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

An Integrative Developmental Genomics and Systems Biology Approach to Identify an In Vivo Sox Trio-Mediated Gene Regulatory Network in Murine Embryos.

Embryogenesis is an intricate process involving multiple genes and pathways. Some of the key transcription factors controlling specific cell types are the Sox trio, namely, Sox5, Sox6, and Sox9, which play crucial roles in organogenesis working in a concerted manner. Much however still needs to be learned about their combinatorial roles during this process. A developmental genomics and systems biology approach offers to complement the reductionist methodology of current developmental biology and provide a more comprehensive and integrated view of the interrelationships of complex regulatory networks that occur during organogenesis. By combining cell type-specific transcriptome analysis and in vivo ChIP-Seq of the Sox trio using mouse embryos, we provide evidence for the direct control of Sox5 and Sox6 by the transcriptional trio in the murine model and by Morpholino knockdown in zebrafish and demonstrate the novel role of Tgfb2, Fbxl18, and Tle3 in formation of Sox5, Sox6, and Sox9 dependent tissues. Concurrently, a complete embryonic gene regulatory network has been generated, identifying a wide repertoire of genes involved and controlled by the Sox trio in the intricate process of normal embryogenesis