Comparison of IRES and F2A-Based Locus-Specific Multicistronic Expression in Stable Mouse Lines

Efficient and stoichiometric expression of genes concatenated by bi- or multi-cistronic vectors has become an invaluable tool not only in basic biology to track and visualize proteins in vivo, but also for vaccine development and in the clinics for gene therapy. To adequately compare, in vivo, the effectiveness of two of the currently popular co-expression strategies - the internal ribosome entry site (IRES) derived from the picornavirus and the 2A peptide from the foot-and-mouth disease virus (FDMV) (F2A), we analyzed two locus-specific knock-in mouse lines co-expressing SRY-box containing gene 9 (Sox9) and enhanced green fluorescent protein (EGFP) linked by the IRES (Sox9IRES-EGFP) or the F2A (Sox9F2A-EGFP) sequence. Both the constructs expressed Sox9 and EGFP proteins in the appropriate Sox9 expression domains, with the IRES construct expressing reduced levels of EGFP compared to that of the F2A. The latter, on the other hand, produced about 42.2% Sox9-EGFP fusion protein, reflecting an inefficient ribosome ‘skipping’ mechanism. To investigate if the discrepancy in the ‘skipping’ process was locus-dependent, we further analyzed the FLAG3-Bapx1F2A-EGFP mouse line and found similar levels of fusion protein being produced. To assess if EGFP was hindering the ‘skipping’ mechanism, we examined another mouse line co-expressing Bagpipe homeobox gene 1 homolog (Bapx1), Cre recombinase and EGFP (Bapx1F2A-Cre-F2A-EGFP). While the ‘skipping’ was highly efficient between Bapx1 and Cre, the ‘skipping’ between Cre and EGFP was highly inefficient. We have thus demonstrated in our comparison study that the efficient and close to equivalent expression of genes linked by F2A is achievable in stable mouse lines, but the EGFP reporter may cause undesirable inhibition of the ‘skipping’ at the F2A sequence. Hence, the use of other reporter genes should be explored when utilizing F2A peptides

Differences in the levels of aminoacylation and contents of modified nucleotides between total tRNAs from N2- and NH4+-grown Azospirillum lipoferum cells

Total tRNAs isolated from N2- and NH4(+)-grown Azospirillum lipoferum cells were compared with respect to amino acid acceptance, isoacceptor tRNA species levels and extent of nucleotide modifications. Amino-acylation of these two tRNA preparations with ten different amino acids indicated differences in the relative acceptor activities. Comparison of aminoacyl-tRNA patterns by RPC-5 column chromatography revealed no qualitative differences in the elution profiles. However, quantitative differences in the relative amounts of some isoacceptors were observed. These results indicate that alterations of relative amounts of functional tRNA species occur to match cellular requirements of the bacterial cells using N2 or NH4+ as nitrogen source. In addition, the content of modified nucleotides in total tRNAs of N2- and NH4(+)-grown cells was determined. In the NH4(+)-grown cells, content of most of the modified nucleotides decreased significantly. Based upon these results, the relationship of chargeability of tRNAs to base modifications is discussed

Azospirillum lipoferum tRNAs: Fractionation and identification of tRNA species and the nucleotide sequence of tRNA(Asn) (QUU)

Transfer RNAs of Azospirillum lipoferum were separated by two- dimensional gel electrophoresis and identified by aminoacylation. Thirty-six tRNA spots were resolved by this technique and twenty-six tRNA species have been identified. There are five tRNAs for Leu, four for Val, three for Pro, two each for Arg, Ile, Lys and Tyr, and one each for Ala, Asp, His, Phe, Ser and Thr. The tRNA(Asn) (QUU) was purified and its nucleotide sequence was determined. The A. lipoferum tRNA(Asn) (QUU) is 92% similar to B. subtilis tRNA(Asn) gene and two hypermodified nucleosides, queuosine (Q) and N-(9-beta-D Ribofuranosylpurine-6-YL) carbamoyl)-threonine (t(6)A) are present in this tRNA

Corrosion behaviour of mild steel in sulphuric acid- Effect of halides

The effect of halide ions on the corrosion behavior of mild steel in sulphuric acid medium was studied. Weight loss and polarization studies were carried out in 1 M sulphuric acid and at various concentration of the halide ions (chloride, bromide and iodide). The results revealed that corrosion of mild steel is more in acid medium without halide ions. Halide ions reduce the rate of corrosion and the inhibition efficiency is found to be in the order iodide > bromide > chlorid

Effect of replacing phenylalanine residues by para-substituted phenylalanines on the aggregation behavior of Aβ16-22

The peptide sequence KLVFFAE that spans the region 16-22 in the amyloid peptide Aβ1-40 has the ability to form fibrils or nanotubes in aqueous medium, depending on the conditions of dissolution. Interaction between the phenylalanine residues is presumed to play an important role in the self-assembly of Aβ16-22. We have investigated the importance of these aromatic residues by substituting them with p-chloro-, p-fluoro- and p-methylphenylalanine. Nanostructures different from the parent peptide were obtained with the substituted analogs, both in methanol as well as aqueous conditions (pH 2 and pH 7). Concentration-dependent effects observed in methanol, suggest that intermediate states occur during fibrillation. A balance between the crucial parameters such as charge, hydrophobicity and steric constraints implicated in self assembly, appear to modulate the nanostructure formation

Molecular targets of CusiRNA.

<p>The scheme displays the possible molecular targets of Ki-67-7 and curcumin when used in combination (CusiRNA). Arrowheads indicate the activation of indicated proteins; hammer heads indicate inhibition and the broken lines represent the possible role for putative intermediates. Further details are provided in the text.</p

Effect of Ki-67-7 and curcumin on cell cycle phases.

<p>AY-27 cells were treated with Ki-67-7, curcumin or with both, as described earlier in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048567#pone-0048567-g003" target="_blank">Figures 3A and 3B</a>. At the end of the incubation period, the cells were treated with PI and the distribution of cells in various cell cycle phases were determined by flow cytometry as described in Methods. Cells were scored as percentage distribution of cells in each of the cell cycle phases (G_o/G_1, S and G2/M). Bars indicate values which are the means ± S.E of three determinations.</p

Dose-dependent effects of curcumin on bladder cancer cell proliferation.

<p>T-24 (2a) and AY-27 (2b) cells were plated in 12-well plates (0.8×10⁵ cells/well) and cultured for 48 hr (40–50% confluence) as described in Methods. Following exposure to various concentrations of curcumin for 24 h, cells were washed with PBS and incubated for another 24 h in curcumin-free medium. Cells incubated with DMSO (0.1%) alone were treated as controls. DMSO alone had little impact on tumor cell growth (data not shown). Cell proliferation was assessed on the basis of cell viability, measured by MTT assay. Cell viability was expressed as percent of viability observed in DMSO-treated control cells. Values are from a representative (of two) (2a) or 3–8 (mean ± S.E) determinations (2b). ^*P<0.05, ^**P<0.01,^***P<0.005.</p

Effect of Ki-67-7 and curcumin on the regulatory proteins of cell cycle phases and apoptosis: Western blot analysis.

<p>Bladder cancer cells (T-24 and AY-27) were cultured and exposed to Ki-67-7 and curcumin as described above in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048567#pone-0048567-g003" target="_blank">Figures 3A and 3B</a>. At the end of the treatment period, total protein was extracted and subjected to Western blotting using appropriate antibodies as described in Methods. For purposes of associating proteins with specific roles, they were grouped in separate categories, such as gene transcription (A), cell-cycle progression (B) and apoptosis (C). β-actin was used as the internal control. Since different proteins (e.g. Cyclin D1 and NF-κB) from the same membrane were identified in separate categories, the same β-actin blot is displayed on more than one occasion. Data presented are western blots from a single experiment, which is representative of 2–3 identical experiments.</p