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D R Edwards - One of the best experts on this subject based on the ideXlab platform.
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improved design of riboprobes from PBluescript and related vectors for in situ hybridization
BioTechniques, 1993Co-Authors: H Witkiewicz, M E Olande, D R EdwardsAbstract:The PBluescript family of plasmids and phagemids are sophisticated multi-purpose cloning vectors that allow convenient production of single-stranded sense and anti-sense RNA probes corresponding to DNA sequences inserted into a large multiple cloning site array. We have observed that in many applications sense (control) probes generated from genes cloned into PBluescript II KS(-) give high background signals on in situ hybridization to human tissue sections. Our studies indicate that this spurious hybridization is due to sequences contained within both strands of the multiple cloning site between the SmaI and SacI sites that are similar to human 28S rRNA. This information is useful in construct design in order to minimize nonspecific background problems, as demonstrated by in situ hybridization of sense and anti-sense probes corresponding to a portion of human stromelysin-3 to sections of human lung carcinoma.
Pe Svoboda - One of the best experts on this subject based on the ideXlab platform.
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The inhibition of luciferase reporter activity is partly dependent on PKR protein level.
2014Co-Authors: Jana Nejepinska, Radek Malik, Susa Wagne, Pe SvobodaAbstract:(A) Stable cell line with shRNA-mediated PKR knock-down. Western blot analysis of HeLa stable cell lines carrying shRNA vector targeting PKR. Ctrl, parental HeLa cells; A1-B6, stably-transfected independent clones carrying antibiotic resistance. Clone B4 showed the highest PKR knock-down and was used for subsequent experiments. (B) Parental HeLa cells (ctrl) or HeLa cells with stably down-regulated PKR expression (KD) were co-transfected with 100 ng/well of each RL (light colors) and FL (dark colors) reporter plasmids and 0 or 50 ng/well of pCAGEGFP-MosIR. Parental plasmid pCAGEGFP was used to maintain a constant amount of transfected DNA. Data are shown as an average of two independent experiments performed in quadruplicates; Error bars = SEM. (C) pCAGEGFP-MosIR transcript associates with PKR. HEK-293 cells were transfected with pCAGEGFP or pCAGEGFP-MosIR. Cell lysates (24 hours post-transfection) were used for immunoprecipitation with anti-PKR antibody or control IgG antibody. Immunoprecipitated RNA was reverse-transcribed and used for real-time PCR analysis. Data are displayed as a percentage of input. Shown is the average of two experiments. Error bars = range of values. (D) pCAGEGFP-MosIR expression activates PKR. Western blotting analysis of HEK-293 cells transfected with increasing amount of pCAGEGFP or pCAGEGFP-MosIR (50–150–250 ng per well). PBluescript was added to maintain the amount of transfected DNA constant. pBS, PBluescript only. UN, untransfected cells; MosIR, pCAGEGFP-MosIR.
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Suppression of co-transfected reporters in mammalian cells is general.
2014Co-Authors: Jana Nejepinska, Radek Malik, Susa Wagne, Pe SvobodaAbstract:(A, B) Suppression of co-transfected reporters in HeLa cells (A) and mouse 3T3 cells (B). Cells were transiently transfected with a constant amount of firefly luciferase (square), Renilla luciferase (triangle) reporter plasmids, and increasing amounts of pCAGEGFP-MosIR or pCAGEGFP. Luciferase activities were measured 48 hours post-transfection. PBluescript was added to maintain a constant amount of transfected DNA. Both luciferase activities are shown relative to cells transfected with 0 ng of the pCAGEGFP-MosIR. Data are shown as an average of at least 3 experiments performed in triplicates. Error bars = SEM. (C) Suppression of co-transfected reporters is independent of the transfection method. HEK-293 cells were transiently transfected with a constant amount of firefly luciferase (black bars), Renilla luciferase (white bars) reporter plasmids, and 50 ng of pCAGEGFP-MosIR using Nanofectin or calcium phosphate transfection. Luciferase activities were measured 48 hours post-transfection. PBluescript was added to maintain a constant amount of transfected DNA. Both luciferase activities are shown relative to cells transfected with 0 ng of the pCAGEGFP-MosIR. Data show a typical experiment measured in triplicates. Error bars = SEM. (D) Suppression of a co-transfected RFP reporter. HEK-293 were transiently transfected with 150 ng of RFP reporter plasmid and 350 ng of pCAGEGFP or pCAGEGFP-MosIR. Shown is FACS analysis of RFP fluorescence 36 h post-transfection. The experiment was performed three times, a representative result is shown.
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Transiently transfected luciferase reporters are inhibited by expressed dsRNA.
2014Co-Authors: Jana Nejepinska, Radek Malik, Susa Wagne, Pe SvobodaAbstract:(A) The inhibition of reporter activity is independent of the hairpin RNA sequence and it is absent when the inverted repeat is placed downstream of non-active ZP3 promoter. HEK-293 cells were transiently transfected with 100 ng/well of each RL (triangle) and FL (square) reporter plasmids and increasing amount (0–250 ng/well) of either pCAGEGFP-Lin28IR (containing an active promoter) or pZP3EGFP-Lin28IR (containing an inactive promoter). Luciferase activity was analyzed 48 hours after transfection. PBluescript was added to maintain the amount of transfected DNA constant. Both luciferase activities are shown relative to cells transfected with 0 ng of the hairpin-expressing plasmid. (B) Similar to (A) except Elavl2IR-expressing plasmids (pCAGEGFP- Elavl2IR or pZP3EGFP-Elavl2IR) were used. Data are shown as an average of at least 3 experiments made in triplicates. Error bars = SEM. (C, D) A hairpin-expressing pCAGEGFP-MosIR plasmid affects the luciferase activity of transiently transfected but not stably integrated reporter plasmids. HEK-293 cells with stably integrated RL and FL reporters (C), or HEK-293 cells with stably integrated RL reporter only (D) were transiently transfected with an increasing amount of pCAGEGFP-MosIR and a constant amount of FL reporter (if not stably integrated). PBluescript was added to maintain the amount of transfected DNA constant. Both FL (squares) and RL (triangles) luciferase activities were analyzed 48 hours post-transfection. Luciferase activities in cells transfected with 0 ng of the pCAGEGFP-MosIR were set to one. Data show an average of at least 3 experiments done in triplicates. Error bars = SEM.
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Expression of hairpin RNA inhibits the luciferase activity of transiently transfected reporter plasmids.
2014Co-Authors: Jana Nejepinska, Radek Malik, Susa Wagne, Pe SvobodaAbstract:(A) Schematic composition of pCAGEGFP-MosIR, pCAGEGFP, and pCAGEGFP-MosMos plasmids. (B) Reporter activity is inhibited by hairpin RNA in a concentration-dependent manner. HEK-293 cells were transiently transfected with a constant amount of firefly luciferase (square), Renilla luciferase (triangle) reporter plasmids, and increasing amount of a tested plasmid. Luciferase activities were measured 48 hours post-transfection. PBluescript was added to maintain a constant amount of transfected DNA. Both luciferase activities are shown relative to cells transfected with 0 ng of the pCAGEGFP-MosIR. Data are shown as an average of at least 3 experiments made in triplicates. Error bars = SEM.
Ji Hye Yoo - One of the best experts on this subject based on the ideXlab platform.
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dynamics of supercoiled and linear PBluescript ii sk phagemids probed with a long lifetime metal ligand complex
Journal of Biochemistry and Molecular Biology, 2005Co-Authors: Jung Sook Kang, Hong Dae Choi, Ji Hye YooAbstract:We extended the measurable time scale of DNA dynamics to microsecond using [Ru(phen)(2)(dppz)](2+)(phen = 1,10-phenanthroline, dppz=dipyrido[3,2-a:2',3'-c]phenazine)(RuPD) , which displays a mean lifetime near 500 ns. To evaluate the usefulness of this luminophore (RuPD) for probing nucleic acid dynamics, its intensity and anisotropy decays when intercalated into supercoiled and linear PBluescript (pBS) II SK(+) phagemids were examined using frequency-domain fluorometry with a blue light-emitting diode (LED) as the modulated light source. The mean lifetime for the supercoiled phagemids ( = 489.7 ns) was somewhat shorter than that for the linear phagemids ( = 506.4 ns), suggesting a more efficient shielding from water by the linear phagemids. The anisotropy decay data also showed somewhat shorter slow rotational correlation times for supercoiled phagemids (997.2 ns) than for the linear phagemids (1175.6 ns). The slow and fast rotational correlation times appear to be consistent with the bending and torsional motions of the phagemids, respectively. These results indicate that RuPD can have applications in studies of both bending and torsional dynamics of nucleic acids.
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comparative dynamics of trna val and PBluescript ii sk phagemid studied with ethidium bromide and a long lifetime metal ligand complex
2004Co-Authors: Jung Sook Kang, Ji Hye YooAbstract:The metal-ligand complex, [Ru(phen)2(dppz)] 2+ (phen=1,10-phenanthroline, dppz=dipyrido[3,2-a:2',3'-c]phenazine) (RuPD), was used as a spectroscopic probe for studying nucleic acid dynamics. The RuPD complex displays a long lifetime and a molecular light switch property upon DNA binding due to shielding of its dppz ligand from water. To show the usefulness of this luminophore (RuPD) for probing nucleic acid dynamics, we compared its intensity and anisotropy decays when intercalated into the tRNA val and PBluescript (pBS) II SK(+) phagemid through a comparison with ethidium bromide (EB), a conventional nucleic acid probe. We used frequency-domain fluorometry with a blue light-emitting diode (LED) as the modulated light source. The mean lifetime for the tRNA val ( = 166.5 ns) was much shorter than that for the pBS II SK(+) phagemid ( = 481.3 ns), suggesting a much more efficient shielding from water by the phagemid. Because of their size difference, the anisotropy decay data showed a much shorter rotational correlation times for the tRNA val (99.9 and 23.6 ns) than for the pBS II SK(+) phagemid (968.7 and 39.5 ns). These results indicate that RuPD can be useful for studying nucleic acid dynamics.
H Witkiewicz - One of the best experts on this subject based on the ideXlab platform.
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improved design of riboprobes from PBluescript and related vectors for in situ hybridization
BioTechniques, 1993Co-Authors: H Witkiewicz, M E Olande, D R EdwardsAbstract:The PBluescript family of plasmids and phagemids are sophisticated multi-purpose cloning vectors that allow convenient production of single-stranded sense and anti-sense RNA probes corresponding to DNA sequences inserted into a large multiple cloning site array. We have observed that in many applications sense (control) probes generated from genes cloned into PBluescript II KS(-) give high background signals on in situ hybridization to human tissue sections. Our studies indicate that this spurious hybridization is due to sequences contained within both strands of the multiple cloning site between the SmaI and SacI sites that are similar to human 28S rRNA. This information is useful in construct design in order to minimize nonspecific background problems, as demonstrated by in situ hybridization of sense and anti-sense probes corresponding to a portion of human stromelysin-3 to sections of human lung carcinoma.
Alexander Steinbuchel - One of the best experts on this subject based on the ideXlab platform.
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molecular characterization of genes of pseudomonas sp strain hr199 involved in bioconversion of vanillin to protocatechuate
Journal of Bacteriology, 1997Co-Authors: Horst Priefert, Jurgen Rabenhorst, Alexander SteinbuchelAbstract:The gene loci vdh, vanA, and vanB, which are involved in the bioconversion of vanillin to protocatechuate by Pseudomonas sp. strain HR199 (DSM 7063), were identified as the structural genes of a novel vanillin dehydrogenase (vdh) and the two subunits of a vanillate demethylase (vanA and vanB), respectively. These genes were localized on an EcoRI fragment (E230), which was cloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The vdh gene was identified on a subfragment (HE35) of E230, and the vanA and vanB genes were localized on a different subfragment (H110) of E230. The nucleotide sequences of fragment HE35 and part of fragment H110 were determined, revealing open reading frames of 1062, 951, and 1446 bp, representing vanA, vanB, and vdh, respectively. The vdh gene was organized in one operon together with a fourth open reading frame (ORF2), of 735 bp, which was located upstream of vdh. The deduced amino acid sequences of vanA and vanB exhibited 78.8 and 62.1% amino acid identity, respectively, to the corresponding gene products from Pseudomonas sp. strain ATCC 19151 (F. Brunel and J. Davison, J. Bacteriol. 170:4924-4930, 1988). The deduced amino acid sequence of the vdh gene exhibited up to 35.3% amino acid identity to aldehyde dehydrogenases from different sources. The deduced amino acid sequence of ORF2 exhibited up to 28.4% amino acid identity to those of enoyl coenzyme A hydratases. Escherichia coli strains harboring fragment E230 cloned in PBluescript SK- converted vanillin to protocatechuate via vanillate, indicating the functional expression of vdh, vanA, and vanB in E. coli. High expression of vdh in E. coli was achieved with HE35 cloned in PBluescript SK-. The resulting recombinant strains converted vanillin to vanillate at a rate of up to 0.3 micromol per min per ml of culture. Transfer of vanA, vanB, and vdh to Alcaligenes eutrophus and to different Pseudomonas strains, which were unable to utilize vanillin or vanillate as carbon sources, respectively, conferred the ability to grow on these substrates to these bacteria.