Post-transcriptional control of bacteriophage T4 gene 25 expression: mRNA secondary structure that enhances translational initiation.

Secondary structure of the mRNA in the translational initiation region is an important determinant of translation efficiency. However, the secondary structures that enhance or facilitate translation initiation are rare. We have previously proposed that such structure may exist in the case of bacteriophage T4 gene 25 translational initiation region, which contains three potential Shine-Dalgarno sequences (SD1, SD2, and SD3) with a spacing of 8, 17, and 27 nucleotides from the initiation codon of this gene, respectively. We now present results that clearly demonstrate the existence of a hairpin structure that includes SD1 and SD2 sequences and brings the SD3, the most typical of these Shine-Dalgarno sequences, to a favourable spacing with the initiation codon of gene 25. Using a phage T7 expression system, we show that mutations that prevent the formation of hairpin structure or eliminate the SD3 sequence result in a decreased level of gp25 synthesis. Double mutation in base-pair V restores the level of gene 25 expression that was decreased by either of the two mutations (C-to-G and G-to-C) alone, as predicted by an effect attributable to mRNA secondary structure. We introduced the mutations into the bacteriophage T4 by plasmid-phage recombination. Changes in the plaque and burst sizes of T4 mutants, carrying single and double mutations in the translational initiation region of gene 25, strongly suggest that the predicted mRNA secondary structure controls (enhances) the level of gene 25 expression in vivo. Hybridization of total cellular RNA with a gene 25 specific probe indicated that secondary structure or mutations in the translational initiation region do not notably affect the 25 mRNA stability. Immunoblot analysis of gp25 in Escherichia coli cells infected by T4 mutants showed that mRNA secondary structure increases the level of gp25 synthesis by three- to fourfold. Since the secondary structure increases the level of gp25 synthesis and does not affect mRNA stability, we conclude that this structure enhances translation initiation. We discuss some features of two secondary structures in the translational initiation regions of T4 genes 25 and 38.

A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR.

Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/µl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).