Journal: Marine Drugs

Article Title: A New Micromonospora Strain with Antibiotic Activity Isolated from the Microbiome of a Mid-Atlantic Deep-Sea Sponge

doi: 10.3390/md19020105

Figure Lengend Snippet: HPLC-ELSD chromatogram of the crude extract of Micromonospora sp. 28ISP2-46 T .

Article Snippet: 16S rRNA gene and whole-genome sequencing of strain 28ISP2-46 T was consistent with its classification as a novel species of the genus Micromonospora , for which the name Micromonospora ferruginea sp. nov. (NCTC number: 14469 T , DSMZ number: 111791 T ) is proposed.

Techniques:

Journal: Marine Drugs

Article Title: A New Micromonospora Strain with Antibiotic Activity Isolated from the Microbiome of a Mid-Atlantic Deep-Sea Sponge

doi: 10.3390/md19020105

Figure Lengend Snippet: Phylogenetic tree for strain 28ISP2-46 T and the other group IA Micromonospora species generated using the TYGS and drawn with iTOL. The tree was constructed using FastME 2.1.6.1 from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values from 100 replications, with an average branch support of 98.4% and a delta statistic of 0.135. The tree was rooted using Micromonospora pallida as the outgroup.

Article Snippet: 16S rRNA gene and whole-genome sequencing of strain 28ISP2-46 T was consistent with its classification as a novel species of the genus Micromonospora , for which the name Micromonospora ferruginea sp. nov. (NCTC number: 14469 T , DSMZ number: 111791 T ) is proposed.

Techniques: Generated, Construct

Journal: Marine Drugs

Article Title: A New Micromonospora Strain with Antibiotic Activity Isolated from the Microbiome of a Mid-Atlantic Deep-Sea Sponge

doi: 10.3390/md19020105

Figure Lengend Snippet: Biosynthetic gene clusters identified in the genome of strain 28ISP2-46 T using AntiSMASH.

Article Snippet: 16S rRNA gene and whole-genome sequencing of strain 28ISP2-46 T was consistent with its classification as a novel species of the genus Micromonospora , for which the name Micromonospora ferruginea sp. nov. (NCTC number: 14469 T , DSMZ number: 111791 T ) is proposed.

Techniques:

Journal: Marine Drugs

Article Title: A New Micromonospora Strain with Antibiotic Activity Isolated from the Microbiome of a Mid-Atlantic Deep-Sea Sponge

doi: 10.3390/md19020105

Figure Lengend Snippet: ( a ) ClusterBLAST hits for the kosinostatin cluster. Homologues of kst genes are coloured as follows: kstA genes red, kstB genes yellow, kstC genes green, kstD genes blue, kstRg genes black, kstRs genes white, and genes uninvolved in kosinostatin biosynthesis grey. A black line at the end of a cluster indicates that it is close to the end of a contig. Gene sizes not to scale. The NCBI protein IDs for first and last coloured genes of each cluster are as follows: Micromonospora sp. TP-A0468 AFJ52719.1 and AFJ52701.1; Micromonospora sp. 28ISP2-46 T QLQ36639.1 and QLQ36600.1; N. valliformis WP_017579213.1 and WP_017579258.1; N. alkaliphila WP_017604198.1 and WP_017604153.1; Streptomyces sp. SM8 PKA38649.1 and PKA38729.1; Streptomyces sp. ScaeMP-6W SCE31237.1 and SCE31955.1; A. bangkokensis OLR89622.1 and OLR89598.1; M. haikouensis SCF11188.1 and SCF11278.1. ( b ) The same gene clusters rearranged for ease of comparison. Coloured as 6a.

Article Snippet: 16S rRNA gene and whole-genome sequencing of strain 28ISP2-46 T was consistent with its classification as a novel species of the genus Micromonospora , for which the name Micromonospora ferruginea sp. nov. (NCTC number: 14469 T , DSMZ number: 111791 T ) is proposed.

Techniques:

Journal: Advanced Science

Article Title: ATM‐Dependent Recruitment of BRD7 is required for Transcriptional Repression and DNA Repair at DNA Breaks Flanking Transcriptional Active Regions

doi: 10.1002/advs.202000157

Figure Lengend Snippet: ATM‐dependent recruitment of BRD7 to DSBs nearby transcriptionally active regions. A) Recruitment of GFP‐BRD7 in laser damage. Representative images after UV laser damage are shown. B) BRD7 recruitment to Fok1‐induced DSBs. GFP‐BRD7 was transfected into U2OS‐265 DSB reporter cells, and cells were damaged by inducing site‐specific DSBs after 24 h transfection. Representative images after DNA damage are shown. C) BRD7 recruitment to I‐SecI‐induced DSBs analyzed by chromatin immunoprecipitation (ChIP) assay. Eight hours after I‐SecI transfection, ChIP assay was performed to detect the enrichment of BRD7 relative to the IgG control. D) The recruitment of BRD7 to DSBs increased throughout the cell cycle. HeLa DR‐GFP cells were treated with double‐thymidine to achieve cells at G1‐S boundary and then left unreleased (G1 phase) or released into the thymidine‐free medium for 3 h (S phase) or 7 h (G2 phase). The cells harvested at indicated phases were subjected to ChIP assay according to the Experimental Procedures. E) The recruitment of BRD7 to DNA damage was not restricted to S/G2 cells. Cyclin A is a S/G2 marker. GFP‐BRD7 was transfected into U2OS‐265 DSB reporter cells, and cells were introduced site‐specific DSBs. Representative images after DNA damage are shown. F) GFP‐BRD7 is preferentially recruited to DSBs induced upstream of active gene. U2OS‐265 DSB reporter cells were first treated with 1 µg mL −1 doxycycline for 2 h to induce nascent transcription of the reporter gene, and then cells were induced site‐specific DSBs after 24 h transfection of GFP‐BRD7. Representative images after DNA damage are shown. G) Quantification of GFP‐BRD7 focus colocalized with Fok1 of panel (F). Data are represented as mean ± SEM. H) ATM and PARP1, but neither ATR nor DNA‐PK is required for the recruitment of BRD7 to DSBs. U2OS‐265 DSB reporter cells were transfected with GFP‐BRD7 for 24 h, then ATM inhibitor (Ku55933, 10 × 10 −6 m ), ATR inhibitor (VE‐821, 10 × 10 −6 m ), DNA‐PK inhibitor (NU7441, 5 × 10 −6 m ), PARP inhibitor (Olaparib, 10 × 10 −6 m ) were added for additional 4 h, followed by introduced site‐specific DSBs. Representative images after DNA damage are shown in (H). All the scale bars are 2 µm. I) Quantification of GFP‐BRD7 relative mean fluorescence intensity (RMFI) from H) calculated from 50 cells using Image J software (NIH) and three independent experiments. Error bars indicate SEM. n.s., not significant; ** P < 0.01, *** P < 0.001, Student's t ‐test.

Article Snippet: [ , ] Briefly, ATM and ATR proteins were immunoprecipitated from HEK293T cells transfected with Flag‐ATM (addgene plasmid #43 907) and Flag‐ATR (addgene plasmid #31 611) constructs using anti‐flag agarose.

Techniques: Transfection, Chromatin Immunoprecipitation, Marker, Fluorescence, Software

Journal: Advanced Science

Article Title: ATM‐Dependent Recruitment of BRD7 is required for Transcriptional Repression and DNA Repair at DNA Breaks Flanking Transcriptional Active Regions

doi: 10.1002/advs.202000157

Figure Lengend Snippet: ATM and ATR directly phosphorylate BRD7 at Ser 263 and Thr 515 sites respectively. A,B) HeLa cells were treated with CPT (1 × 10 −6 m ) for different time intervals followed by lysing with RIPA buffer, and lysates were subjected to immunoprecipitation using either anti‐(pS/pT)Q or anti‐BRD7 antibodies and immunoblotted with the indicated antibodies. C) HeLa cells treated with CPT (1 × 10 −6 m , 1 h) were lysed with RIPA buffer, and lysates were subjected to immunoprecipitation using either anti‐IgG, or ATM or ATR antibodies, and analyzed by Western blot. D) HeLa cells were treated first with DMSO, KU55933 (ATM inhibitor, 10 × 10 −6 m ), VE‐821 (ATR inhibitor, 10 × 10 −6 m ) or NU7441 (DNA‐PK inhibitor, 1 × 10 −6 m ) for 2 h followed by treatment with CPT for another 1 h, and the cells were lysed with RIPA buffer, and subjected to immunoprecipitation using either anti‐IgG, or BRD7 antibodies, and analyzed by Western blot with the indicated antibodies. E) HeLa cells were transfected with indicated siRNAs for 48 h followed by treatment with CPT for another 1 h, and then were lysed with RIPA buffer, and were subjected to immunoprecipitation using indicated antibodies. F) HeLa cells were transfected with BRD7 wild‐type and various BRD7‐mutant plasmids for 24 h followed by treatment with CPT for another 1 h, lysed with RIPA buffer, followed by immunoprecipitation and Western blot with indicated antibodies. G) HeLa cells were transfected with BRD7 wild‐type and BRD7 double mutant plasmids for 24 h followed by CPT treatment for another 1 h, and analyzed by Western blot. H) ATM directly phosphorylates BRD7 at Ser 263. An in vitro ATM assay was performed as described in Experimental Section. I) ATR directly phosphorylates BRD7 at Thr 515. An in vitro ATR assay was performed as described in Supplementary Material and Methods. J) HeLa cells were transfected with either BRD7 wild‐type, BRD7‐S263A or BRD7‐T515A for 24 h, followed by incubation with 10 µg mL −1 cycloheximide (CHX) for the indicated periods of time. Lysates were harvested and analyzed by Western blot. K) Quantification of BRD7 protein levels from panel (J), n = 3. Error bars indicate SEM. Relative amounts normalized to the BRD7 protein level at 0 h.

Article Snippet: [ , ] Briefly, ATM and ATR proteins were immunoprecipitated from HEK293T cells transfected with Flag‐ATM (addgene plasmid #43 907) and Flag‐ATR (addgene plasmid #31 611) constructs using anti‐flag agarose.

Techniques: Immunoprecipitation, Western Blot, Transfection, Mutagenesis, In Vitro, Incubation

Journal: Advanced Science

Article Title: ATM‐Dependent Recruitment of BRD7 is required for Transcriptional Repression and DNA Repair at DNA Breaks Flanking Transcriptional Active Regions

doi: 10.1002/advs.202000157

Figure Lengend Snippet: ATM‐mediated phosphorylation BRD7 at Ser 263 is required for HR repair and transcriptional repression. A) Recruitment of BRD7‐S263A to Fok1‐induced DSBs was remarkedly reduced. GFP‐BRD7 and indicated mutant constructs were transfected into U2OS‐265 DSB reporter cells for 24 h, and cells were induced site‐specific DSBs. Representative images after DNA damage are shown. B) Quantification of GFP‐BRD7 wild‐type and mutants focus colocalized with Fok1 of panel (A). Data are represented as mean ± SEM. Error bars indicate SEM. C) Flag‐BRD7 and mutants BRD7 recruitment to I‐SecI‐induced DSBs analyzed by ChIP assay. HeLa DR‐GFP reporter cells stably expressing indicated constructs were electroporated with I‐SecI plasmid. Eight hours after I‐SecI transfection, ChIP assay was performed to detect the enrichment of Flag‐BRD7 and mutants BRD7 relative to the vector control. D) Recruitment of H2A‐Ub to laser damage was reduced in BRD7‐S263A. Wild‐type and indicated mutant BRD7 were transfected into HeLa cells depleted of endogenous BRD7 and subjected to UV laser, and endogenous H2A‐Ub accumulation was analyzed by immunofluorescence. Representative images after DNA damage are shown. E) Transcription activity was measured in U2OS‐263 DSB reporter cells transfected with indicated BRD7 constructs after depletion of endogenous BRD7, followed by inducing site‐specific DSBs. Ongoing transcription of the reporter gene can be detected by the presence of a YFP‐MS2 foci. Representative images after DSBs induction are shown. F) Quantification of YFP‐MS2 positive cells from experiments as in panel (E). Quantification results are the average of three independent experiments and are shown as mean ± SEM. G) Model of the role of the PBAF subunit BRD7 in coordinating DSB‐induced transcriptional repression and HR repair. Scale bars, 2 µm. n.s., not significant; ** P < 0.01, *** P < 0.001, Student's t ‐test.

Article Snippet: [ , ] Briefly, ATM and ATR proteins were immunoprecipitated from HEK293T cells transfected with Flag‐ATM (addgene plasmid #43 907) and Flag‐ATR (addgene plasmid #31 611) constructs using anti‐flag agarose.

Techniques: Mutagenesis, Construct, Transfection, Stable Transfection, Expressing, Plasmid Preparation, Immunofluorescence, Activity Assay

Journal: Nucleic Acids Research

Article Title: Structural basis for binding the TREX2 complex to nuclear pores, GAL1 localisation and mRNA export

doi: 10.1093/nar/gku252

Figure Lengend Snippet: Mutations in Sac3 that impair binding of the Sac3 CID domain complex to Nup1 in vitro while not impacting on Sac3 CID complex assembly impair Sac3 targeting to the nuclear periphery in vivo . ( A ) Assembly of mutant Sac3 CID domain complexes were monitored by pull-down assays. Clarified bacterial lysates containing GST-Sac3 753–805 wild-type (WT) or mutant derivatives co-expressed with Cdc31 were mixed with an excess of clarified bacterial lysate containing Sus1 and incubated with Glutathione Sepharose resin. Washed resins were analysed by SDS-PAGE and Coomassie staining. ( B ) Mutants were purified in a fragment of Sac3 encompassing residues 723–805 complexed to Sus1A, Sus1B and Cdc31 (the complete Sac3 CID domain complex) and equimolar amounts of each were used as input in pull-down assays using GST-Nup1 191–385 as bait immobilised on Glutathione Sepharose resin ( C ). Washed resins were analysed by SDS-PAGE and Coomassie staining and demonstrated that Nup1 bound only to WT complex (boxed). M; molecular weight markers (kDa). ( D ) Fluorescence micrographs of yeast strains in which the sole genomic copy of SAC3 carried a C-terminal mTurquoise2 tag and was either WT or mutated as indicated. The nuclear periphery was visualiszed by Nup49-GFP. NPC binding was most strongly impaired by sac3 L768A-H774D .

Article Snippet: Plasmid pRS416 ( ) was used as template for amplification of the URA3 marker (nucleotides 182–1298, which contained S. cerevisiae URA3 promoter, coding sequence and terminator), whereas pmTurquoise2-H2A (plasmid 36207, Addgene) was used for amplifying the mTurquoise2 coding sequence (nucleotides 4390–7).

Techniques: Binding Assay, In Vitro, In Vivo, Mutagenesis, Incubation, SDS Page, Staining, Purification, Molecular Weight, Fluorescence