Journal:

Article Title: Different Modes of Diaminopimelate Synthesis and Their Role in Cell Wall Integrity: a Study with Corynebacterium glutamicum

doi:

Figure Lengend Snippet: Strains and selected plasmids used in this study

Article Snippet: The isolation of clones complementing the dapD mutation in E. coli RDD32 was performed as described previously ( 28 ). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant characteristic(s) Reference E. coli DH5 Cloning strain S17-1 Mobilizing donor strain 26 RDD32 dapD ::MuCts 17 C. glutamicum ATCC 13032 Type strain ATCC 13032 dapD dapD ::pEMdapD int Km r This work ATCC 13032 dapE dapE ::pEMdapE int Km r This work ATCC 13032 orf2 orf2 ::pEMorf2 int Km r This work ATCC 13032 Δ orf3 Deletion of the 479-bp Kpn I fragment This work Plasmids pJC1 Shuttle vector, Km r 4 pEM1 Integration vector, mobilizable, nonreplicative in C. glutamicum , Km r 23 pK18mobsacB Replacement vector, mobilizable, nonreplicative in C. glutamicum , Km r 21 pJCdapD1.55 pJC1:: dapD This work pJCorf3/Bam3.4 pJC1:: orf3 /Bam3.4 This work Open in a separate window Strains and selected plasmids used in this study Genetic techniques, sequencing, and hybridization.

Techniques: Plasmid Preparation, Cloning

Journal:

Article Title: Different Modes of Diaminopimelate Synthesis and Their Role in Cell Wall Integrity: a Study with Corynebacterium glutamicum

doi:

Figure Lengend Snippet: Direct comparison of three sequences. The deduced amino acid sequences of the dapD gene product of C. glutamicum (CgDapD) and of E. coli (EcDapD) and of the orf3 gene product of C. glutamicum (CgOrf3) are compared. Amino acids that are identical (black boxes) or similar (shaded boxes) are indicated. Although the DapD polypeptides from C. glutamicum and E. coli both represent functional tetrahydrodipicolinate succinylases, their structural similarity is less than that of the DapD and Orf3 polypeptides from C. glutamicum. The bars represent the imperfect tandem-repeated heptapeptide sequence characteristic of acyltransferases (2).

Article Snippet: The isolation of clones complementing the dapD mutation in E. coli RDD32 was performed as described previously ( 28 ). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant characteristic(s) Reference E. coli DH5 Cloning strain S17-1 Mobilizing donor strain 26 RDD32 dapD ::MuCts 17 C. glutamicum ATCC 13032 Type strain ATCC 13032 dapD dapD ::pEMdapD int Km r This work ATCC 13032 dapE dapE ::pEMdapE int Km r This work ATCC 13032 orf2 orf2 ::pEMorf2 int Km r This work ATCC 13032 Δ orf3 Deletion of the 479-bp Kpn I fragment This work Plasmids pJC1 Shuttle vector, Km r 4 pEM1 Integration vector, mobilizable, nonreplicative in C. glutamicum , Km r 23 pK18mobsacB Replacement vector, mobilizable, nonreplicative in C. glutamicum , Km r 21 pJCdapD1.55 pJC1:: dapD This work pJCorf3/Bam3.4 pJC1:: orf3 /Bam3.4 This work Open in a separate window Strains and selected plasmids used in this study Genetic techniques, sequencing, and hybridization.

Techniques: Comparison, Functional Assay, Sequencing

Journal: Molecular Cell

Article Title: BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation

doi: 10.1016/j.molcel.2021.06.020

Figure Lengend Snippet:

Article Snippet: Drosophila: S2 cell line , ATCC , ATCC CRL-1963.

Techniques: Control, Virus, Recombinant, Transfection, Purification, Extraction, Sequencing, Imaging, Plasmid Preparation, Software

Journal: eLife

Article Title: In vivo identification of GTPase interactors by mitochondrial relocalization and proximity biotinylation

doi: 10.7554/eLife.45916

Figure Lengend Snippet: ( A,B ) Bubble-volcano plots of MitoID with the GTP-locked form of Rac1 (Q60L) or with the same GTPase from which a C-terminal stretch of six basic residues has been deleted (Δ183–188). In each case the GTPase is compared to a background comprising the GDP-locked/empty forms of seventeen GTPases. Known effectors and GAPs are indicated along with proteins whose GO annotations include the term DNA-binding or RNA-binding. The removal of the basic stretch reduces contamination with the latter proteins. ( C ) Immunoblot of strepatividin precipitates from cells in which MitoID had been performed with the indicated versions of Rab5A(Q79L). A representative lysate, and the precipitates were blotted for the indicated Rab5 effectors. Mutation of Ser84 reduces biotinylation of OSBPL9, but has no effect on that of two other effectors, EEA1 and PIK3R4.

Article Snippet: Bacterial expression used E.coli codon-optimized human Rab constructs created by cloning Rab GTPases lacking the C-terminal cysteines and with the relevant activating or inactivating point mutations (see Key resources table) into the vector pGEX6p2 between EcoR1 and XhoI (GE Healthcare Life Sciences).

Techniques: Binding Assay, RNA Binding Assay, Western Blot, Mutagenesis

Journal: Cell & Bioscience

Article Title: Zika virus antagonizes interferon response in patients and disrupts RIG-I–MAVS interaction through its CARD-TM domains

doi: 10.1186/s13578-019-0308-9

Figure Lengend Snippet: Molecular interaction between ZIKV NS4A protein and the type I IFN induction pathway. GAL4-based mammalian two-hybrid screening assays were performed to identify the molecular targets of ZIKV proteins in RIG-I signaling. 293T cells in 24-well plates were co-transfected with a pGL4.31 vector, a pFN11A (BIND) vector expressing a fusion protein of GAL4-BD and individual ZIKV prM, NS4A or DENV NS4A proteins, and a pFN10A (ACT) vector expressing a RIG-I, b MAVS, c TBK1 or d IKKε. The pFN11A (BIND) vector contained a Renilla luciferase gene that was used as an internal control to normalize DNA transfection efficiency. The pBIND and pACT vectors were used as negative controls, and the pBIND-Id and pACT-MyoD vectors were used as positive controls (PCs) according to the manufacturer’s instructions. At 48 h post-transfection, cell lysates were harvested for the luciferase activity assay. The results are shown as relative luciferase activity after normalization with Renilla luciferase activity. Data are shown as the mean ± SD derived from three repeat experiments. * p < 0.05, and ** p < 0.01 (Student’s t test)

Article Snippet: 293T human embryonic kidney cells (the Cell Bank of the Chinese Academy of Sciences, Shanghai, China), HeLa human cervical adenocarcinoma cells (ATCC) and HFF-1 human foreskin fibroblasts cells (the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in DMEM (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (GIBCO, Carlsbad, CA), 2 mM l -glutamine, 100 μg/ml streptomycin and 100 units/ml penicillin (Invitrogen, Carlsbad, CA) at 37 °C under 5% CO 2 .

Techniques: Two Hybrid Screening, Transfection, Plasmid Preparation, Expressing, Luciferase, Control, Activity Assay, Derivative Assay

Journal: Cell & Bioscience

Article Title: Zika virus antagonizes interferon response in patients and disrupts RIG-I–MAVS interaction through its CARD-TM domains

doi: 10.1186/s13578-019-0308-9

Figure Lengend Snippet: ZIKV NS4A co-localizes and interacts with MAVS. a HeLa cells transfected with plasmids expressing Flag-tagged NS4A, Flag-tagged ZIKV prM or influenza Flag-tagged PB1-F2 were stained with anti-Flag and anti-MAVS antibodies as well as DAPI. Secondary antibodies conjugated to rhodamine and FITC dye were used to visualize the indicated proteins. Images are representative of three independent experiments. 293T cells co-transfected with plasmids encoding Myc-tagged MAVS and Flag-tagged NS4A were used in a co-IP assay to address whether ZIKV NS4A protein physically interacts with MAVS. Cell lysates were precipitated with an anti-Flag antibody ( b ), anti-Myc antibody ( c ), or control mouse IgG, and immunocomplexes were analyzed with the indicated antibodies by western blotting. d 293T cells were transfected with plasmids encoding Flag-tagged NS4A, followed by immunoprecipitation using anti-Flag antibody or control IgG. The immunocomplexes were analyzed with anti-MAVS antibody by Western blotting. e HFF-1 cells were infected with ZIKV at an MOI of 5 followed by immunoprecipitation using anti-MAVS antibody or control mouse IgG. The immunocomplexes that were captured by the protein G Dynabeads were analyzed by Western blotting using anti-NS4A, or anti-MAVS antibodies. f SPR analysis of the interactions between MAVS and NS4A. Direct binding was measured by Biacore assays. MAVS was immobilized on a CM5 chip. The analytes consisted of serial dilutions of NS4A proteins ranging between 0 and 2000 nM. The data shown are representative of three independent experiments with similar results

Article Snippet: 293T human embryonic kidney cells (the Cell Bank of the Chinese Academy of Sciences, Shanghai, China), HeLa human cervical adenocarcinoma cells (ATCC) and HFF-1 human foreskin fibroblasts cells (the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in DMEM (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (GIBCO, Carlsbad, CA), 2 mM l -glutamine, 100 μg/ml streptomycin and 100 units/ml penicillin (Invitrogen, Carlsbad, CA) at 37 °C under 5% CO 2 .

Techniques: Transfection, Expressing, Staining, Co-Immunoprecipitation Assay, Control, Western Blot, Immunoprecipitation, Infection, Binding Assay

Journal: Cell & Bioscience

Article Title: Zika virus antagonizes interferon response in patients and disrupts RIG-I–MAVS interaction through its CARD-TM domains

doi: 10.1186/s13578-019-0308-9

Figure Lengend Snippet: ZIKV NS4A interacts with both the CL and TM domains of MAVS and prevents RIG-I from binding MAVS. a A schematic diagram of the MAVS protein and functional domains: CARD-like domain (aa 10 to 77), proline-rich domain (aa 103 to 173) and transmembrane domain (aa 514 to 535). b Co-IP and western blotting analysis of 293T cells transfected with Myc-tagged NS4A along with vectors expressing the indicated Flag-tagged MAVS truncation forms or full-length MAVS. Empty vector was used as a negative control. c 293T cells were co-transfected with Flag-tagged RIG-I and Myc-tagged NS4A or an empty vector control for 24 h. Whole cell lysates were subjected to immunoprecipitation using an anti-MAVS antibody and analyzed by western blotting for RIG-I ( c ). 293T cells were transfected with Myc-tagged NS4A or an empty vector control for 24 h later, transfected with 20 μg/ml of poly(I:C). Whole cell lysates were subjected to immunoprecipitation using an anti-MAVS antibody and analyzed by western blotting for TRAF6 ( d ), or TBK1 ( e ). The expression of precipitated proteins was determined by western blotting analysis using the indicated antibodies (lower panel). The data shown are representative of three independent experiments with similar results

Article Snippet: 293T human embryonic kidney cells (the Cell Bank of the Chinese Academy of Sciences, Shanghai, China), HeLa human cervical adenocarcinoma cells (ATCC) and HFF-1 human foreskin fibroblasts cells (the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in DMEM (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (GIBCO, Carlsbad, CA), 2 mM l -glutamine, 100 μg/ml streptomycin and 100 units/ml penicillin (Invitrogen, Carlsbad, CA) at 37 °C under 5% CO 2 .

Techniques: Binding Assay, Functional Assay, Co-Immunoprecipitation Assay, Western Blot, Transfection, Expressing, Plasmid Preparation, Negative Control, Control, Immunoprecipitation

Journal: Cell & Bioscience

Article Title: Zika virus antagonizes interferon response in patients and disrupts RIG-I–MAVS interaction through its CARD-TM domains

doi: 10.1186/s13578-019-0308-9

Figure Lengend Snippet: ZIKV NS4A negatively regulates IFN production. a Lysates of 293T cells co-transfected with RIG-I(N), IFN-β reporter, together with ZIKV NS4A (250, or 500 ng), DENV NS4A (250, or 500 ng), ZIKV prM (500 ng) or PB1-F2 (500 ng)were analyzed for luciferase activity. 293T cells were transfected with IFN-β reporter, together with ZIKV NS4A (250, or 500 ng), DENV NS4A (250, or 500 ng), ZIKV prM (500 ng) or PB1-F2 (500 ng), b then infected with SeV at 100 HAU/ml for 24 h, c or transfected with 20 μg/ml of poly(I:C) for 24 h followed by analysis of the cell lysates for luciferase activity. d , e HFF-1 cells or f PBMC were transfected with blank control vector or a vector expressing ZIKV NS4A (500 ng), DENV NS4A (500 ng), ZIKV prM (500 ng) or PB1-F2 (500 ng), respectively, and 24 h later were mock-infected or infected with SeV at 100 HAU/ml or transfected with or without 20 μg/ml of poly(I:C) for 24 h, followed by analysis of the supernatant for IFN-β protein levels. g , h Meanwhile, at 24 h post-infection, total cellular RNA was isolated, and real-time PCR was performed to detect IFNB , OAS1 , and IFITM1 mRNA levels in 293T cells. The data are presented as the mean ± SD derived from three repeat experiments. * p < 0.05, and ** p < 0.01 (Student’s t-test)

Article Snippet: 293T human embryonic kidney cells (the Cell Bank of the Chinese Academy of Sciences, Shanghai, China), HeLa human cervical adenocarcinoma cells (ATCC) and HFF-1 human foreskin fibroblasts cells (the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in DMEM (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (GIBCO, Carlsbad, CA), 2 mM l -glutamine, 100 μg/ml streptomycin and 100 units/ml penicillin (Invitrogen, Carlsbad, CA) at 37 °C under 5% CO 2 .

Techniques: Transfection, Luciferase, Activity Assay, Infection, Control, Plasmid Preparation, Expressing, Isolation, Real-time Polymerase Chain Reaction, Derivative Assay

Journal: Cell & Bioscience

Article Title: Zika virus antagonizes interferon response in patients and disrupts RIG-I–MAVS interaction through its CARD-TM domains

doi: 10.1186/s13578-019-0308-9

Figure Lengend Snippet: ZIKV NS4A blocks VSV-induced IFN production. a 293T cells transfected with IFN-β reporter together with ZIKV-NS4A, DENV-NS4A, ZIKV-prM and IVA-PB1-F2 (500 ng/well) were infected with VSV-GFP for 24 h, and cell lysates were then analyzed for reporter luciferase activity. The data are shown as the mean ± SD derived from three repeat experiments. ** p < 0.01 (Student’s t test). b , c 293T cells were transfected with empty vector, and a ZIKV-NS4A-, DENV-NS4A-, ZIKV-prM- or IVA-PB1-F2-expressing plasmid. Twenty-four hours later, the cells were infected with SeV, and the supernatants were UV-treated and used to overlay 293T cells. Twenty-four hours later, the cells were infected with VSV-GFP for 24 h, followed by scoring of the number of GFP-positive cells by flow cytometry ( b ), and cell images were taken under a fluorescence microscope ( c )

Article Snippet: 293T human embryonic kidney cells (the Cell Bank of the Chinese Academy of Sciences, Shanghai, China), HeLa human cervical adenocarcinoma cells (ATCC) and HFF-1 human foreskin fibroblasts cells (the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in DMEM (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (GIBCO, Carlsbad, CA), 2 mM l -glutamine, 100 μg/ml streptomycin and 100 units/ml penicillin (Invitrogen, Carlsbad, CA) at 37 °C under 5% CO 2 .

Techniques: Transfection, Infection, Luciferase, Activity Assay, Derivative Assay, Plasmid Preparation, Expressing, Flow Cytometry, Fluorescence, Microscopy

Journal:

Article Title: Activation of the Cryptic aac(6′)-Iy Aminoglycoside Resistance Gene of Salmonella by a Chromosomal Deletion Generating a Transcriptional Fusion

doi:

Figure Lengend Snippet: Strains and plasmids

Article Snippet: The MICs of aminoglycosides were determined by the method of Steers et al. ( 34 ). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant characteristics a Source or reference E. coli JM83 F − ara Δ( lac-proAB ) rpsL [F80d lac Δ( lacZ )M15] 37 MC1061 F − araD139 Δ( ara-leu ) 7696 gal E15 galK16 Δ( lac )X79 rpsL hsdR2 (r K − m K + ) mcrA mcrB1 36 C600 F − e14 − ( mcrA ) thr-1 leuB6 thi-1 lacY1 supE44 rfbD1 fhuA21 3 Cla F − restriction defective 31 C. freundii ATCC 8090 Reference strain 8 Salmonella BM4361 S. enterica subsp. I serotype Enteridis; clinical isolate This study BM4362 S. enterica subsp. I serotype Enteridis; clinical isolate; Tm This study LT2 S. enterica subsp. I serotype Typhimurium WHOCCS b BM4410 S. enterica subsp. II; 6.8:m.t:1.5 WHOCCS BM4411 S. enterica subsp. IIIa; 21:f.z 51 WHOCCS BM4412 S. enterica subsp. IIIb; 11:1.u:z WHOCCS BM4413 S. enterica subsp. IV; 6.7:z 4 .z 24 WHOCCS BM4414 S. enterica subsp. VI; 1.6.14.25:Q:e.u.x WHOCCS BM4415 S. bongori ; 44:r WHOCCS Plasmids pAT713 2.2-kb Ssp I fragment from BM4361 cloned into pUC18 This study pAT714 1.6-kb Sal I fragment from BM4361 cloned into pUC18 This study pAT715 1.3-kb PCR fragment from BM4361 cloned into pCR-Blunt vector; Tm This study pAT716 438-bp PCR fragment of aac(6′)-Iy from BM4361 cloned into pCR-Blunt vector This study pAT703 2.8-kb Sau 3AI fragment from BM4362 cloned into pUC18; Tm This study pAT711 438-bp PCR fragment of aac(6′)-Iy from BM4362 cloned into pUC19; Tm This study pAT712 6-kb Sal I- Bam HI fragment from BM4362 cloned into pUC18 This study pAT718 1.17-kb Hin dIII PCR fragment of nmpC from BM4361 cloned into pSU19 This study Open in a separate window a Tm, tobramycin resistance.

Techniques: Plasmid Preparation, Clone Assay