rdna  (New England Biolabs)

Journal: Nature structural & molecular biology

Article Title: BRD4 is a Histone Acetyltransferase that Evicts Nucleosomes from Chromatin

doi: 10.1038/nsmb.3228

Figure Lengend Snippet: BRD4 has intrinsic histone acetyltransferase activity (a) HAT assays of H3 and H4 (1μg) with radiolabeled AcCoA with recombinant BRD4 (500 ng), mTAF1 (250 ng) or mock. (b) HAT assays of rHis-mBRD4 (500 μg) from insect sf9 cells, GST-hBRD4 from E.coli , Flag-hBRD4 from HeLa cells and rTAF1 with histone H3 (1μg) and acetyl CoA. Immunoblotting was with anti-AcH3, anti-H3 or anti-BRD4 antibodies. Bottom panel, silver staining as a loading control. (c) Recombinant mBRD4 from sf9 cells, hBRD4 (3.5 μg) and control Sf9 extract resolved on a denaturing gel (left panel), immunoblotted with anti-BRD4 (middle panel) or subjected to an in-gel HAT assay; acetylated H4 peptide visualized with anti-AcH4 (right hand panel). Faster mobility band in hBRD4 lane is a degradation product. (d) Histones H2A, H2B, H3, H4 (1μg) or all four (1μg each) incubated with or without 500ng BRD4 in a HAT assay followed by autoradiography or coomassie staining. (e) Mononucleosomes (equivalent to ~700bp) (10pmol) assembled on 5S rDNA (208 bp), visualized in ethidium bromide stained gels (left panel), subjected to a HAT assay with or without recombinant p300 (500 ng) or BRD4 (500 ng) with radiolabeled AcCoA and visualized by autoradiography or coomassie staining. (f) Histone H3, H4ac and H3ac immunoblots of whole cell extracts (WCE) from HeLa cells grown 14, 16 and 18hr following transfection with 3μg hBRD4 expression plasmid or control. AU: arbitrary units. Data are representative of 3 independent experiments. Uncropped images of gels and immunoblots are shown in Supplementary Data Set 1

Article Snippet: Unmodified recombinant human nucleosomes were assembled on the 5S rDNA with purified human histone H2A/H2B dimers and histone H3/H4 tetramers using the EpiMark Nucleosome Assembly kit (NEB) following the manufacturer’s instructions.

Techniques: Activity Assay, HAT Assay, Recombinant, Silver Staining, Incubation, Autoradiography, Staining, Western Blot, Transfection, Expressing, Plasmid Preparation

Journal: Nature structural & molecular biology

Article Title: BRD4 is a Histone Acetyltransferase that Evicts Nucleosomes from Chromatin

doi: 10.1038/nsmb.3228

Figure Lengend Snippet: BRD4 acetylation of H3K122 mediates histone eviction and nucleosome clearance a) Autoradiogram showing mononucleosomes (10 pmol) assembled in vitro with radiolabeled 5S rDNA and subjected to HAT assays for 15 and 30 minutes with or without 500ng BRD4 or TAF1 in the presence or absence of acetyl CoA or 500 μM JQ1. Free DNA resolved from mononucleosomes by electrophoresis (upper panel) . Immunoblots of identical HAT assays done in parallel with non-radiolabeled mononucleosomes, showing extent of acetylation of H3K122 and H3K9; histone H3 immunoblot served as control (lower panels) . (b) HAT assays and immunoblots as in (a) with or without BRD4, BRD4 HAT mutants or TAF1 (500ng). (c) Ethidium Bromide stained agarose gels showing native chromatin isolated from U2OS cells that was incubated in HAT assays with or without increasing amounts of BRD4 (Lane 3: 1 μg; Lane 4, 5, 6: 0.2, 0.5, 1 μg respectively; Lane 7: 1μg), Acetyl CoA and 500 μM JQ1 (lane 7) and then digested with 0.1U MNase for 3 min (left panel). Quantification of digested bands showing increased MNase digestion correlated with increasing BRD4 (right panel) . (d) Ethidium Bromide stained agarose gels showing native chromatin isolated from U2OS cells transfected with 3μg hBRD4 (BRD4 WT), hBRD4 HAT mutant (Mt BRD4) or pCMV2 vector (control) in the presence of sodium butyrate and incubated with or without 0.1U MNase for 3 min (middle panel). Quantification of digested bands from cells transfected with vector (1), BRD4 WT (2) and Mt BRD4 (3) (right panel) . Immunoblots of WCEs from the transfected cells showing H3K122ac, H3K9ac and BRD4 levels (left panel). Uncropped images of gels and immunoblots are shown in Supplementary Data Set 1

Article Snippet: Unmodified recombinant human nucleosomes were assembled on the 5S rDNA with purified human histone H2A/H2B dimers and histone H3/H4 tetramers using the EpiMark Nucleosome Assembly kit (NEB) following the manufacturer’s instructions.

Techniques: In Vitro, HAT Assay, Electrophoresis, Western Blot, Staining, Isolation, Incubation, Transfection, Mutagenesis, Plasmid Preparation

Journal: Applied and Environmental Microbiology

Article Title: Effect of Field Inoculation with Sinorhizobium meliloti L33 on the Composition of Bacterial Communities in Rhizospheres of a Target Plant (Medicago sativa) and a Non-Target Plant (Chenopodium album)--Linking of 16S rRNA Gene-Based Single-Strand Conformation Polymorphism Community Profiles to the Diversity of Cultivated Bacteria

doi:

Figure Lengend Snippet: Cultivation-independent PCR-SSCP profiles of bacterial communities isolated from rhizospheres of M. sativa (lanes 2 to 7) and C. album (lanes 8 to 13). Results from three replicate field plots are shown for each treatment. The results for plants from noninoculated field plots are shown in lanes 2 to 4 and 8 to 10. The results for plants from S. meliloti -inoculated plots are shown in lanes 5 to 7 and 11 to 13. The results for SSCP species standards, consisting of single-stranded DNA products obtained from PCR-amplified 16S rRNA gene regions (including regions V4 and V5) (see Materials and Methods) of selected bacterial species (Pf, Pseudomonas fluorescens ; Bs, Bacillus subtilis ; Sm, Sinorhizobium meliloti ; Ar, Agrobacterium radiobacter ), are shown in lanes 1 and 14.

Article Snippet: For digestion of the amplified 16S ribosomal DNA fragments, the tetranucleotide-recognizing enzymes Hha I and Hae III (New England Biolabs, Schwalbach/Taunus, Germany) were used.

Techniques: Polymerase Chain Reaction, Isolation, Amplification

Journal: Microorganisms

Article Title: Comparative Studies on the Polymorphism and Copy Number Variation of mtSSU rDNA in Ciliates (Protista, Ciliophora): Implications for Phylogenetic, Environmental, and Ecological Research

doi: 10.3390/microorganisms8030316

Figure Lengend Snippet: The mtSSU rDNA polymorphisms among 13 focal species. ( A ) Bars indicate the most common sequences of each species, bright blue, dark blue, green, and purple dots represent deletion, insertion, transition, and transversion, respectively, while the nucleotides shown in dots represent the polymorphism compared with the common sequence; lengths of sequences are drawn to scale. ( B ) Polymorphic sites between different haplotypes. Clones mean the number of clones for the corresponding haplotype found in total clones (20, 30, or 90); For F. ehrenbergii , the first two individuals and the third one might be cryptic species (i.e., they were indeed different species), so we represented them respectively; matching sites are represented by dots (.) and missing sites are marked with dashes (-); a: Coleps sp.; b: Sterkiella sp.; c: Spirostomum sp.; d: Oxytricha trifallax ; e, f: Favella ehrenbergii ; g: Epistylis sp.; h: Paramecium caudatum ; and i: Trithigmostoma sp.

Article Snippet: Primers mtF (5′-TGT GCC AGC AGC CGC GGT AA-3′) and mtR (5′-CCC MTA CCR GTA CCT TGT GT-3′) were used to amplify the mtSSU rDNA segment of each species [ , ] using Q5 Hot Start High-Fidelity DNA polymerase (Cat. #M0493 L, New England Biolabs, Ipswich, MA, USA).

Techniques: Sequencing, Clone Assay

Journal: Microorganisms

Article Title: Comparative Studies on the Polymorphism and Copy Number Variation of mtSSU rDNA in Ciliates (Protista, Ciliophora): Implications for Phylogenetic, Environmental, and Ecological Research

doi: 10.3390/microorganisms8030316

Figure Lengend Snippet: The maximum likelihood (ML) tree based on mtSSU rDNA sequence alignment, highlighting the 13 species studied. Numbers near nodes represent the bootstrap values of ML and the posterior probability values of Bayesian analysis (BI). Dots (.) mean fully supported node (100/1.00) while asterisks (*) indicate the disagreement between ML and BI topologies. The scale bar corresponds to two substitutions per 100 nucleotide positions. All branches are drawn to scale.

Article Snippet: Primers mtF (5′-TGT GCC AGC AGC CGC GGT AA-3′) and mtR (5′-CCC MTA CCR GTA CCT TGT GT-3′) were used to amplify the mtSSU rDNA segment of each species [ , ] using Q5 Hot Start High-Fidelity DNA polymerase (Cat. #M0493 L, New England Biolabs, Ipswich, MA, USA).

Techniques: Sequencing

Journal: Journal of Clinical Microbiology

Article Title: Identification of a Mutation Associated with Erythromycin Resistance in Bordetella pertussis: Implications for Surveillance of Antimicrobial Resistance

doi: 10.1128/JCM.41.3.1167-1172.2003

Figure Lengend Snippet: Screening for A2058G and A2059G mutations in B. pertussis by PCR-RFLP analysis. (A). Bsa I (lanes 1 to 5) or Bbs I (lanes 6 to 10) digestion of a 521-bp fragment of the 23S rDNA gene of erythromycin-resistant B. pertussis clinical isolates (A228, C353, and MN2531), heterogeneous strain C352, and erythromycin-susceptible strain MN2726. The 521-bp fragment was generated by PCR amplification using primers 1907U and 2408L as described in Materials and Methods. Lanes: M, 100-bp ladder (Life Technologies); 1 and 6, B. pertussis A228; 2 and 7, B. pertussis C352; 3 and 8, B. pertussis C353; 4 and 9, B. pertussis MN2531; 5 and 10, B. pertussis MN2726. (B). Bbs I digestion of the 521-bp fragment of additional isolates of B. pertussis . Lanes: M, 100-bp ladder (Life Technologies); 1 to 7, erythromycin-susceptible clinical isolates B. pertussis MN277, MN973, MN1286, MN1699, MN1773, MN1893, and MN2726; 8, erythromycin-resistant B. pertussis isolate MN253.

Article Snippet: An 8-μl aliquot of the B. pertussis 23S rDNA PCR product was digested with Bsa I or Bbs I (New England Biolabs, Beverly, Mass.), and the products were resolved by electrophoresis on a 2% agarose Tris-borate-EDTA gel.

Techniques: Polymerase Chain Reaction, Generated, Amplification