks. Transcription factors with enriched motifs in the peak are labeled. (middle panel) Genome browser views of ChIP-seq and ATAC-seq results of control and ATRA-treated HL-60 cells; semitransparent cyan color labels the bait site used in 4C-seq, and semitransparent yellow color labels the putative enhancers recognized in 4C-seq. (lower panel) 4C-seq results of control and ATRA-treated HL-60 cells using the bait mentioned above. Red dots indicate 4C-seq RPMs in the ATRA-treated cells, and blue dots indicate RPMs in the control cells; only fragments with significant interactions with the bait are shown. Log2-foldchange of RPMs is shown in the lowest bar graph. b DNA FISH showing the loss of the chromatin loop between the GATA2 promoter and enhancer. BAC probes containing the promoter (red) and enhancer (green) were more separated in the ATRA-treated cells (right) than in the control cells (left). c Loci of promoter and enhancer interacted more in the control cells than in the ATRA-treated cells. p -Value was determined by Mann–Whitney U -test: ** p -value

    Article Snippet: HindIII digestion was performed at 37 °C overnight, and then, a proximity ligation was performed at 4 °C for 4 h. After reversing the cross-linking with a Proteinase K (Ambion, USA) treatment, the DNA was purified using a phenol–chloroform (Solarbio) extraction with ethanol precipitation.

    Techniques: Chromatin Immunoprecipitation, Labeling, Fluorescence In Situ Hybridization, BAC Assay, MANN-WHITNEY

    5′–3′ loop of ZBTB16 was lost upon ATRA induction a Diagram showing ChIP-seq, ATAC-seq and 4C-seq results of ZBTB16. (upper panel) HindIII cutting sites, gene annotation and control-specific ATAC-seq peaks. Transcription factors with enriched motifs in the peak are labeled. (middle panel) Genome browser views of ChIP-seq and ATAC-seq results of ATRA-treated and control HL-60 cells; semitransparent cyan color labels the bait site used in 4C-seq, and semitransparent yellow color labels the site with a strong interaction with the bait recognized in 4C-seq. (lower panel) 4C-seq results of ATRA-treated and control HL-60 cells using the bait mentioned above. Red dots indicate 4C-seq RPMs in the ATRA-treated cells, and blue dots indicate RPMs in the control cells; only fragments with significant interactions with the bait are shown. Log2-foldchange of RPMs are shown in the lowest bar graph. b Model depicting the change in the chromatin architecture around the Gata2 gene. c Model depicting the change in the chromatin architecture around the Zbtb16 gene

    Journal: Cell Death & Disease

    Article Title: Alterations of specific chromatin conformation affect ATRA-induced leukemia cell differentiation

    doi: 10.1038/s41419-017-0173-6

    Figure Lengend Snippet: 5′–3′ loop of ZBTB16 was lost upon ATRA induction a Diagram showing ChIP-seq, ATAC-seq and 4C-seq results of ZBTB16. (upper panel) HindIII cutting sites, gene annotation and control-specific ATAC-seq peaks. Transcription factors with enriched motifs in the peak are labeled. (middle panel) Genome browser views of ChIP-seq and ATAC-seq results of ATRA-treated and control HL-60 cells; semitransparent cyan color labels the bait site used in 4C-seq, and semitransparent yellow color labels the site with a strong interaction with the bait recognized in 4C-seq. (lower panel) 4C-seq results of ATRA-treated and control HL-60 cells using the bait mentioned above. Red dots indicate 4C-seq RPMs in the ATRA-treated cells, and blue dots indicate RPMs in the control cells; only fragments with significant interactions with the bait are shown. Log2-foldchange of RPMs are shown in the lowest bar graph. b Model depicting the change in the chromatin architecture around the Gata2 gene. c Model depicting the change in the chromatin architecture around the Zbtb16 gene

    Article Snippet: HindIII digestion was performed at 37 °C overnight, and then, a proximity ligation was performed at 4 °C for 4 h. After reversing the cross-linking with a Proteinase K (Ambion, USA) treatment, the DNA was purified using a phenol–chloroform (Solarbio) extraction with ethanol precipitation.

    Techniques: Chromatin Immunoprecipitation, Labeling

    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.

    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

    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.

    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

    Control digestions of recombinant plasmid DNA using HindIII (Fermentas) enzyme. (A) pIL253:PptcB:MOG35-55, (B) pIL253:PptcB:MBP85-97, (C) pIL253:PptcB:PLP139-151. Expected DNA fragments after restriction analysis for correct constructs: 3897 bp, 845 bp (marked by red arrows), 1kb DNA ladder (M) 1kb DNA Ladder (Fermentas) used as DNA size reference marker.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Oral Administration of Lactococcus lactis Expressing Synthetic Genes of Myelin Antigens in Decreasing Experimental Autoimmune Encephalomyelitis in Rats

    doi: 10.12659/MSM.892764

    Figure Lengend Snippet: Control digestions of recombinant plasmid DNA using HindIII (Fermentas) enzyme. (A) pIL253:PptcB:MOG35-55, (B) pIL253:PptcB:MBP85-97, (C) pIL253:PptcB:PLP139-151. Expected DNA fragments after restriction analysis for correct constructs: 3897 bp, 845 bp (marked by red arrows), 1kb DNA ladder (M) 1kb DNA Ladder (Fermentas) used as DNA size reference marker.

    Article Snippet: From confirmed proper recombinant cells, plasmid DNA was isolated using the Plasmid Mini Kit (A & A Biotechnology) and subjected to restriction analysis with HindIII enzyme (Fermentas).

    Techniques: Recombinant, Plasmid Preparation, Construct, Marker

    Transient, stable and amplificational replication of the HPV11wt and HPV11E8- genomes in U2OS cells. The mock-transfected cells were used as a negative control (A, lane 7, B, lane 23 and C, lane 10). The linearized HPV11 genome of 100 copies (A, lane 8, B, lane 24 and C, lane 11) and DpnI fragments (C, lane 12) was used as size markers, also indicated with arrows. (A) U2OS cells were transfected with 500 ng of the HPV11wt (lanes 1-3) or E8- (lanes 4-6) genome. Extrachromosomal DNA was extracted via Hirt lysis at 3, 4 and 5 days post-transfection, digested with HindIII and with DpnI. The replication signal was detected via the Southern blot method with a radiolabelled HPV11 genome probe. (B) U2OS cells were transfected with 500 ng of the HPV11wt (lanes 1-11) or HPV11E8- (lanes 12-22) genome, together with 2 μg of the linearized pBabe-Neo construct. The transfected cells were selected with the antibiotic G418, and at 10 days post-transfection, the cells were split and cultivated under either subconfluent (lanes 2-6 and 13-17) or confluent conditions (lanes 7-11 and 18-22). Total DNA was extracted at the time points indicated at top of the figure, and 3 μg of each sample was analyzed as indicated in A . (C) Effect of E8˄E1 and E8˄E2 proteins on viral genome replication. U2OS cells were transfected with 500 ng of the HPV11E8- genome with increasing amounts of either the E8˄E1 or E8˄E2 expression plasmid. Total DNA was extracted at 4 days post-transfection, and 3 μg of each sample was analyzed as indicated in A . (D) The quantitation of HPV11wt and E8- genome DNA replication signals at different time points at subconfluent and confluent culture conditions. The signals were normalized to 10 th day time point. Shown is one of two independent experiments.

    Journal: Virology Journal

    Article Title: The transcription map of HPV11 in U2OS cells adequately reflects the initial and stable replication phases of the viral genome

    doi: 10.1186/s12985-015-0292-6

    Figure Lengend Snippet: Transient, stable and amplificational replication of the HPV11wt and HPV11E8- genomes in U2OS cells. The mock-transfected cells were used as a negative control (A, lane 7, B, lane 23 and C, lane 10). The linearized HPV11 genome of 100 copies (A, lane 8, B, lane 24 and C, lane 11) and DpnI fragments (C, lane 12) was used as size markers, also indicated with arrows. (A) U2OS cells were transfected with 500 ng of the HPV11wt (lanes 1-3) or E8- (lanes 4-6) genome. Extrachromosomal DNA was extracted via Hirt lysis at 3, 4 and 5 days post-transfection, digested with HindIII and with DpnI. The replication signal was detected via the Southern blot method with a radiolabelled HPV11 genome probe. (B) U2OS cells were transfected with 500 ng of the HPV11wt (lanes 1-11) or HPV11E8- (lanes 12-22) genome, together with 2 μg of the linearized pBabe-Neo construct. The transfected cells were selected with the antibiotic G418, and at 10 days post-transfection, the cells were split and cultivated under either subconfluent (lanes 2-6 and 13-17) or confluent conditions (lanes 7-11 and 18-22). Total DNA was extracted at the time points indicated at top of the figure, and 3 μg of each sample was analyzed as indicated in A . (C) Effect of E8˄E1 and E8˄E2 proteins on viral genome replication. U2OS cells were transfected with 500 ng of the HPV11E8- genome with increasing amounts of either the E8˄E1 or E8˄E2 expression plasmid. Total DNA was extracted at 4 days post-transfection, and 3 μg of each sample was analyzed as indicated in A . (D) The quantitation of HPV11wt and E8- genome DNA replication signals at different time points at subconfluent and confluent culture conditions. The signals were normalized to 10 th day time point. Shown is one of two independent experiments.

    Article Snippet: Before Southern blot analysis, half of each extrachromosomal DNA sample or 3 μg of each total DNA sample was treated with the linearizing enzyme HindIII, and non-replicated, bacterially produced dam-methylated input DNA was fragmented with DpnI (Thermo Scientific).

    Techniques: Transfection, Negative Control, Lysis, Southern Blot, Construct, Expressing, Plasmid Preparation, Quantitation Assay

    Identification of recombination vector pET 32b (+)-UL16 by restriction enzymes digestion . Lane M1, DNA marker; Lane 1, PCR products from pET 32b(+)-UL16; Lane 2, recombination plasmid pET 32b(+)-UL16 was digested with two restriction enzymes HindIII and XhoI; Lane M2, DNA marker.

    Journal: Virology Journal

    Article Title: Expression and characterization of UL16 gene from duck enteritis virus

    doi: 10.1186/1743-422X-8-413

    Figure Lengend Snippet: Identification of recombination vector pET 32b (+)-UL16 by restriction enzymes digestion . Lane M1, DNA marker; Lane 1, PCR products from pET 32b(+)-UL16; Lane 2, recombination plasmid pET 32b(+)-UL16 was digested with two restriction enzymes HindIII and XhoI; Lane M2, DNA marker.

    Article Snippet: The T-clone plasmid, pMD18-T- UL16, was digested with the endonucleases HindIII and XhoI, and the UL16 target sequence was subcloned into the same multicloning sites of pET32b (+) (Invitrogen).

    Techniques: Plasmid Preparation, Positron Emission Tomography, Marker, Polymerase Chain Reaction

    Confirmation of genomic composition of 3 independent recombinant VV-ΔTk viruses. ( A ) An ethidium bromide stained DNA gel of genomic HindIII restriction digests of viral DNA isolated from parental VV (Wyeth Strain), 3 clones of VV-ΔTk- yfp-gpt and 3 clones of VV-ΔTk'. Arrows indicate the Tk insertion site (VV-Wyeth), and the unique bands that result from insertion of the yfp-gpt cassette (VV-ΔTk- yfp-gpt ). ( B ) Southern hybridization of the DNA gel in A identifying the yfp insert present in the genome of the VV-ΔTk- yfp-gpt clones, but not in parental VV-Wyeth or the VV-ΔTk' clones. ( C ) DNAStar sequence alignment at the yfp-gpt insertion site of DNA isolated from the 3 VV-ΔTk' clones post Cre passage.

    Journal: PLoS ONE

    Article Title: A Selectable and Excisable Marker System for the Rapid Creation of Recombinant Poxviruses

    doi: 10.1371/journal.pone.0024643

    Figure Lengend Snippet: Confirmation of genomic composition of 3 independent recombinant VV-ΔTk viruses. ( A ) An ethidium bromide stained DNA gel of genomic HindIII restriction digests of viral DNA isolated from parental VV (Wyeth Strain), 3 clones of VV-ΔTk- yfp-gpt and 3 clones of VV-ΔTk'. Arrows indicate the Tk insertion site (VV-Wyeth), and the unique bands that result from insertion of the yfp-gpt cassette (VV-ΔTk- yfp-gpt ). ( B ) Southern hybridization of the DNA gel in A identifying the yfp insert present in the genome of the VV-ΔTk- yfp-gpt clones, but not in parental VV-Wyeth or the VV-ΔTk' clones. ( C ) DNAStar sequence alignment at the yfp-gpt insertion site of DNA isolated from the 3 VV-ΔTk' clones post Cre passage.

    Article Snippet: Briefly, 7 µg of DNA was digested overnight with HindIII restriction enzyme (Invitrogen) at 37°C.

    Techniques: Recombinant, Staining, Isolation, Clone Assay, Hybridization, Sequencing

    Depletion of Mcl-1 downregulates HR and upregulates NHEJ. ( A ) Schematic diagram of HR and NHEJ reporter systems. HR reporter is composed of 2 defective GFP genes that can be rescued only by HR, resulting in GFP fluorescence. In the NHEJ reporter, GFP is interrupted by an adenoviral exon (Ad2) and can be restored upon HindIII digestion and NHEJ repair. ( B ) HR activity was compared in WT and Mcl1 –/– MEFs. Data represent the mean ± SD, n = 3 per group. ** P

    Journal: The Journal of Clinical Investigation

    Article Title: Targeting Mcl-1 enhances DNA replication stress sensitivity to cancer therapy

    doi: 10.1172/JCI92742

    Figure Lengend Snippet: Depletion of Mcl-1 downregulates HR and upregulates NHEJ. ( A ) Schematic diagram of HR and NHEJ reporter systems. HR reporter is composed of 2 defective GFP genes that can be rescued only by HR, resulting in GFP fluorescence. In the NHEJ reporter, GFP is interrupted by an adenoviral exon (Ad2) and can be restored upon HindIII digestion and NHEJ repair. ( B ) HR activity was compared in WT and Mcl1 –/– MEFs. Data represent the mean ± SD, n = 3 per group. ** P

    Article Snippet: First, NHEJ substrate GFP-Pem1-Ad2 plasmids were linearized by restriction enzyme HindIII (Thermo Fisher Scientific).

    Techniques: Non-Homologous End Joining, Fluorescence, Activity Assay

    Representative RFLP patterns of the amplified 1100 bp fragment of actin gene obtained from T. vaginalis isolates. Lane 1, 5 and 8 digested by HindII ; Lane 2, 6 and 9 digested by Tru1 I; Lane 3, 7 and 10 digested by RsaI ; M: DNA ladder (50 bp)

    Journal: Iranian Journal of Parasitology

    Article Title: Genotyping, Drug Susceptibility and Prevalence Survey of Trichomonas vaginalis among Women Attending Gynecology Clinics in Hamadan, Western Iran, in 2014–2015

    doi:

    Figure Lengend Snippet: Representative RFLP patterns of the amplified 1100 bp fragment of actin gene obtained from T. vaginalis isolates. Lane 1, 5 and 8 digested by HindII ; Lane 2, 6 and 9 digested by Tru1 I; Lane 3, 7 and 10 digested by RsaI ; M: DNA ladder (50 bp)

    Article Snippet: RFLP analysis was performed by three restriction enzyme, HindII , Tru1I and RsaI (Fermentas, Thermo Scientific, USA), according to the manufacturer’s instructions.

    Techniques: Amplification

    (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)]

    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

    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.

    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

    U178G/U179G replicates but fails to exit local leaves following rub-inoculation. Total RNA was collected from: (A) inoculated leaves, (B) upper systemically infected leaves, or (C) petioles of inoculated leaves of 10 plants inoculated with U178G/U179G or wild type PSTVd (WT, one plant, positive control). Mock inoculation (M) was a negative control. (A) RNA blot assay indicates U178G/U179G replication in rub-inoculated leaves. (B) RNA blot assay indicates U178G/U179G is unable to traffic to upper leaves following rub inoculation. (C) RT-PCR indicates U178G/U179G is not present in petioles and fails to exit inoculated leaves. In A and B, the region of the blot corresponding to circular progeny genomes is shown. Loading controls were ribosomal RNA (rRNA) (A and B) and RT-PCR of actin mRNA (C), detected by ethidium bromide staining. Images are representative of 10 (A and B) and three (C) independent experiments.

    Journal: PLoS Pathogens

    Article Title: A three-dimensional RNA motif mediates directional trafficking of Potato spindle tuber viroid from epidermal to palisade mesophyll cells in Nicotiana benthamiana

    doi: 10.1371/journal.ppat.1008147

    Figure Lengend Snippet: U178G/U179G replicates but fails to exit local leaves following rub-inoculation. Total RNA was collected from: (A) inoculated leaves, (B) upper systemically infected leaves, or (C) petioles of inoculated leaves of 10 plants inoculated with U178G/U179G or wild type PSTVd (WT, one plant, positive control). Mock inoculation (M) was a negative control. (A) RNA blot assay indicates U178G/U179G replication in rub-inoculated leaves. (B) RNA blot assay indicates U178G/U179G is unable to traffic to upper leaves following rub inoculation. (C) RT-PCR indicates U178G/U179G is not present in petioles and fails to exit inoculated leaves. In A and B, the region of the blot corresponding to circular progeny genomes is shown. Loading controls were ribosomal RNA (rRNA) (A and B) and RT-PCR of actin mRNA (C), detected by ethidium bromide staining. Images are representative of 10 (A and B) and three (C) independent experiments.

    Article Snippet: Plasmid pRZ6-2-Int and mutant derivatives were linearized with Hind III and employed as templates to generate (+)-PSTVd wild type and mutant in vitro transcripts using the T7 Megascript kit (ThermoFisher Scientific).

    Techniques: Infection, Positive Control, Negative Control, Northern blot, Reverse Transcription Polymerase Chain Reaction, Staining

    Stability of U178G/U179G is similar to wild type PSTVd. (A) RNA blot of in vitro degradation assays performed at 28°C in buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM phenylmethylsulfonyl fluoride), or uninfected N . benthamiana leaf extract prepared with the same buffer. (B) Percentage of remaining PSTVd wild type (WT) and U178G/U179G RNA over time was determined using Quantity One software. Data are representative of three independent experiments.

    Journal: PLoS Pathogens

    Article Title: A three-dimensional RNA motif mediates directional trafficking of Potato spindle tuber viroid from epidermal to palisade mesophyll cells in Nicotiana benthamiana

    doi: 10.1371/journal.ppat.1008147

    Figure Lengend Snippet: Stability of U178G/U179G is similar to wild type PSTVd. (A) RNA blot of in vitro degradation assays performed at 28°C in buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM phenylmethylsulfonyl fluoride), or uninfected N . benthamiana leaf extract prepared with the same buffer. (B) Percentage of remaining PSTVd wild type (WT) and U178G/U179G RNA over time was determined using Quantity One software. Data are representative of three independent experiments.

    Article Snippet: Plasmid pRZ6-2-Int and mutant derivatives were linearized with Hind III and employed as templates to generate (+)-PSTVd wild type and mutant in vitro transcripts using the T7 Megascript kit (ThermoFisher Scientific).

    Techniques: Northern blot, In Vitro, Software

    spa -RFLP patterns obtained from Hin dIII digestion of the PCR products. a Human, b bovine and c food S. aureus isolates. Lane L: a 100 bp DNA ladder. The other lanes: RFLP patterns of S. aureus isolates

    Journal: 3 Biotech

    Article Title: Molecular typing of Staphylococcus aureus of different origins based on the polymorphism of the spa gene: characterization of a novel spa type

    doi: 10.1007/s13205-017-1061-6

    Figure Lengend Snippet: spa -RFLP patterns obtained from Hin dIII digestion of the PCR products. a Human, b bovine and c food S. aureus isolates. Lane L: a 100 bp DNA ladder. The other lanes: RFLP patterns of S. aureus isolates

    Article Snippet: The PCR products of the spa gene were digested using two restriction enzymes, Hin dIII (Jena Bioscience, Germany) and Hin fI (Jena Bioscience, Germany).

    Techniques: Polymerase Chain Reaction

    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

    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

    Generation of vPK transgenic mice. FLAG-tagged vPK was cloned, using HinDIII-HF and BamHI-HF, into a plasmid containing a Ub- and hGH-stabilization element. The linearized transgene fragment was microinjected into the embryos of C57BL/6 mice and implanted into pseudopregnant female mice. Each vPK line was developed from breeding a vPK founder to a C57BL/6 mouse. ( A ) Schematic of the linearized construct used to generate the vPK transgenic mice, as well as the cut sites and probe for the Southern blot in D . ( B ) DNA from the tails of 2 WT (wA and wB) and 2 vPK transgenic mice (vA and vB) for lines 1 and 2 was isolated and evaluated by PCR for the vPK transgene. ( C ) Expression of vPK protein in lysates from spleens of the mice in B as determined by SDS-PAGE and Western blot. Lysate from HEK-293 cells that transiently express vPK was used as a positive control for vPK expression. ( D ) Southern blot of WT, vPK1, and vPK2. DNA was isolated from the spleens of WT and vPK lines 1 and 2. D, double digest with HinDIII-HF and BamHI-HF; S, single digestion with AFlII; p-vPK, plasmid Ub.vPK.hGH.

    Journal: The Journal of Clinical Investigation

    Article Title: Human herpesvirus–encoded kinase induces B cell lymphomas in vivo

    doi: 10.1172/JCI97053

    Figure Lengend Snippet: Generation of vPK transgenic mice. FLAG-tagged vPK was cloned, using HinDIII-HF and BamHI-HF, into a plasmid containing a Ub- and hGH-stabilization element. The linearized transgene fragment was microinjected into the embryos of C57BL/6 mice and implanted into pseudopregnant female mice. Each vPK line was developed from breeding a vPK founder to a C57BL/6 mouse. ( A ) Schematic of the linearized construct used to generate the vPK transgenic mice, as well as the cut sites and probe for the Southern blot in D . ( B ) DNA from the tails of 2 WT (wA and wB) and 2 vPK transgenic mice (vA and vB) for lines 1 and 2 was isolated and evaluated by PCR for the vPK transgene. ( C ) Expression of vPK protein in lysates from spleens of the mice in B as determined by SDS-PAGE and Western blot. Lysate from HEK-293 cells that transiently express vPK was used as a positive control for vPK expression. ( D ) Southern blot of WT, vPK1, and vPK2. DNA was isolated from the spleens of WT and vPK lines 1 and 2. D, double digest with HinDIII-HF and BamHI-HF; S, single digestion with AFlII; p-vPK, plasmid Ub.vPK.hGH.

    Article Snippet: Fifteen micrograms of DNA from each mouse was double digested with BamHI-HF and HindIII-HF (New England BioLabs) and single digested using AflII (New England BioLabs) in a total volume of 100 μl at 37°C overnight.

    Techniques: Transgenic Assay, Mouse Assay, Clone Assay, Plasmid Preparation, Construct, Southern Blot, Western Blot, Isolation, Polymerase Chain Reaction, Expressing, SDS Page, Positive Control

    Assay validation. a The goal of the assay in the DGAP230 experimental system is to differentiate the target region (yellow box) on the der(20) chromosome (top) from the target region on the normal chr20 (bottom). The small green bar represents the 3C genomic fragment that contains the target region and the small blue bar represents the digested genomic fragment containing a breakpoint-proximal region from the segment of chr22 translocated to the der(20). Rough gray edges reflect enzymatic digestion at flanking Hin dIII restriction sites. b Schematic of nested PCR amplifications for the predicted ligation product with the target region (green bar above mahogany map) and the chr22 fragment (blue bar above light pink map). c Gel electrophoresis displays products from the first PCR across the breakpoint for experimental and control 3C libraries (left), and the second nested PCR (right, N=3). Key DNA fragment sizes of the markers (M) are indicated on the left. d Sanger sequencing traces of the target variable region from the nested PCR amplicon (top) and genomic DNA from the same cell line (bottom; N=3)

    Journal: Human genetics

    Article Title: 3C-PCR: A novel proximity ligation-based approach to phase chromosomal rearrangement breakpoints with distal allelic variants

    doi: 10.1007/s00439-017-1853-0

    Figure Lengend Snippet: Assay validation. a The goal of the assay in the DGAP230 experimental system is to differentiate the target region (yellow box) on the der(20) chromosome (top) from the target region on the normal chr20 (bottom). The small green bar represents the 3C genomic fragment that contains the target region and the small blue bar represents the digested genomic fragment containing a breakpoint-proximal region from the segment of chr22 translocated to the der(20). Rough gray edges reflect enzymatic digestion at flanking Hin dIII restriction sites. b Schematic of nested PCR amplifications for the predicted ligation product with the target region (green bar above mahogany map) and the chr22 fragment (blue bar above light pink map). c Gel electrophoresis displays products from the first PCR across the breakpoint for experimental and control 3C libraries (left), and the second nested PCR (right, N=3). Key DNA fragment sizes of the markers (M) are indicated on the left. d Sanger sequencing traces of the target variable region from the nested PCR amplicon (top) and genomic DNA from the same cell line (bottom; N=3)

    Article Snippet: Chromatin was digested with Hin dIII-HF (NEB), ligated with T4 DNA ligase (NEB), and reverse crosslinked by incubation with Proteinase K (NEB) and RNase A (EMD Millipore).

    Techniques: Nested PCR, Ligation, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Sequencing, Amplification

    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

    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

    BoHV-4 gB disruption. A) Overall strategy to delete a 1261 bp sequence from the ORF8 coding for gB, via heat inducible homologous recombination. The 2232 bp Kana-GalK selectable DNA stuffer, flanked by ORF8 homologous regions, was introduced between positions 11640 and 12901 of the BoHV-4-A strain cloned as a BAC. The expected ORF8 locus (A, bottom) has an increased size of the HindIII fragment (5280 instead of 4314 bp), generated by HindIII restriction enzyme digestion. The diagrams here presented are not to scale. B) HindIII Restriction profile and corresponding Southern Blotting of six representative targeted clones, compared to the untargeted control. Southern Blotting was performed with a probe spanning Kana sequence and confirmed the above data. C) Clonal stability of the pBAC-BoHV-4-A-ΔgBKanaGalK in Escherichia coli SW102 cells, passaged for 25 consecutive days and analyzed by HindIII digestion and agarose gel electrophoresis.

    Journal: BMC Veterinary Research

    Article Title: Bovine herpesvirus 4 glycoprotein B is indispensable for lytic replication and irreplaceable by VSVg

    doi: 10.1186/1746-6148-9-6

    Figure Lengend Snippet: BoHV-4 gB disruption. A) Overall strategy to delete a 1261 bp sequence from the ORF8 coding for gB, via heat inducible homologous recombination. The 2232 bp Kana-GalK selectable DNA stuffer, flanked by ORF8 homologous regions, was introduced between positions 11640 and 12901 of the BoHV-4-A strain cloned as a BAC. The expected ORF8 locus (A, bottom) has an increased size of the HindIII fragment (5280 instead of 4314 bp), generated by HindIII restriction enzyme digestion. The diagrams here presented are not to scale. B) HindIII Restriction profile and corresponding Southern Blotting of six representative targeted clones, compared to the untargeted control. Southern Blotting was performed with a probe spanning Kana sequence and confirmed the above data. C) Clonal stability of the pBAC-BoHV-4-A-ΔgBKanaGalK in Escherichia coli SW102 cells, passaged for 25 consecutive days and analyzed by HindIII digestion and agarose gel electrophoresis.

    Article Snippet: Restriction enzyme analysis and non isotopic Southern hybridization Fifteen μl of DNA prepared from bacteria containing pBAC-BoHV-4-A and derivatives were restriction enzyme-digested with HindIII, separated by electrophoresis overnight in a 0.8% agarose gel, stained with ethidium bromide, capillary transferred to a positively charged nylon membrane (Roche), and cross-linked by UV irradiation by standard procedures.

    Techniques: Sequencing, Homologous Recombination, Clone Assay, BAC Assay, Generated, Southern Blot, Agarose Gel Electrophoresis

    Sites of Tn 917 insertions. (A) Autoradiogram from a Southern blot analysis of the restriction digests of total DNA from E. faecalis strains following hybridization with 32 P-labeled pTV1-OK. Lane 1, lambda DNA digested with HindIII and EcoRI; lane 2, HindIII-digested

    Journal: Applied and Environmental Microbiology

    Article Title: Characterization of Monolaurin Resistance in Enterococcus faecalis ▿

    doi: 10.1128/AEM.01013-07

    Figure Lengend Snippet: Sites of Tn 917 insertions. (A) Autoradiogram from a Southern blot analysis of the restriction digests of total DNA from E. faecalis strains following hybridization with 32 P-labeled pTV1-OK. Lane 1, lambda DNA digested with HindIII and EcoRI; lane 2, HindIII-digested

    Article Snippet: The presence of single Tn 917 insertions was determined by Southern blot hybridization of HindIII-digested (Roche, Mannheim, Germany) genomic DNA and a radioactively labeled, HindIII-digested pTV1-OK probe ( ).

    Techniques: Southern Blot, Hybridization, Labeling, Lambda DNA Preparation

    Genetic organization of the antibiotic resistance gene cluster of SGI1 and the new SGI1-H variant of serovar Newport strains 01-2174 and 01-5348. The direction of transcription of the genes is indicated by arrows. Black and gray arrows correspond to SGI1 antibiotic resistance genes and chromosomal genes flanking SGI1, respectively. DR-L and DR-R are the left and right direct repeats, respectively, bracketing SGI1. IRi and IRt are 25-bp imperfect inverted repeats defining the left and right ends of complex class 1 integrons. PCRs used to assess the genetic organization of the antibiotic resistance genes (i.e., PCRs floR , A, B, C, D, E, and F) and the SGI1 junctions to the chromosome (i.e., PCRs LJ and RJ for left and right junctions, respectively) are indicated. Restriction sites: X, XbaI; H, HindIII.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Variant Salmonella Genomic Island 1 Antibiotic Resistance Gene Cluster Containing a Novel 3?-N-Aminoglycoside Acetyltransferase Gene Cassette, aac(3)-Id, in Salmonella enterica Serovar Newport

    doi: 10.1128/AAC.48.10.3806-3812.2004

    Figure Lengend Snippet: Genetic organization of the antibiotic resistance gene cluster of SGI1 and the new SGI1-H variant of serovar Newport strains 01-2174 and 01-5348. The direction of transcription of the genes is indicated by arrows. Black and gray arrows correspond to SGI1 antibiotic resistance genes and chromosomal genes flanking SGI1, respectively. DR-L and DR-R are the left and right direct repeats, respectively, bracketing SGI1. IRi and IRt are 25-bp imperfect inverted repeats defining the left and right ends of complex class 1 integrons. PCRs used to assess the genetic organization of the antibiotic resistance genes (i.e., PCRs floR , A, B, C, D, E, and F) and the SGI1 junctions to the chromosome (i.e., PCRs LJ and RJ for left and right junctions, respectively) are indicated. Restriction sites: X, XbaI; H, HindIII.

    Article Snippet: The antibiotic resistance gene organization was also assessed by Southern blot of genomic DNA cut by HindIII (Qbiogene, Illkrich, France) by using as a probe the XbaI fragment of recombinant plasmid pSTF3, comprising nearly the entire serovar Typhimurium DT104 SGI1 antibiotic resistance gene cluster (Fig. ).

    Techniques: Variant Assay

    DNA Gel Blot Hybridization of the Genome-Specific Sequence PSR593 to Genomic DNA from F1 Hybrids and Newly Formed Allopolyploids. (A) Hybridization to genomic DNA from the F1 hybrid between Ae . sharonensis (TH02) and T . monococcum ssp aegilopoides (TMB02), from the S1 generation of the allotetraploid that derived from this hybrid, and from the two parental plants. DNA was digested using EcoRI, EcoRV, DraI, and HindIII. Arrows indicate the bands from the genome of TH02 that disappeared in F1 and in the S1 generation of the allopolyploid. Fragment size is indicated at right in kilobases. (B) Hybridization to genomic DNA from the F1 hybrid between T. turgidum ssp durum (TTR16) and Ae . speltoides (TS01), from the S1 generation of the allohexaploid derived from this hybrid, and from the two parental plants. DNA was digested using HindIII and DraI. Arrows indicate the bands that disappeared in F1 and/or in the S1 generation of the allohexaploid. The upper band of TTR16 is present in F1 of the HindIII digest but is absent in S1. No such difference between F1 and S1 was noted with DraI. This difference between HindIII and DraI probably results from methylation. Fragment size is indicated at right in kilobases.

    Journal: The Plant Cell

    Article Title: Allopolyploidy-Induced Rapid Genome Evolution in the Wheat (Aegilops-Triticum) Group

    doi: 10.1105/TPC.010082

    Figure Lengend Snippet: DNA Gel Blot Hybridization of the Genome-Specific Sequence PSR593 to Genomic DNA from F1 Hybrids and Newly Formed Allopolyploids. (A) Hybridization to genomic DNA from the F1 hybrid between Ae . sharonensis (TH02) and T . monococcum ssp aegilopoides (TMB02), from the S1 generation of the allotetraploid that derived from this hybrid, and from the two parental plants. DNA was digested using EcoRI, EcoRV, DraI, and HindIII. Arrows indicate the bands from the genome of TH02 that disappeared in F1 and in the S1 generation of the allopolyploid. Fragment size is indicated at right in kilobases. (B) Hybridization to genomic DNA from the F1 hybrid between T. turgidum ssp durum (TTR16) and Ae . speltoides (TS01), from the S1 generation of the allohexaploid derived from this hybrid, and from the two parental plants. DNA was digested using HindIII and DraI. Arrows indicate the bands that disappeared in F1 and/or in the S1 generation of the allohexaploid. The upper band of TTR16 is present in F1 of the HindIII digest but is absent in S1. No such difference between F1 and S1 was noted with DraI. This difference between HindIII and DraI probably results from methylation. Fragment size is indicated at right in kilobases.

    Article Snippet: The extracted DNA (10 μg) was digested with the five restriction enzymes EcoRI, EcoRV, HindIII, DraI, and BamHI (Boehringer Mannheim; 1 unit/μg DNA) unless specified otherwise.

    Techniques: Western Blot, Hybridization, Sequencing, Derivative Assay, Methylation

    Equilibrium between circular and linear conformations of 199-bp DNA fragments with various sticky ends. The probability of finding a fragment in circular conformation, f c , can be estimated from its relative mobility in the polyacrylamide gel since the linear (lane M) and circular (lane 1) forms have different mobility in such a gel. The experiments were performed for sticky ends of 4 AT base pairs created by EcoRI restriction enzyme (lane 2), 2 AT and 2 GC base pairs created by HindIII (lane 3) and 4 GC base pairs created by AgeI (lane 4). The gel electrophoresis was run in TBE buffer containing additional 10 mM of MgCl 2 at 5°C ( A ) and 25°C ( B ).

    Journal: Nucleic Acids Research

    Article Title: Temperature dependence of DNA persistence length

    doi: 10.1093/nar/gkq932

    Figure Lengend Snippet: Equilibrium between circular and linear conformations of 199-bp DNA fragments with various sticky ends. The probability of finding a fragment in circular conformation, f c , can be estimated from its relative mobility in the polyacrylamide gel since the linear (lane M) and circular (lane 1) forms have different mobility in such a gel. The experiments were performed for sticky ends of 4 AT base pairs created by EcoRI restriction enzyme (lane 2), 2 AT and 2 GC base pairs created by HindIII (lane 3) and 4 GC base pairs created by AgeI (lane 4). The gel electrophoresis was run in TBE buffer containing additional 10 mM of MgCl 2 at 5°C ( A ) and 25°C ( B ).

    Article Snippet: The sequences of sticky ends correspond to the restriction sites of EcoRI (AATT), HindIII (AGCT) and AgeI (CCGG) endonucleases.

    Techniques: Nucleic Acid Electrophoresis

    The j -factor measured for λ phage DNA fragments with EcoRI sticky ends at 5°C (top) and HindIII sticky ends at 42°C (bottom). The lines correspond to the theoretical fit of the data. The best fit shown by the solid lines, correspond to DNA persistence length of 53.2 nm and γ of 10.43 for 5°C and 42.5 nm and γ of 10.56 for 42°C; the value of C was equal to 3.10 −19 erg·cm for both temperatures. The dotted lines correspond to the theoretical equation with a reduced/increased by 1 nm from the best fits.

    Journal: Nucleic Acids Research

    Article Title: Temperature dependence of DNA persistence length

    doi: 10.1093/nar/gkq932

    Figure Lengend Snippet: The j -factor measured for λ phage DNA fragments with EcoRI sticky ends at 5°C (top) and HindIII sticky ends at 42°C (bottom). The lines correspond to the theoretical fit of the data. The best fit shown by the solid lines, correspond to DNA persistence length of 53.2 nm and γ of 10.43 for 5°C and 42.5 nm and γ of 10.56 for 42°C; the value of C was equal to 3.10 −19 erg·cm for both temperatures. The dotted lines correspond to the theoretical equation with a reduced/increased by 1 nm from the best fits.

    Article Snippet: The sequences of sticky ends correspond to the restriction sites of EcoRI (AATT), HindIII (AGCT) and AgeI (CCGG) endonucleases.

    Techniques:

    Dependence of the measured C/D on the concentration of T4 DNA ligase. The shown data correspond to the fragments with EcoRI (5°C, filled circle) and HindIII (42°C, filled triangle) sticky ends. The data show that the ratio does not change if the ligase concentration is below 100 U/ml.

    Journal: Nucleic Acids Research

    Article Title: Temperature dependence of DNA persistence length

    doi: 10.1093/nar/gkq932

    Figure Lengend Snippet: Dependence of the measured C/D on the concentration of T4 DNA ligase. The shown data correspond to the fragments with EcoRI (5°C, filled circle) and HindIII (42°C, filled triangle) sticky ends. The data show that the ratio does not change if the ligase concentration is below 100 U/ml.

    Article Snippet: The sequences of sticky ends correspond to the restriction sites of EcoRI (AATT), HindIII (AGCT) and AgeI (CCGG) endonucleases.

    Techniques: Concentration Assay

    Agarose gel electrophoresis showing the presence of HBcAg VLP in the pUC57 vector and (pET28a/VLP). ( A ) The nucleotide sequence coding for HBcAg VLP from the pUC57 vector. Lane 1: uncut pUC 57 vector containing the HBcAg VLP sequence; Lane 2: pUC57 vector containing the HBcAg VLP sequence digested with NheI and HindIII enzymes; Lane 3: 1-kb DNA ladder; Lane 4: uncut pET28a vector; Lane 5: pET28a vector linearized with NheI and HindIII enzymes; ( B ) Confirmation of the presence of the hybrid HBcAg VLP nucleotide sequence in (pET28a/VLP). Lane 1: uncut (pET28a/VLP); Lanes 2–3: pET28a HBcAg VLP digested with NheI and HindIII enzymes; Lane 4: 1-kb DNA ladder; Lane 5: uncut pET28a vector; Lane 6: pET28a vector with NheI and HindIII enzymes.

    Journal: International Journal of Molecular Sciences

    Article Title: Production and Evaluation of Virus-Like Particles Displaying Immunogenic Epitopes of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

    doi: 10.3390/ijms16048382

    Figure Lengend Snippet: Agarose gel electrophoresis showing the presence of HBcAg VLP in the pUC57 vector and (pET28a/VLP). ( A ) The nucleotide sequence coding for HBcAg VLP from the pUC57 vector. Lane 1: uncut pUC 57 vector containing the HBcAg VLP sequence; Lane 2: pUC57 vector containing the HBcAg VLP sequence digested with NheI and HindIII enzymes; Lane 3: 1-kb DNA ladder; Lane 4: uncut pET28a vector; Lane 5: pET28a vector linearized with NheI and HindIII enzymes; ( B ) Confirmation of the presence of the hybrid HBcAg VLP nucleotide sequence in (pET28a/VLP). Lane 1: uncut (pET28a/VLP); Lanes 2–3: pET28a HBcAg VLP digested with NheI and HindIII enzymes; Lane 4: 1-kb DNA ladder; Lane 5: uncut pET28a vector; Lane 6: pET28a vector with NheI and HindIII enzymes.

    Article Snippet: The codon-optimized nucleotide sequence was artificially synthesized and cloned into a pUC57 vector at BamHI and HindIII by Genscript Corporation (Piscataway, NJ, USA).

    Techniques: Agarose Gel Electrophoresis, Plasmid Preparation, Sequencing

    pL102 presentation and shortening description. pL102 is composed of a 13.5-kb DNA fragment (▪) inserted in the pUC19 vector (□).The mclC structural gene, the mclI immunity gene, and the mclA and mclB export genes constitute the MccL genetic system. The restriction sites used for the insert shortening were XbaI/SacI on one side and HindIII/PauI on the other side. The HindIII-PauI fragment was deleted. The dotted arrows indicate the start points and the direction of the shortening.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Mechanism of Bactericidal Activity of Microcin L in Escherichia coli and Salmonella enterica ▿

    doi: 10.1128/AAC.01217-10

    Figure Lengend Snippet: pL102 presentation and shortening description. pL102 is composed of a 13.5-kb DNA fragment (▪) inserted in the pUC19 vector (□).The mclC structural gene, the mclI immunity gene, and the mclA and mclB export genes constitute the MccL genetic system. The restriction sites used for the insert shortening were XbaI/SacI on one side and HindIII/PauI on the other side. The HindIII-PauI fragment was deleted. The dotted arrows indicate the start points and the direction of the shortening.

    Article Snippet: This plasmid (5.7 μg) then was digested with 30 U HindIII (Eurogentec) in Fermentas buffer R (required for the second restriction digest) for 3 h at 37°C.

    Techniques: Plasmid Preparation

    Cloning of SFD1 . (A) Alignment of the SFD1 sequence with the consensus sequence from COG0240 and Pfam0210.8. Basic Local Alignment Search Tool (BLASTp) analysis of the predicted SFD1 protein sequence revealed homology to DHAP reductases/G3P dehydrogenases in the COG2040 and Pfam0210.8 protein family data sets. The alignment of SFD1 amino acids 89 to 419 to the consensus sequence from COG2040 and Pfam0210.8 is shown. Solid and dashed lines above the SFD1 sequence mark the predicted NAD + and substrate binding domains, respectively. The arrow indicates the Ala 381 that is mutated to yield Thr 381 in sfd1-2 . Amino acids that are identical to SFD1 are shown in red and those that are similar are shown in blue. (B) Complementation of the sfd1-1 and sfd1-2 mutants by SFD1 . Comparison of the morphology of 4-week-old, soil-grown wild-type sfd1-1 , ssi2 npr1 , and sfd1-1 ssi2 npr1 plants and the sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a 5-kb HindIII fragment containing the genomic SFD1 clone. The photographs were taken from the same distance. (C) Restoration of the ssi2 -conferred cell death by SFD1 . Leaves from 4-week-old, soil-grown ssi2 npr1 , sfd1-1 ssi2 npr1 , sfd1-1/SFD1 ssi2 npr1 , and sfd1-2/SFD1 ssi2 npr1 plants were stained with trypan blue. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a 5-kb HindIII fragment containing the genomic SFD1 clone. Trypan blue–stained leaves from the transgenic plants and from the ssi2 npr1 plants show intensely stained dead cells (yellow arrows). All of the photographs were taken at the same magnification. (D) Restoration of the ssi2 -conferred constitutive PR1 expression by SFD1 . PR1 expression was monitored in 4-week-old, soil-grown sfd1-1 ssi2 npr1 ( sfd1-1 ) and sfd1-2 ssi2 npr1 ( sfd1-2 ) plants transformed with pBI121 (Vector) or with pBI121 containing a 5-kb HindIII genomic fragment spanning SFD1 (SFD1). Blots were hybridized with a radiolabeled PR1 probe. Gel loading was monitored by photographing the ethidium bromide (EtBr)–stained gel before transfer to the membrane.

    Journal: The Plant Cell

    Article Title: The Arabidopsis thaliana Dihydroxyacetone Phosphate Reductase Gene SUPPRESSOR OF FATTY ACID DESATURASE DEFICIENCY1 Is Required for Glycerolipid Metabolism and for the Activation of Systemic Acquired Resistance W⃞

    doi: 10.1105/tpc.016907

    Figure Lengend Snippet: Cloning of SFD1 . (A) Alignment of the SFD1 sequence with the consensus sequence from COG0240 and Pfam0210.8. Basic Local Alignment Search Tool (BLASTp) analysis of the predicted SFD1 protein sequence revealed homology to DHAP reductases/G3P dehydrogenases in the COG2040 and Pfam0210.8 protein family data sets. The alignment of SFD1 amino acids 89 to 419 to the consensus sequence from COG2040 and Pfam0210.8 is shown. Solid and dashed lines above the SFD1 sequence mark the predicted NAD + and substrate binding domains, respectively. The arrow indicates the Ala 381 that is mutated to yield Thr 381 in sfd1-2 . Amino acids that are identical to SFD1 are shown in red and those that are similar are shown in blue. (B) Complementation of the sfd1-1 and sfd1-2 mutants by SFD1 . Comparison of the morphology of 4-week-old, soil-grown wild-type sfd1-1 , ssi2 npr1 , and sfd1-1 ssi2 npr1 plants and the sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a 5-kb HindIII fragment containing the genomic SFD1 clone. The photographs were taken from the same distance. (C) Restoration of the ssi2 -conferred cell death by SFD1 . Leaves from 4-week-old, soil-grown ssi2 npr1 , sfd1-1 ssi2 npr1 , sfd1-1/SFD1 ssi2 npr1 , and sfd1-2/SFD1 ssi2 npr1 plants were stained with trypan blue. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a 5-kb HindIII fragment containing the genomic SFD1 clone. Trypan blue–stained leaves from the transgenic plants and from the ssi2 npr1 plants show intensely stained dead cells (yellow arrows). All of the photographs were taken at the same magnification. (D) Restoration of the ssi2 -conferred constitutive PR1 expression by SFD1 . PR1 expression was monitored in 4-week-old, soil-grown sfd1-1 ssi2 npr1 ( sfd1-1 ) and sfd1-2 ssi2 npr1 ( sfd1-2 ) plants transformed with pBI121 (Vector) or with pBI121 containing a 5-kb HindIII genomic fragment spanning SFD1 (SFD1). Blots were hybridized with a radiolabeled PR1 probe. Gel loading was monitored by photographing the ethidium bromide (EtBr)–stained gel before transfer to the membrane.

    Article Snippet: A 5-kb HindIII fragment then was further subcloned into the HindIII site of the binary vector pBI121 (Clonetech, San Fransisco, CA).

    Techniques: Clone Assay, Sequencing, Binding Assay, Transformation Assay, Staining, Transgenic Assay, Expressing, Plasmid Preparation

    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.

    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

    Schematic illustration of PCR-RFLP assays using HindIII endonuclease, indicating that attB and attP sequences are heterogeneous hybrids of the fixed attL and attR sequences. The att sequences are shown as white bars, flanked by DNA of ICE backbone ORFs

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Characterization of a Streptococcus suis tet(O/W/32/O)-Carrying Element Transferable to Major Streptococcal Pathogens

    doi: 10.1128/AAC.00629-12

    Figure Lengend Snippet: Schematic illustration of PCR-RFLP assays using HindIII endonuclease, indicating that attB and attP sequences are heterogeneous hybrids of the fixed attL and attR sequences. The att sequences are shown as white bars, flanked by DNA of ICE backbone ORFs

    Article Snippet: The four att sequences were investigated by PCR-restriction fragment length polymorphism (RFLP) analysis using HindIII endonuclease (Roche Applied Science, Basel, Switzerland).

    Techniques: Polymerase Chain Reaction