Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 1. Silver-stained 16.4% (w/v) T (total acrylamide), 1.9% (w/v) C (bisacrylamide) Tricine SDS-polyacrylamide gel show- ing the LPS profiles of E. coli K12 strains. 1, x705; 2, x711; 3, x711(pJB2); 4, Y10; 5, D21e7 (rfa-1); 6, CS2051 (has a deletion elimi- nating rfaG, rfaP, rfaM, rfaN, and rfaB); 7, D31m4 (rfa-229, rfa-230).

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: Staining

Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 2. Physical map of the lpcA region. Vector sequences are not shown. pE4021 is a cosmid clone containing chromosomal DNA from E. coli W3110, including the region from 4.9 to 5.8 min. RNHQ, PEPD, GPTA, PHOE, and PROAB indicate the location of sequenced genes. pJB1 contains a 14-kb EcoRI fragment cloned from pE4021. pJB2 and pJB8 contain a 3-kb BamHI fragment cloned from pJB1 into different vectors. pJB2-9 to pJB2-34 indicate the various deletions of pJB2 span- ning the lpcA region. pJB15 indicates the DNA insert used for construc- tion of DIG-labeled riboprobes. ORF1 and ORF2 are two open reading frames found on opposite strands of the DNA. The direction of tran- scription of lpcA is indicated by the arrow beneath ORF2. The comple- mentation of the novobiocin supersensitivity phenotype by the deletion clones is indicated: R, successful complementation; S, unsuccessful complementation. Restriction enzymes indicated are: A, AvaII; B, BamHI; Bs, BstEII; E, EcoRI; Ev, EcoRV; Hc, HincII; P, PvuI.

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: Plasmid Preparation, Clone Assay, Labeling

Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 4. A schematic representation of the proposed events leading to the chromosomal deletion of the lpcA locus in E. coli strain x711. Panel A, chromosomal map of E. coli K12 strain x705. RNHQ, LPCA, PROAB, IS30A, IS5A, IS1B, and IS30 indicate the location of sequenced genes. Panel B, Southern blot showing chromo- somal DNA profiles of E. coli strains x711 and x705 probed with a 14-kb EcoRI DIG-11-dUTP-labeled DNA probe. M, l HindIII molecular weight markers; 1, x711 DNA digested with EcoRI; 2, x705 DNA di- gested with EcoRI; 3, pJB2 digested with BamHI; 4, pJB1 digested with EcoRI. Panel C, restriction maps of pJB1 and pJB16 showing identical nucleotide sequence (hatched box) and the IS5 element (open box). Panel D, transposition of the IS5A insertion element from approximately 5.9 min to 5.2 min followed by replication of the element, and chromosomal map of E. coli strain x711 showing the resulting deletion of the lpcA locus. Restriction endonucleases indicated are: E, EcoRI.

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: Southern Blot, Labeling, Molecular Weight, Sequencing

Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 5. Reversed-phase high performance liquid chromatogra- phy analyses of carbohydrates synthesized by E. coli strains x711 and x711(pJB2) cell extracts following incubation with 1.0 mmol of sedoheptulose 7-phosphate. Panel A, x711 incubated 60 min. Panel B, x711(pJB2) incubated 2 min. Panel C, x711(pJB2) incu- bated 60 min without sedoheptulose 7-phosphate. Panel D, x711(pJB2) boiled extract incubated 60 min. Large arrow indicates the retention peak of the phosphorylated product. Small arrow indicates the reten- tion peak of sedoheptulose 7-phosphate.

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: Synthesized, Incubation

Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 6. Effect of alkaline phosphatase treatment in the reac- tion products analyzed by reversed-phase high performance liquid chromatography. Upper panel, HPLC profile of x711(pJB2) extract incubated with 1.0 mmol of sedoheptulose 7-phosphate (SED- 7-P) and treated with alkaline phosphatase (4 units) prior to derivat- ization with ABEE. Arrow indicates the location of the reaction peak of the reaction product in the absence of alkaline phosphatase treatment. Lower panel, HPLC profile of authentic glyceromannoheptose derivat- ized with ABEE. ABEE, p-aminobenzoic ethyl ester; AP, alkaline phos- phatase; GMH, glyceromannoheptose.

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: High Performance Liquid Chromatography, Incubation

Journal: Molecular plant

Article Title: DET1-mediated COP1 regulation avoids HY5 activity over second-site gene targets to tune plant photomorphogenesis.

doi: 10.1016/j.molp.2021.03.009

Figure Lengend Snippet: Figure 1. DET1- and COP1-associated proteins. (A) Schematic representation of proteins found to associate with DET1 and COP1 in TAP assays. Color code represents the maximum number of peptides for each represented protein found in a TAP assay as detailed in Supplemental Table 1. DET1 and COP1 proteins were expressed in Arabidopsis cell cultures. Five independent TAP experiments were performed for DET1 and two for COP1 (Supplemental Table 2). (B) MBP-COP1 and MBP-HY5 recombinant proteins expressed in E. coli pulled-down MYC-DET1 from 7-day-old Arabidopsis seedlings. MBP re- combinant protein was used as a control. Anti-MYC and anti-MBP antibodies were used for the immunoblots. (C and D) F€orster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET–FLIM) analysis of the interaction between COP1 or HY5 fused to GFP and DET1 (C), and HY5 or COP1 fused to RFP (D). Box plots show the distribution of 5–9 measurements ±SD. (E) FRET–FLIM analysis of the interaction between GFP-COP1 and RFP-HY5 upon cluc-DET1 co-expression. Box plots show the distribution of 10 measurements ±SD. All FRET assays were performed following transient expression in N. benthamiana leaves. Free RFP was used as negative control. FE, FRET efficiency. Asterisks indicate statistically significant differences according to Student’s t-test (****p < 0.0001; ***p < 0.001; *p < 0.01). For all FRET experiments three independent biological replicates were performed, and results from one replicate are shown.

Article Snippet: Pull-down assays MBP recombinant protein fusions were expressed in the Escherichia coli BL21 (DE3) strain carrying the corresponding coding sequence cloned into the pKM596 plasmid, a gift from David Waugh (Addgene plasmid #8837).

Techniques: Recombinant, Control, Western Blot, Förster Resonance Energy Transfer, Imaging, Microscopy, Expressing, Negative Control