СПЕКТРОСКОПІЯ ЯДЕРНОГО МАГНІТНОГО РЕЗОНАНСУ В АНАЛІЗІ ОБ'ЄКТІВ ДОВКІЛЛЯ

Additional file 3. Multiplication rate

Evidence for translational regulation of <i>var2csa</i> in cultured, wildtype parasites.

<p>A. The parasite population NF54 VAR2CSA was selected using anti-VAR2CSA rabbit antibodies. Flow analysis indicates that a majority of the population displays VAR2CSA on the cell surface (red) with a MFI value of 14.2 compared to the background (black) with a MFI value of 5.2 (arbitrary units). Flow analysis of NF54-239 indicates that the majority of the population displays low levels of VAR2CSA on the cell surface with a MFI value of 5.0 compared to the background value of 3.6 (arbitrary units). B. Selection for VAR2CSA expression results in recognition by antibodies predominantly from female sera whereas the NF54-239 line is poorly recognized. C. <i>var</i> transcript analysis shows that <i>var2csa</i> is the dominant transcript in both the NF54 VAR2CSA (Black) and Nf54-239 (white) parasite lines.</p

The <i>var2csa</i> uORF is a repressive element.

<p>A. Constructs used in transient transfections. Constructs drive expression of either the firefly <i>luciferase</i> reporter gene, <i>Renilla luciferase</i> (in the case of control plasmid HRH) or both (in the case of V2RLH). VLH contains the un-modified promoter and upstream regulatory region from <i>var7b</i>. V2LH contains the un-modified promoter and upstream regulatory region from <i>var2csa</i>. V2LHm is identical to V2LH, except a single base pair mutation has altered the start codon of the upstream open reading (uORF) from ATG to ACG. In V2BLH, the uORF has been replaced with the <i>bsd</i> coding region while in V2RLH the uORF has been replaced by the coding region for <i>Renilla luciferase</i>. In V2ΔLH the entire region upstream of the uORF, including the transcription start site, has been deleted. In SURF the uORF has been shortened to 48 bp by the introduction of a premature stop codon, while in uORFL it has been lengthened to 450 bp by eliminating the endogenous stop codon. ICL contains the intact upstream regulatory region, including the uORF, however the intercistronic region has been duplicated. B. Levels of firefly luciferase expression from each construct shown in A. C. Levels of <i>Renilla</i> luciferase expression. V2RLH supports translation of robust levels of <i>Renilla</i> luciferase indicating that the uORF can be translated. The plasmid HRH, containing the strong <i>hrp3</i> promoter, was employed as a positive control for <i>Renilla</i> luciferase expression, while V2LH, which does not contain the <i>Renilla</i> luciferase gene, was a negative control. All assays were done simultaneously in triplicate.</p

Growth rates of parasites translating different ORFs.

<p>Constructs are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000256#ppat-1000256-g004" target="_blank">Figure 4</a>. A. Parasites that have been selected for translation of the second open reading frame encoding <i>blasticidin-S-deaminase</i> (V2B+blasticidin) grow at the same rate as parasites not under blasticidin selection (V2B−blasticidin) or those in which translation of the uORF has been disrupted (V2mB+blasticidin). Parasites in which the uORF has been replaced with the coding region of <i>Renilla luciferase</i> (V2RB+blasticidin) grow at a slower rate. B. Parasites actively translating the second ORF encoding <i>blasticidin-S-deaminase</i> continue to display resistance to the drug if grown without selection pressure very briefly. C. When parasites are grown in the absence of blasticidin in the media for 21 days, they rapidly begin to repress translation of the second ORF and revert to the drug sensitive phenotype as demonstrated by slower growth when placed back under drug pressure (V2B+blasticidin). The histogram at the right shows that reversion to drug sensitivity is not due to a switch in promoter activity. Parasites grown without blasticidin pressure (V2B−BSD) for three weeks continue to express <i>bsd</i> mRNA at levels equal to or greater than those grown continuously under blasticidin pressure (V2B+BSD). <i>bsd</i> mRNA levels are shown as copy number normalized to <i>seryl-tRNA synthetase</i>.</p

Translational repression in stably transformed parasites.

<p>Parasites were stably transfected with either pVLH or pV2BLH (A). V2BLH contains the <i>bsd</i> selectable marker and VLH contains <i>hdhfr</i> for maintaining the episomes in transfected parasites (not shown). Integration into the genome occurred spontaneously and was selected for by alternate growth with or without drug. <i>luciferase</i> mRNA levels were determined using Q-RT-PCR (B) with three different primer pairs (1–3), whose corresponding location on the <i>luciferase</i> ORF is shown in (A). Levels of protein expression were assayed by measuring luciferase activity (C).</p

Selection for reversal of translational repression by the uORF of <i>var2csa</i>.

<p>A. Constructs used for selection of parasites that translate the second open reading frame of the <i>var2csa</i> gene. The drug selectable marker <i>blasticidin-S-deaminase</i> (BSD) was used to select parasites translating the downstream cistron (equivalent to exon I of <i>var2csa</i>). All three constructs also contain the <i>hdhfr</i> selectable marker for maintaining the episomes in transfected parasites prior to blasticidin selection (not shown). B. A drug resistant population appeared after six days suggesting that a subset of parasites is capable of translating the second ORF. Replacing the uORF with <i>Renilla luciferase</i> (V2RB) led to severely retarded growth in presence of blasticidin. Analysis of episomes recovered from these parasites indicated that they had undergone recombination (not shown).</p

Changes in gene mRNA levels during erythrocytic development in two phenotypically distinct parasites-4

<p><b>Copyright information:</b></p><p>Taken from "Changes in gene mRNA levels during erythrocytic development in two phenotypically distinct parasites"</p><p>http://www.malariajournal.com/content/6/1/78</p><p>Malaria Journal 2007;6():78-78.</p><p>Published online 12 Jun 2007</p><p>PMCID:PMC1904452.</p><p></p>(A) and (B) and as pie charts in (C) at different time points during the intra-erythrocytic life cycle of NF54 and NF54VAR2CSA. (C) shows only expression of full-length genes. (A) and (B):blue – (was used for monitoring the different stages), green – PFL0935c (dominant transcript in NF54), grey – , red diamonds – (DBL4 primers), red circles – (DBL2 primers), red rectangles – (5' UTR primers). (C): light green – PFL0935c, red – (DBL2 primers). pi – post invasio

Changes in gene mRNA levels during erythrocytic development in two phenotypically distinct parasites-1

<p><b>Copyright information:</b></p><p>Taken from "Changes in gene mRNA levels during erythrocytic development in two phenotypically distinct parasites"</p><p>http://www.malariajournal.com/content/6/1/78</p><p>Malaria Journal 2007;6():78-78.</p><p>Published online 12 Jun 2007</p><p>PMCID:PMC1904452.</p><p></p>on of both populations corresponds to the expression of the most abundant transcript, PFL0935c and (PFL0030c), respectively. The most striking difference in transcription is the slightly earlier distinctive peak for (PFL0030c) in NF54VAR2CSA compared to the broader wave-like appearance of transcription in NF54. All measurements at the different time points were normalized against

Changes in gene mRNA levels during erythrocytic development in two phenotypically distinct parasites-3

<p><b>Copyright information:</b></p><p>Taken from "Changes in gene mRNA levels during erythrocytic development in two phenotypically distinct parasites"</p><p>http://www.malariajournal.com/content/6/1/78</p><p>Malaria Journal 2007;6():78-78.</p><p>Published online 12 Jun 2007</p><p>PMCID:PMC1904452.</p><p></p>SA, and the unselected control, NF54. The median of FITC fluorescence as measured by flow cytometry is shown for each serum sample. Pools of human sera from exposed females and males, and unexposed Danes (DK-pool) were used as primary antibodies. VAR2CSA expression was confirmed by staining with VAR2CSA DBL5ε-specific rabbit antibodies and irrelevant rabbit immune serum was used as negative control. Samples were processed and run simultaneously at individual time points

Changes in gene mRNA levels during erythrocytic development in two phenotypically distinct parasites-0

<p><b>Copyright information:</b></p><p>Taken from "Changes in gene mRNA levels during erythrocytic development in two phenotypically distinct parasites"</p><p>http://www.malariajournal.com/content/6/1/78</p><p>Malaria Journal 2007;6():78-78.</p><p>Published online 12 Jun 2007</p><p>PMCID:PMC1904452.</p><p></p>(A) and (B) and as pie charts in (C) at different time points during the intra-erythrocytic life cycle of NF54 and NF54VAR2CSA. (C) shows only expression of full-length genes. (A) and (B):blue – (was used for monitoring the different stages), green – PFL0935c (dominant transcript in NF54), grey – , red diamonds – (DBL4 primers), red circles – (DBL2 primers), red rectangles – (5' UTR primers). (C): light green – PFL0935c, red – (DBL2 primers). pi – post invasio