Schematic of the microarray experiment to determine half-lives through the life cycle

Four separate time course experiments were conducted at 12-hour intervals using a single source culture of synchronized parasites. Numbers in red represent the hour after invasion when actD was added in relationship to the previously published transcriptome experiment. Total RNA was subsequently harvested at the indicated time points. These samples were reverse transcribed into cDNA and hybridized to DNA microarrays. Specific spiked controls were included to determine correct normalization during microarray scanning.<p><b>Copyright information:</b></p><p>Taken from "Whole-genome analysis of mRNA decay in reveals a global lengthening of mRNA half-life during the intra-erythrocytic development cycle"</p><p>http://genomebiology.com/2007/8/7/R134</p><p>Genome Biology 2007;8(7):R134-R134.</p><p>Published online 7 Aug 2007</p><p>PMCID:PMC2323219.</p><p></p

Examples of mRNA decay profiles for each stage determined by microarray analysis

Four example genes were chosen to demonstrate the range of half-lives that can be measured in this experiment. The black dots represent data points from each of the microarray replicates for that time point, including the 0 time point with and without actinomycin D treatment. The colored lines represent the fitted decay curve. The half-life (t) for each example is listed.<p><b>Copyright information:</b></p><p>Taken from "Whole-genome analysis of mRNA decay in reveals a global lengthening of mRNA half-life during the intra-erythrocytic development cycle"</p><p>http://genomebiology.com/2007/8/7/R134</p><p>Genome Biology 2007;8(7):R134-R134.</p><p>Published online 7 Aug 2007</p><p>PMCID:PMC2323219.</p><p></p

Parasite Culturing and Data Characteristics of the P. falciparum IDC Transcriptome Analysis

<div><p>(A) Giemsa stains of the major morphological stages throughout the IDC are shown with the percent representation of ring-, trophozoite-, or schizont-stage parasites at every timepoint. The 2-h invasion window during the initiation of the bioreactor culture is indicated (gray area).</p> <p>(B–D) Example expression profiles for three genes, encoding EBA175, DHFR-TS, and ASL, are shown with a loess fit of the data (red line).</p> <p>(E) MAL6P1.147, the largest predicted ORF in the <i>Plasmodium</i> genome, is represented by 14 unique DNA oligonucleotide elements. The location of each of the oligonucleotide elements within the predicted ORF and the corresponding individual expression profiles are indicated (oligo 1–14). A red/green colorimetric representation of the gene expression ratios for each oligonucleotide is shown below the graph. The pairwise Pearson correlation for these expression profiles is 0.98 ± 0.02.</p> <p>(F) The percentage of the power in the maximum frequency of the FFT power spectrum was used as an indicator of periodicity. A histogram of these values reveals a strong bias toward single-frequency expression profiles, indicating that the majority of P. falciparum genes are regulated in a simple periodic manner. This bias is eliminated when the percent power was recalculated using random permutations of the same dataset (inset). For reference, the locations of EBA175 (peak B), DHFR-TS (peak C), and ASL (peak D) are shown.</p></div

The Centromeric Enhancer Is Active at an Ectopic Location

<div><p>The endogenous centromere on CHRIII was removed, and <i>CEN6</i> was inserted at an ectopic location (SGD coordinate approximately 260 kb), producing yeast strain PMY318, as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020260#s4" target="_blank">Materials and Methods</a>. Cells containing the ectopic centromere and isogenic wild-type cells (1829-15B) were staged in G1 using αF, and then released into fresh medium containing nocodazole to arrest cells in mitosis. Cells were then fixed in formaldehyde and processed for ChIP using epitope-tagged Mcd1-6HAp as a marker for the cohesin complex.</p> <p>(A) The Mcd1p binding profiles at the ectopic location on endogenous CHRIII (black squares) and in the presence of the ectopic centromere (gray circles) are shown. The location of the ectopic centromere is indicated by the black oval.</p> <p>(B) The levels of Mcd1p binding in the region flanking the ectopically placed centromere were divided by those observed in the isogenic wild-type control strain to determine the fold increases in Mcd1p binding in the presence of the centromere. Data are plotted as a function of the SGD coordinates for this region.</p></div

Coregulation of Gene Expression along the Chromosomes of P. falciparum Is Rare, While Plastid Gene Expression Is Highly Coordinated

<p>Expression profiles for oligonucleotides are shown as a function of location for Chromosome 2 ([A], Oligo Map). With the exception of the SERA locus (B), coregulated clusters of adjacent ORFs are seldom observed, indicating that expression phase is largely independent of chromosomal position. (C) In contrast to the nuclear chromosomes, the polycistronic expression of the circular plastid genome is reflected in the tight coregulation of gene expression. This is an expanded view of the plastid-encoded genes from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000005#pbio-0000005-g002" target="_blank">Figure 2</a>J. Genomic differences between strain 3D7, from which the complete genome was sequenced, and strain HB3 were measured by CGH. The relative hybridization between the gDNA derived from these two strains is shown as a percent reduction of the signal intensity for 3D7 ([A],<b> </b> CGH Data). Differences between the two strains are predominately located in the subtelomeric regions that contain the highly polymorphic <i>var, rifin,</i> and <i>stevor</i> gene families. Intrachromosomal variations, as observed for the <i>msp2</i> gene, were rare.</p

Mcd1p Binding within the Endogenous CHRIII Pericentric Region after Centromere Excision or Centromere Movement

<p>The Mcd1p binding profiles in the endogenous CHRIII pericentric region are shown in cells in which the centromere is absent, either because of centromere excision (gray circles, PMY185) or because of the movement of the centromere to an ectopic location on the right arm of CHRIII (black triangles, PMY318). PMY185 and PMY318 are highly related strains; PMY185 was one of the parental strains used to generate PMY318. In the centromere excision strain, Mcd1p binding was examined in the same cell cycle in which the centromere was lost, whereas in the ectopic centromere strain, Mcd1p association was determined many generations after centromere relocation (see text for further discussion). Mcd1p binding data from the centromere excision experiment are the same as those shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020260#pbio-0020260-g002" target="_blank">Figure 2</a>B, but are replotted here for clarity. <i>CEN3</i> normally occupies the interval between SGD coordinates 114382-114498.</p

A Functional Centromere–Kinetochore Complex Is Essential for Enhanced Pericentric Cohesin Association

<p>Cultures of isogenic wild-type (1846-15A) and <i>ndc10-42</i> mutant (1846-15C) cells were arrested in αF at 23 °C and then released into fresh medium containing nocodazole at 37 °C. After the cells arrested in mitosis (approximately 3 h), the cultures were crosslinked with formaldehyde and processed for ChIP using a monoclonal antiserum against epitope-tagged Mcd1p (Mcd1-6HAp) as an indicator of the cohesin complex. The cohesin association profiles in the pericentric regions of CHRIII (A) and CHRI (B) are shown for <i>NDC10</i> (black squares) and <i>ndc10-42</i> (gray circles) cultures. The positions of the centromeres are indicated by ovals (not drawn to scale). The dashed line in (B) indicates a region containing a Ty element. (C) To identify chromosomal regions depleted for cohesin binding in the absence of a functional kinetochore, the Mcd1p-ChIP-to-input fluorescence ratio obtained for each ORF and intergenic region in genomic microarray analyses of CHRV, CHRVI, and CHRIX in <i>ndc10-42</i> cells was divided by the ratio obtained for <i>NDC10</i> cells and plotted on a map of the chromosomes. Regions that demonstrated 2.5-fold or greater reduction in Mcd1p binding in the <i>ndc10-42</i> mutant are shaded dark green, while lighter green hues represent further fold reductions in Mcd1p binding. Regions where the magnitude of Mcd1p binding was similar in <i>NDC10</i> and <i>ndc10-42</i> cells are shown in gray. Gaps in the chromosomal maps are genomic regions not represented on the microarrays, while regions shaded blue were present on the arrays but gave no data during hybridizations for reasons described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020260#s4" target="_blank">Materials and Methods</a>. The location of the centromere on each chromosome is indicated by an asterisk.</p

Centromere Excision

<div><p>(A) <i>CEN1</i> on CHRI was replaced with a <i>CEN3-URA3</i> cassette flanked by head-to-tail-oriented site-specific recombination target sites (red arrows) for the R recombinase from <i>Zygosaccharomyces rouxii,</i> as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020260#s4" target="_blank">Materials and Methods</a>. This strain (1824-23B) contained the R recombinase under the control of a galactose-inducible promoter. Genomic DNA samples, taken prior to the addition of galactose to the culture medium (0) and at 0.5-h intervals for 4.5 h after the galactose addition, were digested to completion with PvuII (black arrows) and analyzed by Southern blot analysis using a 1.25-kb probe corresponding to CHRI SGD coordinates 151823 to 153080.</p> <p>(B) The percentage of centromere excision was determined for the timecourse shown in (A). Briefly, a phosphorimage of the Southern blot and ImageQuant software were used to determine the pixel intensities of the unexcised and excised bands (top and bottom bands, respectively). The percent excision was then calculated as the pixel intensity present in the excised band divided by the total pixel intensities of both bands at each timepoint.</p> <p>(C) A Southern blot analysis of centromere excision from CHRIII. The endogenous <i>CEN3</i> on CHRIII was replaced by R-recombinase target-site-flanked <i>CEN3</i> in strain 1829-15B, as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020260#s4" target="_blank">Materials and Methods</a>. The efficiency of centromere excision from CHRIII was determined by Southern blot analysis in two independent experiments using genomic DNA samples digested with SnaBI and a probe corresponding to CHRIII SGD coordinates 113799-114336. Lanes 1 and 3 represent uninduced controls, and lanes 2 and 4 represent the extent of centromere excision after 2 h of recombinase induction. The percent excision was determined as in (B).</p></div

Shearing of Centromere-Proximal Chromatin by Sonication

<p>As a control for the shearing of chromatin, a precipitation of chromatin was performed in each experiment using a polyclonal antiserum specific for the kinetochore protein Mif2p, which has been shown to interact with centromeric DNA (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020260#pbio-0020260-Meluh1" target="_blank">Meluh and Koshland 1997</a>). DNA crosslinked to Mif2 was then subjected to PCR analysis throughout a 2-kb region spanning <i>CEN3,</i> using a series of primer pairs that amplify 240 ± 21–bp fragments. <i>CEN3</i> DNA spans SGD coordinates 114382 to 114498, indicated with the ovals. Data were plotted on a scale similar to cohesin subunit ChIP data for comparison, and the inset shows in detail the magnitude of binding within a 2-kb centromere-flanking region.</p

Mcd1p Binding Profiles in Centromere-Proximal and -Distal Regions

<p>Cells containing Mcd1-6HAp were first staged in G1 using αF, and then released from G1 into medium containing nocodazole to arrest the cells in mitosis. For the centromere excision experiments (B–D), the cultures were divided in half after G1 arrest, and one half of each culture was treated with galactose for 2 h to induce centromere excision (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020260#s4" target="_blank">Materials and Methods</a>). Both the induced (acentric) and uninduced (centric) control cultures were then released from the G1 arrest into fresh medium and rearrested in mitosis. Once arrested in mitosis, cells were fixed in formaldehyde and then processed for ChIP using antiserum against epitope-tagged Mcd1p (Mcd1-6HAp) as an indicator of the cohesin complex. DNA isolated from the ChIPs and diluted input DNA not subject to immunoprecipitation were then subjected to PCR analysis using oligonucleotide primer pairs that amplify approximately 300-bp fragments within the indicated regions. Quantitation of DNA in the Mcd1p ChIPs, expressed as a percentage of the input DNA, is plotted as a function of the locations of the midpoints of those DNA fragments based on the SGD coordinates. Centromere position is indicated by an oval (not drawn to scale). (A) The Mcd1p association profile for the CHRI pericentric region in strain 1377A1-4B is shown. Mcd1p binding adjacent to <i>CEN1</i> is difficult to assess fully because of the presence of a moderately repetitive Ty element in the region from approximately 160 to 166 kb, indicated with the dashed line. Similarly, the Mcd1p binding profiles in the pericentric regions of CHRIII (B) and CHRXIV (C) are shown in the presence (black squares) and absence (gray circles) of <i>CEN3</i> and <i>CEN14</i> using strains PMY185 and PMY206, respectively. (D) The Mcd1p binding profiles for a centromere-distal region of CHRIII are shown for comparison in the presence (black squares) and absence (gray circles) of <i>CEN3</i>.</p