Journal: Nucleic Acids Research

Article Title: Single-molecule observations of topotecan-mediated TopIB activity at a unique DNA sequence

doi: 10.1093/nar/gkn035

Figure Lengend Snippet: Binding of the TFO–TPT construct and site-specific cleavage by TopIB. ( a ) A triple helix-forming oligonucleotide (red), covalently linked to a topotecan (TPT) molecule binds in the major groove of DNA (blue). ( b ) Synthetic route for the preparation of 10-(6-aminohexyloxy)-topotecan NTPT (referred to as TPT) and 10-(6-aminohexyloxy)-camptothecin NCPT (referred to as CPT). The chemical structure of the conjugates TFO–TPT and TFO–CPT. ( c ) The target site on the duplex is underlined and the topoisomerase I-mediated DNA cleavage site is indicated by an arrow. M , 5-methyl-2′-deoxycytidine; P , 5-propynyl-2′-deoxyuridine. ( d ) PAGE analysis of triple helix formation: the duplex target RY* ([RY*] = 20 nM), radiolabeled at the 5′ end of the pyrimidine strand Y*, is incubated at 37°C in 50 mM HEPES pH = 7.2, 100 mM NaCl, 10 mM MgCl 2 in the presence of decreasing concentrations of: TFOs (1, 0.5, 0.3, 0.2 and 0.1 µM), TFO–TPT (2, 1, 0.8, 0.5 and 0.1 µM) and TFO–CPT (5, 2, 1 and 0.5 µM). Aliquots were removed after 2 h of incubation at 37°C and analyzed on a 15% nondenaturating acrylamide gel. ( e ) A radiolabeled 324-bp DNA fragment containing the duplex target (lane 1) was incubated at 37°C and pH 7.2 in the presence of topoisomerase (lane 2) and of the TFO at 5 µM (lane 3), CPT at 10 µM (lane 4), TFO–CPT at 5 µM (lane 5), TPT at 10 µM (lane 6) and TFO–TPT at 5 µM (lane 7). Adenine/guanine-specific Maxam–Gilbert chemical cleavage reactions were used as markers (G + A). The position of the cleavage site of the conjugates is indicated by an arrow. The region corresponding to the triplex site is indicated (TFO), as the orientation.

Article Snippet: Oligonucleotide TFO (sequence reported in c) was purchased from Eurogentec and purified using quick spin columns and sephadex G-25 fine (Boehringer, Mannheim).

Techniques: Binding Assay, Construct, Incubation, Acrylamide Gel Assay

Journal: Nucleic Acids Research

Article Title: Single-molecule observations of topotecan-mediated TopIB activity at a unique DNA sequence

doi: 10.1093/nar/gkn035

Figure Lengend Snippet: Experimental strategy to detect sequence-specific uncoiling by TopIB at the single-molecule level. ( a ) A supercoiled double-stranded and continuous DNA molecule (blue) is tethered between a magnetic bead (gray sphere) and a glass surface. The TFO (red), connected to the TPT (black) binds in a sequence-specific manner to the DNA molecule. TopIB enzyme molecules (blue C-shaped structures) diffuse freely in buffer solution. ( b ) TopIB can only remove DNA supercoils in a drug-dependent manner at a site on the DNA that is dictated by the sequence-specific binding of the TFO–TPT.

Article Snippet: Oligonucleotide TFO (sequence reported in c) was purchased from Eurogentec and purified using quick spin columns and sephadex G-25 fine (Boehringer, Mannheim).

Techniques: Sequencing, Binding Assay

Journal: Nucleic Acids Research

Article Title: Single-molecule observations of topotecan-mediated TopIB activity at a unique DNA sequence

doi: 10.1093/nar/gkn035

Figure Lengend Snippet: The presence of the TFO does not influence quasi-static mechanical properties of DNA. ( a ) Using the magnetic tweezers, positive (+) or negative (–) supercoils can be readily introduced in the DNA molecule by either twisting the magnets along with (green arrow) or against (red arrow) the natural helicity of the DNA. ( b ) Under the appropriate conditions of stretching force and buffer, the response of the DNA molecule to twisting is symmetrical: both (+) and (–) supercoils cause the DNA extension to decrease in an equal manner, as plectonemic supercoils form. ( c ) A positively supercoiled molecule will relax, when cleaved by a nicking enzyme. Consequently, the DNA extension will increase in time. ( d ) The dynamics of the uncoiling is in thermal equilibrium, and is well-predicted by a model that is described in ref. 33. ( e ) Cleavage of a supercoiled DNA molecule, in the presence of a bound TFO construct. The sequence of the TFO is chosen such that cleavage occurs at a distance of 5 bp from the 3′-end of the TFO. ( f ) The dynamics of supercoil removal after cleavage with a nicking enzyme in the presence of TFO is well described by the quasi-static model, similar to (d).

Article Snippet: Oligonucleotide TFO (sequence reported in c) was purchased from Eurogentec and purified using quick spin columns and sephadex G-25 fine (Boehringer, Mannheim).

Techniques: Construct, Sequencing

Journal: Nucleic Acids Research

Article Title: Single-molecule observations of topotecan-mediated TopIB activity at a unique DNA sequence

doi: 10.1093/nar/gkn035

Figure Lengend Snippet: Lifetime of the covalent TopIB–DNA complex. ( a ) The DNA molecule is mechanically coiled by twisting the magnets, leading to a decrease in DNA extension (green lines). In this trace, positive supercoils are immediately removed by TopIB in a TPT-dependent fashion, with TPT bound to the TFO. Supercoil removal manifests itself as a slow increase in DNA extension (red dots). The total time duration of slow continuous supercoil removal, < Δt> , is measured for a number of uncoiling events for both sequence-specific uncoiling using the TFO–TPT construct ( b , red histogram, n = 20) and for nonsequence-specific uncoiling using free TopIB in solution ( c , blue histogram, n = 122). The distributions have similar widths.

Article Snippet: Oligonucleotide TFO (sequence reported in c) was purchased from Eurogentec and purified using quick spin columns and sephadex G-25 fine (Boehringer, Mannheim).

Techniques: Sequencing, Construct

Journal: Translational Neurodegeneration

Article Title: Inflammation-induced PINCH expression leads to actin depolymerization and mitochondrial mislocalization in neurons

doi: 10.1186/s40035-020-00211-4

Figure Lengend Snippet: PINCH is transcriptionally regulated by MEF2A under inflammatory conditions. a Quantification of TNFα production by neurons exposed to Tat at different time points. b Quantification of PINCH mRNA levels in neurons untreated or exposed to Tat or TNFα for 48 h. c Bioinformatic analysis of lims1/pinch promoter sequence predicted a conserved putative binding site for different transcription factors (TF). Inset: Sequences show conserved binding sites for MEF2A, Cc-FOS, Cc-Jun, and AP-1. d ChIP-assay was performed in neurons untreated or exposed to Tat or TNFα. Antibodies specific for MEF2A, c-Jun, Foxd1, and HoxA9 were used to immunoprecipitate the chromatin and the fold enrichment of lims1/pinch promoter relative to the matched input control was quantified by qPCR. e Representative Western blot of lysates from neurons untreated or exposed to Tat or TNFα and probed with antibodies specific for phospho-CamkII, CamkII, phospho-P38, P38, phospho-MEF2A, MEF2A, PINCH and GAPDH. f-i Quantification of relative protein abundance of phospho-CamkII/CamkII ( f ), phospho-P38/P38 ( g ), phospho-MEF2A/MEF2A ( h ), and PINCH/GAPDH ( i ) from ( e ). j Schematic representation of the lims1/pinch promoter-luciferase constructs. The MEF2A consensus response element at − 169-175 base pairs (bp) (TATTATA) is shown in the oval. k Neurons transfected with control and lims1/pinch luciferase constructs were untreated or exposed to Tat or TNFα for 48 h and luciferase activity was measured. Data represent mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001; n = 3–5 (one-way ANOVA)

Article Snippet: Light Switch Promoter Reporter GoClone plasmid with lims1/pinch (PINCH) (NM_004987) promoter sequence (SwitchGear Genomics; S712264) and the corresponding control plasmids were used for the luciferase assay.

Techniques: Sequencing, Binding Assay, Control, Western Blot, Quantitative Proteomics, Luciferase, Construct, Transfection, Activity Assay

Journal: eLife

Article Title: Concentration dependent chromatin states induced by the bicoid morphogen gradient

doi: 10.7554/eLife.28275

Figure Lengend Snippet: ( A ) Quanitification of Knirps intensity in wild-type, triple mutant ( hb nos tsl ) and quadruple mutant ( bcd hb nos tsl ). Bicoid activates patterned expression of Knirps. In embryos in which Bicoid is the only source of maternal patterning information ( hb nos tsl ), a broad domain of Kni is expressed in the posterior of the embryo. In quadruple mutant embryos, a low level of uniform Knirps is expressed ubiquitously, suggesting that Bcd is required for activating expression of knirps above a background level. Heat-fixed embryos from wild-type (Oregon-R) mothers, hunchback nanos torso-like germline clones and bicoid hunchback nanos torso-like germline clones were pooled and immunostained in a single tube with a rat anti-Knirps primary antibody and Alexa-647 rat antibody. Embryos were mounted on a single slide and imaged by confocal microscopy. Representative embryos for each genotype are shown. Fluorescence intensity of Knirps was extracted from dorsal profiles of midsagittal sections of embryos and plotted using MATLAB. Data are fluorescence intensity minus background, and error bars are standard error of the mean for n = 5 wild-type, n = 8 hb nos tsl, and n = 6 bcd hb nos tsl embryos. ( B ) Smear plot generated in EdgeR showing the log transformed fold-change in Bcd binding between mutant and wild-type embryos for each Bcd peak, vs. the average log transformed sequencing read counts per million (CPM). Bcd binding shows no significant changes between wild-type and nos tsl mutant embryos. Significance was determined using EdgeR to perform a pairwise exact test with a cutoff of FDR ≤ 0.05, comparing binding between eGFP-Bcd;;bcd E1 and eGFP-Bcd;; bcd E1 hb FB nos L7 tsl 4 in the 1,027 Bcd peaks. ( C ) Schematic of the uniform Bcd transgene. The uniform Bcd transgene contains an N-terminal GFP-tagged Bcd driven by the various maternal promoters discussed in the text. Downstream of the bcd coding sequence is a cassette containing the endogenous bcd 3'UTR and a 3xP3-hsp70 promoter driving promoter of RFP. This cassette is flanked by FRT sites. The sqh 3'UTR lies downstream of the FRT cassette. Flies expressing this version of the transgene can be identified by RFP expression in their eyes, and females produce embryos in which Bcd is distributed in a gradient. Males from this transgenic stock are crossed to females expressing a heat shock inducible flippase ( hsFLP) , and heat shocking the F1 larvae results in recombination and excision of the cassette at the FRT sites, bringing the sqh 3'UTR directly downstream of the bcd coding sequence. This initially results in mosaic F1 flies with a mosaic graded/uniform Bcd germline. The F1 are further outcrossed to bcd E1 mutants and F2 individuals producing embryos with uniform Bcd distributions can be identified by the lack of RFP expression in the eyes. ( D ) Expression levels of uniform Bcd constructs measured by western blots. Western blots for GFP-Bcd were performed on embryos at NC14. Representative gels and quantifications are shown for the bcd promoter-driven transgene ( A ), mtrm promoter-driven transgene ( B ) and αTub67C promoter-driven transgene ( C ). In the barplots, band intensities are reported relative to wild-type (GFP-Bcd). All lanes are normalized to an α-tubulin loading control. Error bars are standard deviation between two biological replicates for each sample. MW = molecular wt marker, *=skipped lane.

Article Snippet: A sequence containing the bcd 3'UTR and a 3xP3-RFP reporter flanked by FRT sites was synthesized by GenScript and cloned by Gibson Assembly into the pBABR GFP-Bcd3'sqh plasmid.

Techniques: Mutagenesis, Expressing, Clone Assay, Confocal Microscopy, Fluorescence, Generated, Transformation Assay, Binding Assay, Sequencing, Transgenic Assay, Construct, Western Blot, Control, Standard Deviation, Marker

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

Article Title: A draft genome of field pennycress ( Thlaspi arvense ) provides tools for the domestication of a new winter biofuel crop

doi: 10.1093/dnares/dsu045

Figure Lengend Snippet: Comparative genomics of pennycress and other Brassicaceae species. (A) Syntenic path assembly dot plots comparing pennycress scaffolds >75 kilobases long to the seven Eutrema salsugineum pseudochromosomes fromYang et al. (B) BLASTp analysis of the 27,390 predicted pennycress peptides against predicted peptide sets from Capsella rubella , Brassica rapa , Arabidopsis thaliana (Bevan and Initiative, 2000), Arabidopsis lyrata , and Eutrema salsugineum . Highly similar is defined as pennycress predicted peptide having at least one BLASTp hit e < 1 × 10 −5 and positive sequence similarity >70%. (C) BLASTp analysis of predicted pennycress peptides against a protein database containing the predicted peptides of the five Brassicaceae species listed. Predicted peptides with top hits ( e ≤ 1 × 10 −5 and >70% hit length) to a predicted protein from the corresponding species are shown, with pennycress peptides with hits falling below this threshold shown in the lower right half of the pie chart.

Article Snippet: FASTQ files from all sequencing runs were imported and subjected to quality control using the Sequencing QC Report tool in CLC Genomics Workbench Version 6.5 (CLC Bio, www.clcbio.com ).

Techniques: Sequencing