Untranslated regions detecting

A prototype bDNA assay was developed for quantification of HGV/GBV-C RNA in serum (Pessoa et al, 1997). The assay employed target probes based on the relatively conserved sequence in the 5 untranslated region of the HGV/GB V-C genome. Preamplifier molecules and incorporation of isoC and isoG into the sequences common to bDNA assays were used to enhance the analytical sensitivity. The provisional limit of detection was 32,500 genome equivalents/ml based on dilutions of a 700-nucleotide synthetic HGV/GBV-C RNA transcript. The run-to-run variance of the assay was <15%. 

Fig. 24.1 Variant alleles at the human TPMT locus. Grey boxes are exons containing mutations. White boxes are untranslated regions and black boxes represent exons in the ORF. Dashed box represents exon 2, which was detected in one of 16 human liver cDNAs (adapted from [30]).

Regulation of expression may occur at both the transcriptional and post-transcriptional levels. The mRNA for GM-CSF contains (in common with those of some other cytokines) conserved regulatory sequences in the 3 untranslated region, which may affect its rate of translation. The gene is constitutively transcribed in monocytes, endothelial cells and fibroblasts, but the mRNA is unstable and so does not accumulate to levels sufficient to allow translation into significant amounts of protein. Activation of these cells results in the increased expression of GM-CSF protein, which arises from both an enhanced rate of transcription (as detected in nuclear runoff experiments) and also an increased stability of the mRNA, perhaps by mechanisms analogous to those described above during activation of G-CSF expression ( 2.2.3.1). 

For Agrobacterium transformation, the gene of interest must be carried in a binary vector with appropriate bacterial T-DNA elements. For other transformation methods the requirements maybe less stringent. However, even in these cases, the DNA of interest is typically amplified in bacteria. The choice of a promoter is of particular importance, as this element will dictate both the expression level and the time course of protein expression. The inclusion of additional elements, such as enhancers, 5 and 3 untranslated regions, and fusion elements (e.g. fluorescent or 6-histidine tags), should also be considered for improved expression, detection, and downstream processing. 

Figure 43-3 Thiopurine S-methyltransferase (TPMT) allele variants. Gray boxes represent mutations that result in amino acid changes.TPMT 4 is a 5 splice site mutation for exon 10 that does not alter an amino acid. White boxes represent untranslated regions. Black boxes represent exons in the open reading frame.The dashed box represents exon 2, which was detected in 6.25% of human liver cDNAs during initial evaluation. (From McLeod HL, Siva C. The thiopurine S-mefhy/transferose gene locus—implications for clinical pharmacogenomics. Pharmacogenomics 2002 3 89-98. Reproduced by permission from future Medicine Ltd [London].)...

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