Transcriptional efficiency

Lipoxygenase AVNTR polymorphism in 100 bp upstream from the ATG start codon is associated with transcription efficiency. Differences in response to 5-lipoxygenase inhibitors and leucotriene receptor antagonists. 

A technical challenge with this step is to achieve RNA extraction of uniform quality and efficiency for each fraction. This is because the amount of RNA in each sucrose gradient fraction varies considerably and the high concentration of sucrose in the bottom fractions interferes with phase separation in typical phenol-based extraction steps. To address these problems, we spike each fraction with an aliquot of a foreign (control) RNA, which can be used later to correct for differences in RNA recovery (and reverse transcription efficiency) between samples. We then remove sucrose from the samples by precipitation of total nucleic acid and protein with ethanol. To purify RNA, a standard Trizol (Invitrogen) extraction is performed as outlined later (also see product insert). 

DNA, since proximity eflfects demand that the DNA or nascent RNA closest to the histones at the point of disruption will be the polyanion for which those histones will preferentially reassociate. Ten Heggeler-Bordier et al. [95] have verified these observations. They used immuno-electron microscopy to determine what happens to histones after transcription with T7 RNA polymerase of a multi-nucleosomal template and also observed transfer to the nascent RNA. In contrast, Kirov et al. [96] have reported that no histones displace during transcription with this polymerase. However, as described above, transcriptional efficiency and ultimately histone displacement is not efficient in very low ionic strength conditions. 

The hybrid tac promoter, a combination of lac and trp promoters, combines the high transcription efficiency of the trp promoter with the easy inducibility of the lac promoter. The lac promoter, like the other IPTG-inducible promoters, does not have tight repressor control, laclq E. coli strains with a large quantity of lacl repressor should always be used with this promoter possibly extra copies of lacl should even be provided (such as rep4 plasmid from Qiagen). 

Angotti E, Mele E, Costanzo F, Avvedimento EV. A polymorphism (G->A transition) in -78 position of apolipoprotein A-I promoter increases transcription efficiency. J Biol Chem. 1994, 269 17371-17374. 

In the case of non-viral gene therapy, efficacy depends on the intrinsic properties of plasmid DNA and the delivery system used. The topology of plasmid DNA is known to affect its in-vitro transcriptional efficiency. Higher gene expression... 

Vural, I., Memi oglu-Bilensoy, E., Renoir, J. M., Bochot, A., Duchene, D., and Hincal, A. A. (2005), Transcription efficiency of tamoxifen citrate loaded P-cyclodextrin nanoparticles,/. Drug Deliv. Sci. Technol., 15(5), 339-342. 

Vector/gene copy number Transcription efficiency of promoter Efficiency of mRNA maturation and transport Translational efficiency mRNA stability... 

Figure 31.35. Attenuation. (A) In the presence of adequate concentrations of tryptophan (and, hence, Trp-tRNA), translation proceeds rapidly and an RNA structure forms that terminates transcription. (B) At low concentrations of tryptophan, translation stalls awaiting Trp-tRNA, giving time for an alternative RNA structure to form that does not terminate transcription efficiently.

The human D4 dopamine receptor exhibits a number of genetic variants affecting both transcription efficiency and protein sequence, making it one of the most variable human genes. Most prominent among these variants is a 48-bp variable... 

For optimal transcriptional efficiency of T7 polymerase a few points should be considered. The initial base in the transcript must be a purine and preferably a G (the optimal sequence is (5 -Gp(G/C)).8 However, the polymerases accept a wide range of modifications at the 5 positions such as GpppG, dinucleotides and biotinylated dinucleotides (provided the 3 -nucleotide is a G, Section 2.3.2). If transcriptions are performed under conditions where one of the nucleotides is limiting the sequence of the first ten nucleotides is crucial for a high yield. If [a-32P] UTP is limiting in cotranscriptional labelling of RNA uridines within the initial ten nucleotides of the transcript will result in a high frequency of short abortive transcripts. This has been rationalised as a result of a transition from a labile initiation complex (the initial ten nucleo-... 

The transcription efficiency is greatly reduced or abolished if the 5 -nucleotide is not a guanosine. Some transcripts require nucleotides different from pppG at the 5 -end, and in these cases special precautions should be taken. If the second nucleotide is a G, ribodinucleo-tides, such as ApG, 4SUpG, etc. can be added to the reaction to cap the transcript. The ribodinucleotide will be incorporated at the 5 -end at the same ratio as between the dinucleotide and GTP in the reaction mixture. An alternative method of generating any 5 -end is given in Section 2.3.2.1. 

Then, when delivered into the cytosol, the gene has to find its way to the ceU nucleus, the site where transcription and replication wiU occur. A detailed recent smdy showed the crucial importance of nuclear transcription efficiencies. In fact there are many individual steps that make up the gene transfection and mechanistic studies are performed to identify these steps and to find ways to facilitate these steps. 

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