Journal: Journal of Bacteriology

Article Title: PhaQ, a New Class of Poly-?-Hydroxybutyrate (PHB)-Responsive Repressor, Regulates phaQ and phaP (Phasin) Expression in Bacillus megaterium through Interaction with PHB

doi: 10.1128/JB.186.10.3015-3021.2004

Figure Lengend Snippet: DNase I footprinting analysis of PhaQ binding to the phaQ promoter region. (A) A 0.4-kb SmaI-HindIII DNA fragment containing the phaQ promoter region (positions −356 to +39) and labeled with 32 P at its HindIII site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. (B) A 0.36-kb BamHI-EcoRI DNA fragment containing the phaQ promoter region (positions −105 to + 249) and labeled with 32 P at its BamHI site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. The numbers on the left indicate the positions of bases relative to the transcriptional initiation site of phaQ . Solid brackets on the right denote the protected regions.

Article Snippet: To construct plasmid pGS1144, the above-mentioned 443-bp DNA fragment carrying the coding sequence of phaQ and flanked by BamHI and HindIII sites was cloned between the BamHI and HindIII sites of pMAL-c2 (New England Biolabs, Inc.) to generate a maltose-binding protein (MalE)-PhaQ fusion protein.

Techniques: Footprinting, Binding Assay, Labeling, Incubation

Journal: Nucleic Acids Research

Article Title: Differences in silencing of mismatched targets by sliced versus diced siRNAs

doi: 10.1093/nar/gky287

Figure Lengend Snippet: Proposed function of sli-RISCs versus si-RISCs. ( A ) Northern blotting to detect chemically synthesized sli-887 and si-887. Processed products of sli-887 are longer than the 21-mer si-887. ( B ) Northern blotting to detect sli-887 and the 25-nt form of sli-887 (nts 1 to 25). Processed products of sli-887 are shorter than the 25-mer form. ( C ) Northern blotting to detect processed sli-1148 from a stably expressed sli-1148 driven by a U6 promoter, which mainly exists in an approximately 30-mer isoform, in HCT116 cells. ( D ) Proposed model of sli-RISC versus si-RISC function: si-RISCs can involve three non-slicing Agos (Ago1, Ago3, and Ago4) and slicing Ago2 loaded with uniform 21-mer guide RNAs; sli-RISCs contain the slicing Ago2 loaded with guide RNAs with various 3′ end lengths, and silencing may couple with 3′ end trimming/tailing by PARN. After the passenger strand is sliced, the hairpin might open during target binding, allowing nts 19 to 22 to base pair (open hairpin hypothesis), or the loop from nts 19 to 22 might remain, allowing only nts 2 to 14 to be used for target binding (partial hairpin hypothesis).

Article Snippet: We first cloned FLAG/HA-Ago2 as a Hind III and Spe I fragment into Hind III and Xba I sites in pcDNA3.1-neo (Thermo Fisher Scientific, Grand Island, NY, USA) to generate pcDNA-FLAG/HA-hAgo2; then we used gBlocks (Integrated DNA Technologies [IDT], San Diego, CA, USA) or PCR to generate hAgo2-dLS (truncated before the N domain) and hAgo2-LS-mut (with five amino acids in hAgo2 mutated to match the amino acids of hAgo1 at the same locations: P27R, D30G, R36K, Q41L, F45Y).

Techniques: Northern Blot, Synthesized, Stable Transfection, Binding Assay

Journal: Applied and Environmental Microbiology

Article Title: A Genetic System for Clostridium ljungdahlii: a Chassis for Autotrophic Production of Biocommodities and a Model Homoacetogen

doi: 10.1128/AEM.02891-12

Figure Lengend Snippet: Restriction and PCR analyses of various plasmid DNAs from C. ljungdahlii . (A) Plasmid pCL1. Plasmid DNA was digested with HindIII and analyzed by agarose gel electrophoresis. Lane 1, plasmid preparation from E. coli which was transformed with a plasmid preparation from a C. ljungdahlii transformant; lane 2, plasmid used to transform C. ljungdahlii ; lane L, 1-kb DNA ladders (New England BioLabs). (B) HindIII-digested pQexp. Lane 1, plasmid preparation from E. coli which was transformed with a plasmid preparation from a C. ljungdahlii transformant; lane 2, plasmid used to transform C. ljungdahlii ; lane L, 1-kb DNA ladders. (C) Colony PCR amplification of the catP gene from pJIR750ai. Lane 1, a C. ljungdahlii transformant; lane 2, plasmid preparation from E. coli as the positive control; lane 3, C. ljungdahlii genomic DNA as a negative control; lane 4, no DNA as a negative control for the PCR amplification.

Article Snippet: The upstream region of the fliA gene, the downstream region of the fliA gene, and the ermC gene were prepared by digesting the plasmids with XbaI and EcoRI, HindIII and XhoI, and EcoRI and HindIII, respectively, and were cloned in the XbaI and XhoI sites of pBluescript II KS(−) (Stratagene).

Techniques: Polymerase Chain Reaction, Plasmid Preparation, Agarose Gel Electrophoresis, Transformation Assay, Amplification, Positive Control, Negative Control