43 research outputs found
Co-localization of TF target genes positively affects co-regulation.
<p>(<b>a</b>). Comparison of expression profiles of genes showing both co-localization patterns and recognition patterns by a specific DLBCL annotated transcription factor (Status = 0) from those that although being co-localized were not recognized by the specified transcription factor (Status = 1). The comparison of the gene expression profiles between the two groups was done using the Mann-Whitney U test at a threshold of p<0.05 and results were illustrated as boxplot. Only TF co-localized target genes differing in their expression patterns with respect to those not recognized by the specific TFs have been detailed (Oct-1 and IK-1). (<b>b</b>). Both co-regulation by a common TF and co-localization patterns determine co-expression of TF target genes. The expression profiles of Oct-1 co-localized target genes (Status = 0) were compared to those that although recognized by Oct-1did not cluster together (Status = 1); notably Oct-1 co-localized target genes differed in their expression from those that did not cluster as shown by the Mann-Whitney U test at a threshold of p<0.05.</p
Analysis of co-operative regulatory effect of Oct-1, c-REL, NF-KappaB, IK-1, BSAP and CP2 in shared DLBCL genomic sites.
<p>Co-operative regulatory regions in target genes were identified using the functional distance metric weighted by local density and TF similarity. The panels refer to an investigation aimed at identifying TFs appearing more often together in clusters. The values in the contingency tables (right panels) are the frequencies of clusters featuring either both TFs of a given pair (true), or only one of them (false). The tables contain also the p-values of the Fisher’s exact test, indicating whether there is some statistically significant difference between the co-occurrences of TF pairs within clusters. Panels refer specifically to the (a). Oxphos; (b). BRC and (c). HR subsets. Only statistically significant results are shown. These results emphasize the role of c-REL, CP2 and Oct-1 association in shared diffuse large B-cell lymphoma regulatory regions.</p
Percentage of of co-localized positive annotated B-cell transcription factors.
<p>Percentage of Oct-1, NF-KAPPA B, IK-1, c-REL, BSAP and CP2 target genes found in clusters obtained with the functional distance metric weighted by local gene density and TF similarity and with the simple positional distance metric, out of the total amount of recognized TF target genes.</p
Different clustering behaviours of the DLBCL subsets subjected to sequential clustering and different distance metrics.
<p>(<b>a</b>). The one sample t-test was used to validate the statistical significance of the number of clusters found in the test datasets, vs. number of clusters found in random datasets. (<b>b</b>). % target genes found in clusters with: simple positional distance metric, functional with local density and functional with local density and TF-similarity. (<b>c</b>) A histogram showing the frequency distribution of clusters is shown.</p
Characterization of the immune/inflammatory response genes of the HR subset.
<p>(<b>a</b>). Total and average number of TFBSs present in the promoters of genes expressed in the HR subset. Frequencies were calculated on the genes clustered using the functional distance metric weighted by local density and TF similarity. In bold are shown TFs (Oct-1 and NF-KAPPA B) with an average number value exceeding 2 sites/promoter. (<b>b</b>). Analysis of localization within clusters of HR genes having the signature of immune/inflammatory response. The <i>Show dataset into clusters</i> utility of CluGene (top panel), allows to identify which clusters (red) out of the total (blue) contain a specified list of genes (immune annotated genes). The list of genes contained in clusters and their location is given on the right part of the panel. The tables (bottom) include the identified clustered immunity genes (bold), their function and the size and what other genes are grouped within the same cluster.</p
Schematic representation and alignment of wild type and transgenic <i>P.berghei</i> circumsporozoite proteins.
<p>(A) Schematic representation of the CS proteins present in each of the four transgenic <i>P. berghei</i> parasite lines generated. The corresponding names of the transgenic parasite lines are shown on the right. PbCS<sup>DHFR</sup> parasites carry the wildtype PbCS coding sequence (white boxes, RI and RII indicated with light and dark green respectively) and, similar to all transgenic lines, contain the <i>T. gondii</i> DHFR drug selectable marker inserted in the CS locus. PgCS<sup>SX</sup> parasites carry the full PgCS coding sequence (grey boxes, RI and RII indicated with light and dark blue respectively), but with the <i>Spe</i>I (S) and <i>Xho</i>I (X) restriction endonuclease sites inserted on either side of the repeat region. PgCS/Pb<sup>RR</sup> parasites contain the PgCS N-terminal and C-terminal regions (grey boxes) and the PbCS repeat region (white box). PbCS/Pg<sup>CT</sup> parasites carry the PbCS N-terminal and repeat regions (white boxes) and the PgCS C-terminal region (grey box). (B) Alignment of the wild type PbCS and transgenic PgCS<sup>SX</sup> and PgCS/Pb<sup>RR</sup> amino acid sequences. Shaded boxes represent areas of amino acid identity. Regions I and II (black lines), the repeat region (blue line) and the <i>Spe</i>I and <i>Xho</i>I restriction sites (red lines) are labelled. The <i>Spe</i>I and <i>Xho</i>I sites were introduced into the PgCS sequence to mediate exchange of the PgCS repeat region with the PbCS repeat region.</p
Generation and southern blot analysis of transgenic PgCS/Pb<sup>RR</sup> parasite lines.
<p>(<b>A</b>) Schematic representation of (i) the PgCS/Pb<sup>RR</sup> targeting construct, (ii) the wt <i>PbCS</i> locus and (iii) the targeted locus after recombination between the <i>PbCS</i> 5′UTR and 3′UTR sequences. The vertically dashed box indicates the 1.13 kb 5′UTR sequence used in the construct and the horizontally dashed boxes indicate the 0.3 kb and 0.85 kb 3′UTR sequences between which the TgDHFR-TS selectable marker cassette (light grey) was inserted in the construct. The PgCS/Pb<sup>RR</sup> chimeric gene contains the <i>PgCS</i> N- and C-terminal regions (dark grey boxes) and the <i>PbCS</i> repeat region (white box) flanked by the <i>Spe</i>I (<i>S</i>) and <i>Xho</i>I (<i>X</i>) sites. In the wt PbCS locus the white box indicates the full <i>PbCS</i> gene. Thick black lines indicate the probes used in southern blots. E: <i>EcoR</i>V site. (<b>B</b>) Southern blot of <i>EcoR</i>V/<i>Spe</i>I (E/S) and <i>EcoR</i>V/<i>Xho</i>I (E/X) digested genomic DNA from Pbwt parasites and transgenic PgCS/Pb<sup>RR</sup> parasites (clones 4 and 5), hybridised with the <i>PbCS</i> 3′UTR probe. Two bands of 4.0 and 1.5 kb were present in Pbwt DNA in both digestions. E/S digested DNA from the PgCS/Pb<sup>RR</sup> clones revealed two bands of 1.1 and 2.0 kb, while E/X digested DNA revealed two bands of 0.6 and 2.0 kb, demonstrating the replacement of the endogenous <i>PbCS</i> gene with the targeted construct. (<b>C</b>) The same membrane was then hybridised with the <i>PgCS</i> N-terminal probe (PgCS-Nt), encompassing the entire N-terminal region of <i>PgCS</i>. No band was present in Pbwt DNA. However, a band of 3.1 kb or 3.6 kb was present in the E/S or E/X digested transgenic DNA, respectively.</p
Infectivity of midgut sporozoites for the vertebrate host.
a<p>Pre-patent period is the number of days between injection and first appearance of the parasites in the peripheral blood.</p><p>C57BL/6 mice were injected intravenously with Pbwt or transgenic midgut sporozoites collected on day 21 p.i.. The number of mice that became infected and the pre-patent period were both recorded.</p
Transgenic parasite development in the <i>Anopheles stephensi</i> mosquito vector.
a<p>Significant difference compared to Pbwt and PbCS<sup>DHFR</sup> transgenic parasites; p<0.01.</p>b<p>Significant difference compared to PgCS<sup>SX</sup>; p<0.03.</p><p><i>A. stephensi</i> mosquitoes were fed on mice infected with one of the transgenic clones or Pbwt parasites. Values represent the mean ± S.D. of at least three independent experiments, each with a minimum of 50 mosquitoes.</p
Western blot analysis of CSP expression and processing in transgenic midgut sporozoites 18 days post-infection.
<p>CSP expression was revealed by incubation with either a monoclonal antibody directed against the PbCSP repeat region (PbCS-RR) or a serum directed against the PgCSP N-terminal region (PgCS-Nt). As a control, midgut lysates from uninfected mosquitoes were also analysed. Antibody against the PbCSP repeat region reveals a higher molecular weight precursor polypeptide and a lower molecular weight processed polypeptide. A ladder of degradation products is also visible. Antibody against the PgCSP N-terminal region reveals only the higher molecular weight protein.</p