The 5′-UTR of enhances translation when cap-dependent scanning is inhibited in a monocistronic construct

<p><b>Copyright information:</b></p><p>Taken from "Cryptic promoter activity in the DNA sequence corresponding to the 5′-UTR"</p><p>Nucleic Acids Research 2005;33(7):2248-2258.</p><p>Published online 20 Apr 2005</p><p>PMCID:PMC1083428.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Schematic diagram of the constructs used. The 5′-UTR of was introduced immediately upstream of the luciferase open reading frame in the control plasmid pF to create pF-PIM. The stable hairpin structure with a free energy (Δ) of −55 kcal/mol was introduced upstream of the firefly luciferase start site in pF to create pHpF. The 5′-UTR of was introduced between the hairpin and the luciferase start site to create pHpF-PIM. () Relative luciferase activities of the monocistronic reporter constructs. Cos-7 cells were transfected with constructs pF, pF-PIM, pHpF and pHpF-PIM in combination with pSV-β-gal plasmid. Cell lysates were prepared 30 h post-transfection, and the activity of firefly luciferase was measured and normalized to that of β-galactosidase. The data were from four independent assays

Northern blot analysis of RNA transcripts derived from the 5′-UTR promoter

<p><b>Copyright information:</b></p><p>Taken from "Cryptic promoter activity in the DNA sequence corresponding to the 5′-UTR"</p><p>Nucleic Acids Research 2005;33(7):2248-2258.</p><p>Published online 20 Apr 2005</p><p>PMCID:PMC1083428.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Cos-7 cells were transfected with pRF (lane 1), pR-PIM-F (lane 2), R-PIM-F (lane 3), pF (lane 4), pR-EMCV-F (lane 5) and pR-HRV-F (lane 6). Poly(A) mRNAs were isolated from transfected cells 48 h later, electrophoresed in the presence of formaldehyde, transferred onto Hybond N nylon membrane and probed with P-labeled firefly luciferase probe (). After stripping, the membrane was subsequently re-probed with P-labeled luciferase specific probe (). The arrowhead and arrow indicate the dicistronic and monocistronic mRNA transcripts, respectively. The open arrowhead shows unknown RNAs hybridized with luciferase probes, which have been consistently observed in previous studies (,,). The migrations of 28S and 18S rRNA are indicated

Stimulation of the second-cistron expression by the 5′-UTR sequence of the

<p><b>Copyright information:</b></p><p>Taken from "Cryptic promoter activity in the DNA sequence corresponding to the 5′-UTR"</p><p>Nucleic Acids Research 2005;33(7):2248-2258.</p><p>Published online 20 Apr 2005</p><p>PMCID:PMC1083428.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Schematic diagram of the dicistronic constructs without inserts (pRF), or with the IRES of EMCV (pR-EMCV-F), the IRES of HRV (pR-HRV-F) and the 5′-UTR of (pR-PIM-F). () Relative luciferase activity generated from the dicistronic constructs. Cos-7 cells were transfected with dicistronic constructs together with plasmid pSV-β-gal. Lysates were prepared from cells 30 h post-transfection, the and firefly luciferase activities were measured and the relative ratios were calculated and normalized to that of the vector-transfected cells (pRF). The data were from four independent assays performed in triplicates

The 5′-UTR is still able to direct second cistron expression in the presence of a stable hairpin

<p><b>Copyright information:</b></p><p>Taken from "Cryptic promoter activity in the DNA sequence corresponding to the 5′-UTR"</p><p>Nucleic Acids Research 2005;33(7):2248-2258.</p><p>Published online 20 Apr 2005</p><p>PMCID:PMC1083428.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Schematic diagram of the dicistronic constructs. The stable hairpin structure with a free energy of −55 kcal/mol was introduced upstream of the open reading frame in the vectors pHpRF and pHpR-PIM-F. () Relative luciferase activity conferred by the dicistronic constructs. Cos-7 cells were transfected with pRF, pHpRF, pR-PIM-F or pHpR-PIM-F in combination with plasmid pSV-β-gal. Cells were harvested 30 h post-transfection, and and firefly luciferase activities were measured and normalized to those of the control plasmid (pRF). The data were from three independent experiments performed in triplicates

Demonstration of elevated amounts of PIM1 and HMGA1 proteins in PECs after adherence by <i>T. vaginalis</i> (<i>Tv</i>).

<p>In this experiment, trichomonads were added to T25 confluent monolayers of PECs (lane labeled+) using a parasite to PEC ratio of 10∶1. PECs without added organisms are labeled with a minus sign (−). This ratio of 10∶1 was chosen because it has been shown to yield at least one parasite attached per epithelial cell in adherence assays and to optimally signal epithelial cells for up-regulation of expression of genes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Kucknoor1" target="_blank">[13]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Garcia1" target="_blank">[27]</a>. After visible attachment to PECs, non-adherent organisms were decanted and fresh PEC medium added followed by incubation at 37°C for an additional 30 min. The flask was then placed directly in ice for detachment of organisms, after which PECs were washed and removed from the flask for preparation of total proteins for immunoblotting using established protocols, polyclonal rabbit antibodies produced in our laboratories, and equal loading of protein onto gels, as detailed previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Hu1" target="_blank">[28]</a>. Under conditions of exposure of PECs with or without <i>T. vaginalis</i>, no change in the amount of other cellular proteins was detected, as evidenced by no changes in the amounts of Akt and Bad, and this served to show equal amounts of total proteins loaded onto gels for SDS-PAGE and immunoblotting <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Hu1" target="_blank">[28]</a>. Prebleed rabbit serum was used as the negative control and gave no reactivity.</p