Journal: Thoracic cancer

Article Title: S- and R-Carvedilol Prevent Benzo(a)pyrene-Induced Lung Carcinogenesis.

doi: 10.1111/1759-7714.70109

Figure Lengend Snippet: FIGURE 3 | HPLC analysis of R- and S-Carvedilol: Chiral high-performance liquid chromatography (HPLC) was performed to verify the enantio- meric purity of R- and S-carvedilol obtained from Chem-Impex International Inc. Analysis was conducted using a Phenomenex Lux 5 μm Cellulose-4 LC column (250 × 4.6 mm; Part No. 00G-4491-E0; Serial No. H21-389311; Batch No. 5599–0063). Racemic carvedilol was used as a reference standard (Figure 3A). The results confirmed the high purity and correct identity of both enantiomers (Figure 3B,C).

Article Snippet: These findings indicate that both forms of carvedilol effectively FIGURE 3 | HPLC analysis of R- and S- Carvedilol: Chiral high- performance liquid chromatography (HPLC) was performed to verify the enantiomeric purity of R- and S- carvedilol obtained from Chem- Impex International Inc.

Techniques: High Performance Liquid Chromatography

Journal:

Article Title: In vivo selection of spectinomycin-binding RNAs

doi:

Figure Lengend Snippet: Functional spectinomycin interactions with E.coli 16S rRNA. (A) Structure of spectinomycin. (B) Secondary structure of folded three-arm junction within 16S rRNA containing the spectinomycin-binding site (8,24). The vertical stem in this view is helix 34. Guanine 1064 (indicated) is protected from N-7 alkylation by dimethyl sulfate in the presence of spectinomycin. The violet dot indicates the 5′ terminus of this fragment. Color coding of RNA strands is for clarity and is consistent between panels (B) and (D). (C) Molecular model of the corresponding 16S rRNA fragment structure extracted from the X-ray crystal structure of the small ribosomal subunit of T.thermophilus (7). The white arrow indicates the N-7 position of guanine 1064. (D) Same view as (C), but including the spectinomycin molecule (rendered as space-filling atoms) observed by Carter et al. (7) after soaking crystals of the small ribosomal subunit of T.thermophilus with antibiotic. Molecular graphics were created with InsightII using Protein Data Bank coordinates extracted from file 1FJG (7).

Article Snippet: For studies of spectinomycin effects on mung bean nuclease reactivity, radiolabeled RNAs (600 000 c.p.m., 1 µM final, achieved by mixing radiolabeled and unlabeled RNA) were incubated at 22°C in 10 mM Tris–HCl buffer with the indicated concentrations of spectinomycin for 15 min. Mung bean nuclease (1 U; New England Biolabs, Beverly, MA) was then added and reactions were allowed to proceed for 2 min prior to addition of urea loading buffer and freezing on dry ice.

Techniques: Functional Assay, Binding Assay

Journal:

Article Title: In vivo selection of spectinomycin-binding RNAs

doi:

Figure Lengend Snippet: Plasmids used in this work

Article Snippet: For studies of spectinomycin effects on mung bean nuclease reactivity, radiolabeled RNAs (600 000 c.p.m., 1 µM final, achieved by mixing radiolabeled and unlabeled RNA) were incubated at 22°C in 10 mM Tris–HCl buffer with the indicated concentrations of spectinomycin for 15 min. Mung bean nuclease (1 U; New England Biolabs, Beverly, MA) was then added and reactions were allowed to proceed for 2 min prior to addition of urea loading buffer and freezing on dry ice.

Techniques: RNA Expression, Plasmid Preparation, Variant Assay, Derivative Assay, Clone Assay

Journal:

Article Title: In vivo selection of spectinomycin-binding RNAs

doi:

Figure Lengend Snippet: RNA fragments of pJ653 that confer spectinomycin resistance in vivo. (A) Schematic diagram of plasmid pJ653 fragments tested in strain BL452. Promoter and terminator sequences of pJ456 are indicated in black. Inserts are color coded as in Figure ​Figure3.3. ‘Sector’ indicates the plating sector shown below. Asterisks in the schematic of plasmid pJ703 indicate a change in the flanking restriction sites used during cloning. Plasmid pJ708 is identical to pJ697, reassembled to confirm that no extraneous sequences within the vector had contributed to spectinomycin resistance. (B) Spectinomycin resistance phenotype after plating on 80 µg/ml spectinomycin. No cells were plated in sector 1.

Article Snippet: For studies of spectinomycin effects on mung bean nuclease reactivity, radiolabeled RNAs (600 000 c.p.m., 1 µM final, achieved by mixing radiolabeled and unlabeled RNA) were incubated at 22°C in 10 mM Tris–HCl buffer with the indicated concentrations of spectinomycin for 15 min. Mung bean nuclease (1 U; New England Biolabs, Beverly, MA) was then added and reactions were allowed to proceed for 2 min prior to addition of urea loading buffer and freezing on dry ice.

Techniques: In Vivo, Plasmid Preparation, Clone Assay

Journal:

Article Title: In vivo selection of spectinomycin-binding RNAs

doi:

Figure Lengend Snippet: Escherichia coli genetic selection intended to identify RNA inhibitors of DNA-binding proteins. The genetic manipulation strategy of Whipple (14) was modified to produce test strain BL452 containing an E.coli promoter under the regulation of the phage λ repressor (constitutively expressed from a linked cI gene), driving expression of tandem aadA (spectinomycin resistance) and lacZ (β-galactosidase) reporter genes, all in single copy on an F′ episome. Strain BL452 is then transformed with a plasmid library expressing stable, non-coding RNA transcripts containing a random 60 nt RNA insert driven by a strong lpp promoter (16). Selection for derepression of aadA and lacZ seeks RNAs that antagonize the repressor protein, in this case λ repressor. This selection unexpectedly also identified RNAs that directly antagonize spectinomycin.

Article Snippet: For studies of spectinomycin effects on mung bean nuclease reactivity, radiolabeled RNAs (600 000 c.p.m., 1 µM final, achieved by mixing radiolabeled and unlabeled RNA) were incubated at 22°C in 10 mM Tris–HCl buffer with the indicated concentrations of spectinomycin for 15 min. Mung bean nuclease (1 U; New England Biolabs, Beverly, MA) was then added and reactions were allowed to proceed for 2 min prior to addition of urea loading buffer and freezing on dry ice.

Techniques: Selection, DNA Binding Assay, Modification, Expressing, Transformation Assay, Plasmid Preparation

Journal:

Article Title: In vivo selection of spectinomycin-binding RNAs

doi:

Figure Lengend Snippet: Selected RNAs conferring spectinomycin resistance in selection strain BL452. (A) Growth of BL452 cells harboring the indicated RNA expression plasmids in the absence (left) or presence (right) of 80 µg/ml spectinomycin. 1, no inoculation; 2, pJ753 (single insert); 3, pJ744 (single insert); 4, pJ653 (three tandem inserts); 5, pJ697 (deletion mutant of pJ653 carrying two tandem inserts); 6, pJ456 (no insert). (B–D) Predicted secondary structures of RNA transcripts from pJ456 (B, no insert), pJ744 (C, single insert shown in purple) and pJ653 (D, three tandem inserts shown in blue, green and red).

Article Snippet: For studies of spectinomycin effects on mung bean nuclease reactivity, radiolabeled RNAs (600 000 c.p.m., 1 µM final, achieved by mixing radiolabeled and unlabeled RNA) were incubated at 22°C in 10 mM Tris–HCl buffer with the indicated concentrations of spectinomycin for 15 min. Mung bean nuclease (1 U; New England Biolabs, Beverly, MA) was then added and reactions were allowed to proceed for 2 min prior to addition of urea loading buffer and freezing on dry ice.

Techniques: Selection, RNA Expression, Mutagenesis

Journal:

Article Title: In vivo selection of spectinomycin-binding RNAs

doi:

Figure Lengend Snippet: Analysis of mutations that inactive spectinomycin resistance induced by RNA expressed from pJ697. (A) Mapping of 15 point mutations that reduce spectinomycin resistance. Insert segments of pJ697 are color coded as in Figure ​Figure3D.3D. Point mutants are numbered on the left, with the mutation shown within vertical red bars. (B) Spectinomycin gradient (0–80 µg/ml) growth assay of strain BL452 cells harboring the indicated RNA expression vectors: pJ456 (no insert); pJ697 (two of the three inserts found in pJ653); pJ744 and pJ753 (single inserts); mut 1 and mut 5 [inactivating point mutations mapped in (A)]. (C) Predicted RNA secondary structures for RNA transcripts from pJ697 compared with mut 1 (right), indicating predicted refolding of one subdomain (boxed). Inactivating mutations are indicated by red circles within the trapezoidal region. The position of the inactivating point mutation in mut 1 is indicated by an asterisk within the trapezoidal region and within the inset at right. (D) Native PAGE of radiolabeled in vitro transcripts corresponding to pJ697 and mut 1, confirming that the point mutation in mut 1 alters the distribution of RNA conformers.

Article Snippet: For studies of spectinomycin effects on mung bean nuclease reactivity, radiolabeled RNAs (600 000 c.p.m., 1 µM final, achieved by mixing radiolabeled and unlabeled RNA) were incubated at 22°C in 10 mM Tris–HCl buffer with the indicated concentrations of spectinomycin for 15 min. Mung bean nuclease (1 U; New England Biolabs, Beverly, MA) was then added and reactions were allowed to proceed for 2 min prior to addition of urea loading buffer and freezing on dry ice.

Techniques: Mutagenesis, Growth Assay, RNA Expression, Clear Native PAGE, In Vitro

Journal:

Article Title: In vivo selection of spectinomycin-binding RNAs

doi:

Figure Lengend Snippet: Analysis of mechanism of spectinomycin resistance induced by RNA. (A) RNAs that induce spectinomycin resistance do not alter steady-state levels of λ cI mRNA probed by primer extension analysis. Strains and plasmids are listed above. Strain BL445 expresses neither library RNA nor λ cI mRNA. Strain BL452 expresses only λ cI mRNA. Plasmids pJ697, pJ653, pJ744 and pJ753 were selected for their ability to induce spectinomycin resistance in BL452. (B) RNAs that induce spectinomycin resistance do not alter steady-state levels of λ repressor protein assayed by western blotting. (C) Absence of derepression of linked lacZ gene in the presence of RNAs that induce resistance to spectinomycin.

Article Snippet: For studies of spectinomycin effects on mung bean nuclease reactivity, radiolabeled RNAs (600 000 c.p.m., 1 µM final, achieved by mixing radiolabeled and unlabeled RNA) were incubated at 22°C in 10 mM Tris–HCl buffer with the indicated concentrations of spectinomycin for 15 min. Mung bean nuclease (1 U; New England Biolabs, Beverly, MA) was then added and reactions were allowed to proceed for 2 min prior to addition of urea loading buffer and freezing on dry ice.

Techniques: Western Blot

Journal:

Article Title: In vivo selection of spectinomycin-binding RNAs

doi:

Figure Lengend Snippet: Selected RNAs confer direct spectinomycin resistance. The indicated strains were spotted onto an increasing spectinomycin gradient from 0 to 40 µg/ml. The top two rows show the indicated plasmids in strain BL452 containing the aadA gene repressed by λ repressor. The remaining rows show the results of RNA expression from the indicated plasmids in strain DH5α, lacking aadA.

Article Snippet: For studies of spectinomycin effects on mung bean nuclease reactivity, radiolabeled RNAs (600 000 c.p.m., 1 µM final, achieved by mixing radiolabeled and unlabeled RNA) were incubated at 22°C in 10 mM Tris–HCl buffer with the indicated concentrations of spectinomycin for 15 min. Mung bean nuclease (1 U; New England Biolabs, Beverly, MA) was then added and reactions were allowed to proceed for 2 min prior to addition of urea loading buffer and freezing on dry ice.

Techniques: RNA Expression

Journal:

Article Title: In vivo selection of spectinomycin-binding RNAs

doi:

Figure Lengend Snippet: In vitro mapping of a spectinomycin-binding site in selected RNA. (A) RNAs encoded by plasmids pJ456 (no insert; lanes 1–6) and pJ744 (single insert that causes resistance to spectinomycin; lanes 7–12) were transcribed in vitro, end-labeled and subjected to partial mung bean nuclease digestion in the presence of increasing concentrations of spectinomycin (0, 0.4 or 4 mg/ml). Partial alkaline hydrolysis ladders are shown in lanes 1 and 7. Partial digestions with RNase T1 provide the guanosine ladders shown in lanes 2 and 8. Untreated samples are present in lanes 3 and 9. The insert sequence within the pJ744 RNA is indicated by a purple line to the left of lane 7. The site of mung bean nuclease hypersensitivity caused by spectinomycin is indicated in red. (B) Predicted fold of RNA transcript from pJ456 showing no spectinomycin-induced mung bean nuclease hypersensitive sites. (C) Predicted fold of RNA transcript from pJ744. Insert sequence is shown in purple. The spectinomycin-induced mung bean nuclease hypersensitive site is indicated in red numbering.

Article Snippet: For studies of spectinomycin effects on mung bean nuclease reactivity, radiolabeled RNAs (600 000 c.p.m., 1 µM final, achieved by mixing radiolabeled and unlabeled RNA) were incubated at 22°C in 10 mM Tris–HCl buffer with the indicated concentrations of spectinomycin for 15 min. Mung bean nuclease (1 U; New England Biolabs, Beverly, MA) was then added and reactions were allowed to proceed for 2 min prior to addition of urea loading buffer and freezing on dry ice.

Techniques: In Vitro, Binding Assay, Labeling, Sequencing