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One way to test whether gene expression differences between species accumulate at a rate consistent with neutral expectation is to compare them to the expression differences observed for a class of genes that can reasonably be expected to not be the direct targets of positive or negative selection. Since expressed pseudogenes do not produce any functional gene products, they can be viewed as such a class of genes. Thus, if a substantial proportion of intact genes accumulate expression differences faster than pseudogenes, this would indicate that they are positively selected. Such an observation would falsify a neutral model. | 15138501_p9 | 15138501 | A Test for Neutrality | 4.184958 | biomedical | Study | [
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To test this, we considered the expression patterns in four regions of the brain in three humans and three chimpanzees using the Affymetrix U95 array set interrogating approximately 40,000 genes (Philipp Khaitovich, unpublished data). In order to identify all probe sets on these arrays that interrogate expressed pseudogenes, we aligned the probe sequences, as well as published lists of human pseudogenes, to the human genome ( see Materials and Methods ). In total, 889 probe sets that overlap with pseudogenes were identified. Thirty-three of these were detected (detection p -value < 0.05) in at least one of four brain regions in either the chimpanzees or the humans after masking all probes carrying DNA sequence differences between the species. Of these, 28 contained at least one mutation that leads to a loss of function in both humans and chimpanzees. We therefore assumed that these pseudogenes were nonfunctional in the common ancestor of humans and chimpanzees. Finally, we checked whether these probe sets may crosshybridize with any intact genes by aligning them to the human genome. This left us with 23 expressed pseudogenes. | 15138501_p10 | 15138501 | A Test for Neutrality | 4.17389 | biomedical | Study | [
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We compared the distributions of the squared differences between the mean expression levels of each gene in humans and in chimpanzees for the 23 pseudogenes and 12,647 intact genes for each of the four brain regions. In each case, only the genes detected in a given brain region were used for the calculation. In all four brain regions the distribution of expression distances among intact genes did not differ significantly from that among pseudogenes in either a Kolmogorov-Smirnov test or a Wilcoxon rank sum test. These tests would have been significant if more than 5% (1/23) of the genes had a distribution radically different from that of the pseudogenes. When the data for four brain regions were combined, no visual difference between the two distributions was apparent ( p = 0.16 and p = 0.69, respectively) . | 15138501_p11 | 15138501 | A Test for Neutrality | 4.11399 | biomedical | Study | [
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Thus, we failed to detect any significant excess of intact genes that diverged faster in expression than pseudogenes. This indicates that the fraction of gene expression differences between the species that are fixed by positive selection is small. Interestingly, there was also no detectable excess of intact genes that diverged slower than pseudogenes. This may seem unexpected, since the expression of many intact genes might be thought to be stabilized by negative selection and therefore to change more slowly than pseudogenes. This may indicate that purifying selection as well is a weak force affecting gene expression. However, it should be noted that the small number of expressed pseudogenes analyzed limits the power to detect positive and negative selection. A targeted effort to study expressed pseudogenes in closely related species would be a worthwhile undertaking. | 15138501_p12 | 15138501 | A Test for Neutrality | 4.095333 | biomedical | Study | [
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The fact that the overall accumulation of expression differences conforms to a selectively neutral model does not mean, of course, that all expression differences between species are selectively neutral. As for nucleotide changes, some changes in gene expression will have had phenotypic consequences and some of these will have become fixed due to positive selection. To identify such gene expression differences, we propose to use the ratio of divergence between species to diversity within species, akin to the tests suggested for quantitative genetic traits and in agreement with recent suggestions by Rifkin et al. or Hsieh et al. . However, to do this it is necessary for each gene considered to distinguish the gene expression diversity caused by genetic differences between individuals from the diversity caused by environmental factors. This is crucial since the environmental component is likely to be much larger than the genetic component. For example, under strict neutrality and no environmental influence, we expect a divergence to diversity ratio that is equal to the ratio of time of divergence of the species to the average time to the common ancestors of the individuals sampled within a species. This would be about 1:10 for humans and chimpanzees . However, the observed ratio is approximately 1:3, suggesting that the environmental component is on the order of three times bigger than the genetic component. Studies of gene expression differences among individuals with different genetic relatedness will eventually allow an estimation of the genetic component of expression variation. | 15138501_p13 | 15138501 | A Test for Positive Selection | 4.325393 | biomedical | Study | [
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Since we are unable to tease apart genetic and environmental contributions to expression diversity, we instead used pseudogenes to estimate the distribution of divergence to diversity ratios observed in the absence of selection and compared these ratios to intact genes. No significant difference was found (Kolmogorov-Smirnov test, p = 0.388; Wilcoxon rank sum test, p = 0.134), and both distributions appeared to center around roughly the same values . Note that this observation has to be taken cautiously since it is based on a small number of pseudogenes and the gene expression diversity is calculated from only three human individuals. Nevertheless, this result indicates that there is no drastic difference between the expression patterns of intact genes and expressed pseudogenes, since our tests would have been significant if 5% or more of the genes had had a radically different divergence to diversity ratio than that observed among the pseudogenes. | 15138501_p14 | 15138501 | A Test for Positive Selection | 4.10301 | biomedical | Study | [
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Different anatomical brain structures appeared at different times during vertebrate evolution. These time points can be viewed as divergence times between brain regions extending millions of years back in the past . If gene expression changes between different brain regions have a large random component, gene expression differences between brain regions within species could potentially be used as a molecular clock to time the divergences of tissues. To investigate whether this may be the case, we compared expression patterns for Brodmann's area 44, the prefrontal cortex, the anterior cingulate cortex, the primary visual cortex, the caudate nucleus, and the cerebellum in three adult human and three adult chimpanzee males (Philipp Khaitovich, unpublished data). All comparisons were performed between brain regions within the same individual. This has two advantages. First, such comparisons are unaffected by nucleotide sequence variation between and within species. Second, environmental differences and post mortem changes have little effect when expression differences within one individual are studied. In Figure 5 B, we plot the average squared distances between the six brain regions in humans and chimpanzees against the time when these brain regions emerged during vertebrate evolution for 2,297 and 2,525 genes detected in all human and all chimpanzee samples, respectively. It can be seen that the expression differences increase approximately linearly with time over more than half a billion years. To investigate if this finding holds also in another mammalian species, we used published expression data for 1,346 genes with detectable expression in eight brain regions in the mouse . In this case as well there is an approximately linear relationship between transcriptome differences and evolutionary divergence times . | 15138501_p15 | 15138501 | Transcriptome Evolution among Brain Regions | 4.226161 | biomedical | Study | [
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If gene expression differences between the brain regions were largely adaptive, one would expect them to correlate with tissue function and not with evolutionary divergence time. Our data show that tissues that diverged recently have very similar gene expression profiles irrespective of the differences in function. For instance, the transcriptome of Brodmann's area 44 in the left hemisphere (Broca's area) is very similar to that of the prefrontal cortex in both humans and chimpanzees, although it is known to be involved in speech processing in humans while it must have another function in chimpanzees . This is what we would expect if the time since divergence rather than the extent of functional differences determined the magnitude of transcriptome change. Thus, although a number of expression differences between brain regions surely correspond to functional differences, our findings suggest that a sizeable proportion of the differences are functionally neutral. | 15138501_p16 | 15138501 | Transcriptome Evolution among Brain Regions | 4.182488 | biomedical | Study | [
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A noteworthy finding is that the accumulation of expression differences between brain regions within a species is much slower than the accumulation of expression differences within a brain region between species. In fact, the expression differences that have accumulated among the primate species over 20 million years are approximately as extensive as those that have accumulated among brain regions over 500 million years . This is likely to result from the fact that all expression differences seen between brain regions within an individual are caused by changes in regulatory networks established during development by cells that carry the same genome. In addition, expression differences between brain regions reflect the different cell-type compositions of these regions. In contrast, transcriptome differences between species are the result of changes in regulatory networks and cellular composition of tissues, as well as nucleotide sequence differences between species that affect promoters and other genomic elements that determine transcript levels. Our results show that the latter type of changes are much more common than the former. | 15138501_p17 | 15138501 | Transcriptome Evolution among Brain Regions | 4.245424 | biomedical | Study | [
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A possible alternative explanation for the correlation between differences in gene expression and evolutionary divergence time among brain regions could be that differences in gene expression do not correlate with evolutionary divergence time, but instead with divergence time during fetal development. Our observations would then result from the fact that both developmental divergence times and expression differences correlate with evolutionary divergence. A correlation between developmental and evolutionary divergence times has been hypothesized before . In fact, gene expression analyses now provide a quantitative approach to address this question and may also provide a tool to date the evolutionary emergence of brain regions that cannot be discerned in the fossil record. | 15138501_p18 | 15138501 | Transcriptome Evolution among Brain Regions | 4.200582 | biomedical | Study | [
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We show that a neutral model of evolution can predict the main features of transcriptome evolution in the brains of primates and mice. A neutral model is also in agreement with published observations in Drosophila and fish . Although selective scenarios that explain some or even most of these observations can be found, the combined evidence presented leads us to conclude that a neutral model is the most adequate null model for transcriptome evolution. This suggests that the majority of gene expression differences within and between species are not functional adaptations, but selectively neutral or nearly neutral. The main challenge now is to develop a mathematical model of transcriptome evolution that allows quantitative predictions of transcriptome changes. Such a model, combined with experimental data estimating the normal variation of gene expression within a species and the relative contributions of genetic and environmental factors to this variation, should allow adaptive gene expression changes to be identified. Further work is also needed to reveal whether proteome evolution is also dominated by changes that are largely selectively neutral. | 15138501_p19 | 15138501 | Conclusions | 4.247097 | biomedical | Study | [
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Finally, the finding that gene expression differences can be used as a molecular clock to date tissue divergences opens the prospect of reconstructing the evolutionary history of organs and tissues based on gene expression measurements in a single species. | 15138501_p20 | 15138501 | Conclusions | 3.890893 | biomedical | Study | [
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For the primate samples, approximately 200 mg of gray matter was collected from post mortem brain samples from prefrontal cortex region corresponding to Brodmann's area 9 in the left hemisphere from six male humans who were 45, 45, 63, 65, 70, and 70 years old; five male chimpanzees that were 7, 12, 12, 12, and approximately 40 years old; one 16-year-old male orangutan; five approximately 10-year-old male rhesus macaques; and five approximately 15-year-old male crab-eating macaques. All individuals had no history of brain-related diseases and suffered sudden deaths without associated brain damage. For the mouse samples, approximately 50 mg of gray matter was collected from the frontal cortex regions of six M. musculus , three M. spretus, and one M. caroli individuals. All mice were outbred, older than 14 weeks, and healthy. Total RNA was isolated using the TRIzol reagent (GIBCO, San Diego, California, United States) according to manufacturer's instructions and purified with Quiagen RNeasy kit (Quiagen, Valencia, California, United States) following the “RNA cleanup” protocol. RNAs were of high and comparable quality as gauged by the ratio of 28S to 18S ribosomal RNAs visualized on agarose gels and by the signal ratios between the probes for the 3′ and 5′ ends of the mRNAs of GAPDH and β-actin genes used as quality controls on Affymetrix microarrays (Affymetrix, Santa Clara, California, United States). | 15138501_p21 | 15138501 | Tissue samples and microarray data collection | 4.145786 | biomedical | Study | [
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For Affymetrix microarrays, labeling of 5 μg of the RNA, hybridization, staining, washing steps, and array scanning were carried out following Affymetrix protocols. Expression data were collected using Affymetrix HG U95Av2 arrays for the primate samples and Affymetrix MG U74Av2 arrays for the mice samples. The Affymetrix CEL files containing expression data for the different regions of the mouse brain, including amygdala, cerebral cortex, hippocampus, hypothalamus, cerebellum, olfactory bulb, and two regions of spinal cord were provided by John Hogenesch. | 15138501_p22 | 15138501 | Tissue samples and microarray data collection | 3.998848 | biomedical | Study | [
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Arrays containing 51,000 cDNAs corresponding to approximately 40,000 UniGene clusters were manufactured in the laboratory of W.A. as described elsewhere . Labeling, hybridization, staining, washing, and array scanning were carried out as described by Cortes-Canteli et al. with slight modifications. All samples were hybridized twice with dye reversal, using a mixture of all samples as a common reference. All primary expression data were submitted to the Array Express database ( http://www.ebi.ac.uk/arrayexpress/ ). | 15138501_p23 | 15138501 | Tissue samples and microarray data collection | 4.0308 | biomedical | Study | [
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In order to exclude all oligonucleotide probes that did not match perfectly between humans and chimpanzees, we aligned all Affymetrix target sequences ( http://www.affymetrix.com/analysis/index.affx ) first to the human genome (build 33) and then to a draft version of the chimpanzee genome . Using BLAT , we matched chimpanzee sequences with Affymetrix target sequences containing the 16 oligonucleotide probes and determined the best hit using a scoring function. The chimpanzee sequence was then aligned to the human genome to determine whether the best match coincided with the match obtained from alignment of Affymetrix target sequences with the human genome. To identify insertion and deletions (indels), we compared the alignment of the Affymetrix target sequence to the human genome and to the chimpanzee genome, and differences in the indel structure relative to the target sequence were identified as indels. We then identified all oligonucleotide probes within target sequences that matched the chimpanzee sequence perfectly. These probes were used for the analysis while the rest of the probes were masked. | 15138501_p24 | 15138501 | Masking of sequence differences between humans and chimpanzees | 4.138158 | biomedical | Study | [
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Affymetrix microarray image data were analyzed with Affymetrix Microarray Suite v5.0 using default parameters. Arrays were scaled to the same average intensity using all probes on the array. Detected genes were defined as those with a detection p -value less than or equal to 0.05. For calculation of the expression values, data were processed with the Bioconductor “affy” software package using the quantile normalization procedure . cDNA arrays were analyzed using the TM4 software package . Detected genes were defined as those with a spot intensity exceeding the background intensity by more than 2-fold. All slides were normalized to the common reference using the LOWESS normalization algorithm. For calculation of diversity and divergence, signal to reference ratio measurements were transformed into standardized signal intensities by multiplying them by the average reference intensity for each gene. Divergence was defined as the squared difference between the mean expression of two groups of samples averaged over (all detected) genes. Diversity was defined as the expression variance within a group of samples. | 15138501_p25 | 15138501 | Microarray data analysis | 4.208042 | biomedical | Study | [
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We measured the divergence between human and chimpanzee by looking at the squared difference between the mean expression values in humans and chimpanzees. This estimate of divergence includes the errors in our estimates of the two means, which is proportional to the variance in each of the species, and thus to the diversity in each species. Therefore, even if no correlation between divergence and diversity existed, our measured divergence and diversity estimates would correlate, and the smaller the divergence is relative to diversity, the stronger the correlation would be. To estimate if the observed correlation is larger than that expected from this effect alone, we performed a randomization test, in which we computed how much correlation between diversity and divergence would be generated from the above effect even if no correlation between diversity and divergence exists. To be conservative, we first generated a distribution that deliberately underestimated the real divergence between humans and chimpanzees. This was done by first generating a distribution of the expected observed differences (X) in gene expression between humans and chimpanzees if the real divergence is zero. Then using this distribution and the observed distribution of differences (Z), we generated a distribution (Y) that-added to values from X-would give Z. In order to underestimate the divergence, we generated Y assuming that the correlation of X and Y is one. We then generated random samples in the following way: For each gene (g), we chose a random difference of expression (d) from our generated distribution. We then drew six samples from a normal distribution whose mean is zero and whose variance is the diversity in humans for gene g, and three samples from a normal distribution whose mean is d and whose variance is the diversity of chimps for gene g. For these expression values we then calculated the correlation between diversity and divergence. We repeated the whole procedure 1,000 times. None of these randomizations generated a correlation that is as strong as the observed one. To make sure that the whole test is conservative, we generated 100 datasets of three types, all of which had a similar diversity, but had a “real” divergence distribution of (1) zero, (2) the underestimated divergence, or (3) the measured divergence, and had uncorrelated diversity and divergence. We then performed the whole test described above, doing just one randomization test. If the test was not conservative, one would expect the correlation in the dataset to be higher than the correlation after randomization in 50% of the cases. Instead, the correlation after randomization was higher in 98, 98, and 99 cases respectively-showing that our test is indeed conservative. | 15138501_p26 | 15138501 | Correlation significance test | 4.253573 | biomedical | Study | [
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We retrieved sequences of all pseudogenes as determined by Torrents et al. , Zhang et al. , and the VEGA project ( http://vega.sanger.ac.uk ). These sequences, as well as the Affymetrix target sequences, were mapped to the human genome (build 34) using BLAT , and the best hit was determined using the following parameters: match, +1; mismatch, −3; gap-opening penalty only for gaps ≤ 20, −5; and gap extension, −1. Next, using BLAT, we determined the Affymetrix target sequences where the best-matching sequence did not overlap with the genomic region of a known gene ( http://genome.ucsc.edu ). Thus, we identified 889 probe sets that overlapped with a pseudogene, but not with a known gene. Combined with gene expression data collected in four brain regions (anterior cingulate cortex, Broca'a area, caudate nucleus, cerebellum; Philipp Khaitovich, unpublished data) in three humans and three chimpanzees, 33 of these probe sets had detectable expression levels in at least one brain region in either three chimpanzees or three humans. For these probe sets, we checked whether at least one of the identified interruptions of the human pseudogene was also present in the chimpanzee, indicating that the pseudogene was already nonfunctional at the time of the chimpanzee–human divergence. This left us with 28 probe sets that were checked for crosshybridization with other genes by aligning oligonucleotide probes from these probe sets to the human genome. Finally, we were left with 23 expressed pseudogenes that did not match perfectly to any other gene by more than seven out of 16 probes in the probe set. | 15138501_p27 | 15138501 | Expressed pseudogenes | 4.211842 | biomedical | Study | [
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Biochemical studies performed in higher eukaryotes have shown that DNA replication initiates at specific sites, or within initiation zones, suggesting the involvement of particular DNA sequences called replicators . In contrast, functional studies, as well as studies of DNA replication performed in early embryos of various vertebrates and invertebrates, have suggested that initiation of DNA replication can take place with limited sequence specificity . | 15208711_p0 | 15208711 | Introduction | 4.18052 | biomedical | Study | [
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The presence of specific initiation sites and of initiation zones has also been proposed to explain the latent replication of the Epstein-Barr virus (EBV) genome in human cell lines. During latent replication, the EBV genome is maintained as a circular episome (∼175 kb in size), and the host cell provides both the replication machinery and the licensing apparatus that limit the genome's duplication to once per cell cycle . Initiation site oriP was the first initiation site identified in the EBV genome. In the presence of the viral protein EBNA1, this DNA sequence confers autonomous replication to plasmids transfected into human cell lines . In addition, initiation of DNA replication at oriP was recently shown to be regulated by geminin, and to correlate with the binding of various cellular components of the replication complex (Orc1, Orc2, Orc3, Orc4, Orc6, Mcm2, Mcm3, and Mcm7) . These and other reports have been interpreted as evidence that oriP contains a replicator . However, other initiation sites have also been described , and a study performed by two-dimensional (2D) gel electrophoresis at neutral pH has suggested the presence of a large initiation zone . In addition, reports from different laboratories have shown that various portions of the EBV genome, including oriP, can be deleted without affecting the maintenance of the episomes in replicating cells (see Discussion and references therein). Therefore, the presence of specific replicator sequences and their relationship with the sites of initiation of DNA replication also remain to be demonstrated in this system. | 15208711_p1 | 15208711 | Introduction | 4.681689 | biomedical | Study | [
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We recently began to study the replication of individual EBV episomes using fluorescence microscopy . In a previous study, we collected various images of the Raji EBV genome . The analysis of those molecules demonstrated that the duplication of different EBV episomes begins at different initiation sites located within the initiation zone identified by 2D gel electrophoresis. However, the number of molecules analyzed was not sufficient to infer the precise dynamics of activation of the initiation sites (i.e., to detect events having a short life or occurring infrequently during the duplication of the episomes). | 15208711_p2 | 15208711 | Introduction | 4.061748 | biomedical | Study | [
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In the present study, we performed an extensive analysis of the replication dynamics of the EBV genome in two human Burkitt's lymphoma cell lines (Raji and Mutu I). By utilizing a different procedure to stretch DNA molecules we were able to collect a large number of images of the EBV genome representative of different stages of duplication. This allowed us to determine how DNA replication initiates, progresses, and terminates throughout the EBV genome and to precisely measure the duplication time of specific portions of the EBV genome. | 15208711_p3 | 15208711 | Introduction | 4.139605 | biomedical | Study | [
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These improvements allowed us to obtain important new results as well as to extend previous observations. Here we show that initiation events are not limited to a specific portion of the EBV genome (namely the initiation zone detected by 2D gel electrophoresis), but, unexpectedly, take place throughout the EBV genome. Multiple initiation events were also detected in individual EBV episomes. Hence, if the initiation sites do correspond to replicators, the latter must necessarily be highly redundant (present at a frequency of one or more every 20 kb). | 15208711_p4 | 15208711 | Introduction | 4.096342 | biomedical | Study | [
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Our new results also indicate that, in these two EBV strains, both the frequency and the order of activation of the initiation sites vary considerably throughout the viral genome. This variation involves initiation sites such as oriP, the sequence of which is highly conserved in the two EBV strains . Hence, the utilization of the initiation sites is largely independent of their DNA sequence, and it is affected by the genomic context (i.e., the presence/absence of initiation sites activated earlier or the presence of transcription). Finally, we noticed that the initiation sites that tend to be activated earlier during the duplication of each episome are located in clusters, each of which spans several kilobases. The locations of these clusters are different in the Raji and Mutu I strains. Therefore, the utilization of the initiation sites (particularly their order of activation) appears to be regulated at the level of genomic regions rather than at the level of individual initiation sites. | 15208711_p5 | 15208711 | Introduction | 4.21562 | biomedical | Study | [
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] | [
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In order to study DNA replication, we used a procedure that we call single molecule analysis of replicated DNA (SMARD). This procedure labels the replicating DNA in a way that allows us to determine the position, the direction, and the density of the replication forks in a steady-state population of replicating molecules (in this case, EBV episomes). This in turn allows us to determine how DNA replication initiates, progresses, and terminates throughout the genomic region analyzed. SMARD presents several advantages over procedures that utilize different labeling schemes and allows us to overcome most of the limiting factors that have traditionally affected studies of replication performed on DNA fibers (see Materials and Methods ). | 15208711_p6 | 15208711 | Fluorescent Hybridization Immunostaining of Individual EBV Episomes Stretched on Microscope Slides | 4.1419 | biomedical | Study | [
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] | [
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] | en | 0.999998 |
In our procedure, an asynchronous population of cells is sequentially labeled with 5′-iodo-2′-deoxyuridine (IdU) and 5′-chloro-2′-deoxyuridine (CldU) . The length of each labeling period is longer than the time required to completely replicate the EBV genome (3.5–4 h; see Materials and Methods ). This allows some of the replicating EBV episomes to become substituted with the halogenated nucleotides along their entire length . The incorporation of these nucleotide analogs is later detected by immunofluorescence of individual DNA molecules stretched on microscope slides. In these molecules, the transitions from IdU to CldU mark the positions of the replication forks at the time of the switch from the first to the second labeling period (see below). Hence, the results of this analysis are presented as a series of images of EBV episomes representative of the different stages of duplication that were present at the end of the first labeling period. In addition, the use of long labeling periods makes the data collected by SMARD suitable for quantitative analysis, allowing us to calculate the duplication time of different genomic regions. | 15208711_p7 | 15208711 | Fluorescent Hybridization Immunostaining of Individual EBV Episomes Stretched on Microscope Slides | 4.166544 | biomedical | Study | [
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] | [
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0.000377628137357533,
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0.00006895251863170415
] | en | 0.999999 |
In the experiments described in this study, agarose-embedded total DNA was prepared from cells labeled with halogenated nucleotides. The circular EBV episomes were converted to linear molecules by digestion with a restriction endonuclease (PacI or SwaI). After pulsed field gel electrophoresis, the EBV DNA was recovered by agarase treatment and stretched on microscope slides by capillary action (see Materials and Methods ). Using this procedure we obtained relatively high numbers of stretched molecules even when the starting amount of purified DNA was very small. The hybridization of specific biotinylated probes was used to identify the EBV molecules and their orientation . In addition, the halogenated nucleotides were visualized using specific monoclonal antibodies and secondary antibodies conjugated with Alexa Fluor 568 and Alexa Fluor 488 . The detection procedure and the analysis of the images are described in Materials and Methods and in Figures 1 and 2 . The use of long labeling periods, and the analysis of molecules substituted with the halogenated nucleotides along their entire length, present several advantages. In particular this procedure provides multiple internal controls that could not have been performed if short labeling times had been used (see Materials and Methods ). | 15208711_p8 | 15208711 | Fluorescent Hybridization Immunostaining of Individual EBV Episomes Stretched on Microscope Slides | 4.122641 | biomedical | Study | [
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] | [
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0.00030931824585422873,
0.00005838099241373129
] | en | 0.999998 |
In order to define precisely how the Raji EBV genome replicates, we recovered the images of 245 PacI-linearized EBV episomes that incorporated halogenated nucleotides along their entire length (112 fully stained in red, 84 fully stained in green, and 49 stained in both red and green). The results of this experiment are shown in Figure 3 . In the episomes that incorporated both kinds of halogenated nucleotides, the red to green transitions define the position of the replication forks at the time of the switch from the first to the second labeling period. The red portions of these molecules are nested around the ends of the PacI-linearized episomes . This indicates that DNA replication proceeded in a similar manner in most of episomes. | 15208711_p9 | 15208711 | The Raji EBV Genome Contains a Region That Usually Replicates First and a Region That Usually Replicates Last | 4.134166 | biomedical | Study | [
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] | [
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0.000282646797131747,
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] | en | 0.999997 |
However, the progression of DNA replication throughout the EBV genome is better described by the replication profile of the molecules analyzed . This profile was obtained by dividing the map of the episomes into intervals of 5 kb (horizontal axis) and then indicating the percentage of molecules stained in red within each interval (vertical axis). From this profile we can easily identify a genomic region that usually replicates first (RRF; more frequently stained in red), a genomic region that usually replicates last (RRL; less frequently stained in red), and two transition regions. | 15208711_p10 | 15208711 | The Raji EBV Genome Contains a Region That Usually Replicates First and a Region That Usually Replicates Last | 4.12683 | biomedical | Study | [
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] | [
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The RRF contains the initiation sites most frequently utilized to begin the duplication of the Raji EBV episomes. More than 80% of the molecules analyzed were stained in red throughout intervals 1–7 and 31–35 . In the molecules representing early stages of episomal duplication , initiation events took place either within the RRF (molecules 2–21) or in adjacent portions of the EBV genome (i.e., molecule 1). Interestingly, low levels of replication bubbles had been previously detected by 2D gel electrophoresis within various restriction fragments located in the same portions of the EBV genome . Therefore, the initiation sites activated earlier during the duplication of each episome are located within what appears to be an initiation zone that spans several tens of kilobases . | 15208711_p11 | 15208711 | The Raji EBV Genome Contains a Region That Usually Replicates First and a Region That Usually Replicates Last | 4.258297 | biomedical | Study | [
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] | [
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] | en | 0.999998 |
The RRL appears in the replication profile of the Raji EBV episomes as a large valley . The bottom of the valley spans about 40 kb (intervals 11–18), and its flat appearance indicates that throughout this region the episomes terminate their duplication with similar probability. Note, however, that termination events can also occur within the transition regions . Interestingly, the level of transcription across the RRL is higher than in the rest of the Raji EBV genome, while across the RRF it is either very low or absent . | 15208711_p12 | 15208711 | The Raji EBV Genome Contains a Region That Usually Replicates First and a Region That Usually Replicates Last | 4.231851 | biomedical | Study | [
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] | [
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] | en | 0.999998 |
The presence of RRF and RRL was confirmed by a second SMARD experiment in which we digested the EBV episomes with SwaI. This enzyme cleaves twice in the viral genome, producing fragments of 105 and 70 kb. The larger fragment was expected to contain most of the RRF (now located near the center of DNA molecules), and a small portion of the RRL. We recovered 209 fully substituted 105-kb fragments (94 red, 79 green, and 36 red and green). These molecules were analyzed as described for the PacI-linearized EBV episomes . We found that both the RRF (intervals 1–13), and the RRL (intervals 17–21) encompass the same genomic sequences occupied in the previous SMARD experiment . Initiation events located within the RRF are visible in molecules 1–4 . We conclude that the results obtained by SMARD are reproducible and do not depend on the particular restriction enzyme used for digesting the DNA molecules. | 15208711_p13 | 15208711 | The Raji EBV Genome Contains a Region That Usually Replicates First and a Region That Usually Replicates Last | 4.174006 | biomedical | Study | [
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] | [
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0.0003034064138773829,
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] | en | 0.999998 |
The movement of the replication forks throughout the EBV genome is described by the profiles of replication fork abundance . These profiles were obtained by dividing the map of the EBV genome into intervals of 5 kb (horizontal axis) and then indicating the percentage of molecules containing red-to-green transitions within each interval (vertical axis). As seen earlier, these transitions indicate the position, and the direction, of the replication forks at the time of the switch from the first to the second labeling period. The significant accumulation of red-to-green transitions visible within interval 8 indicates that replication forks were not moving freely across this portion of the EBV genome. A similar accumulation is visible for the SwaI-digested molecules in the same portion of the EBV genome . This result was expected since four different pausing sites had been previously described within and near oriP . However, no other major accumulation of forks is visible. Therefore, replication forks move mostly unimpeded across the Raji EBV genome. | 15208711_p14 | 15208711 | Replication Forks Move Without Significant Pausing throughout the Raji EBV Genome with the Exception of the Genomic Region near oriP | 4.246817 | biomedical | Study | [
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] | [
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] | en | 0.999998 |
From the profiles of replication fork abundance we can also determine the prevalent direction of the replication forks throughout specific portions of the EBV genome. For example, throughout most of the RRL, replication forks move in both directions at similar frequencies . The bidirectional movement of the replication forks also characterizes the central portion of the RRF . However, this was not evident from the profile of replication fork abundance of the PacI-linearized EBV episomes because the extremities of the molecules can be distorted or not fully stretched . As a consequence, in the PacI experiment, the position of the replication forks could not always be observed properly within the RRF. From this we conclude that within the central portions of RRF and RRL, replication forks move in both directions with a similar probability. | 15208711_p15 | 15208711 | Replication Forks Move Without Significant Pausing throughout the Raji EBV Genome with the Exception of the Genomic Region near oriP | 4.139754 | biomedical | Study | [
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] | [
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0.000342866696882993,
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] | en | 0.999997 |
Within the rest of the EBV genome, however, the movement of the replication forks is mostly unidirectional . For example, replication forks move mainly rightward from interval 11 throughout oriP and beyond . This direction bias is compatible with a previous 2D gel analysis of the oriP region in Raji cells and is not affected by the pausing of the replication fork. We conclude that the direction of movement of the replication forks is mainly a consequence of the dynamics of initiation of DNA replication throughout the viral genome. | 15208711_p16 | 15208711 | Replication Forks Move Without Significant Pausing throughout the Raji EBV Genome with the Exception of the Genomic Region near oriP | 4.256543 | biomedical | Study | [
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] | [
0.99901282787323,
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] | en | 0.999997 |
Early studies performed by electron microscopy identified Raji EBV episomes with multiple replication bubbles but could not identify the position of these initiation events . In order to detect the presence of these events and to determine their location we analyzed the immunostaining patterns of the DNA molecules described above. Multiple initiation events should produce molecules containing multiple red patches, each surrounded by green. The qualitative examination of the replication patterns shown in Figure 3 B revealed some of these immunostaining patterns. In molecules 17 and 43, for example, an early initiation event apparently took place within the RRF (large region stained in red). However, shorter red regions are also present, indicating the occurrence of initiation events at later times. Throughout this manuscript, when we refer to multiple initiation events, we will mean that they occur on the same DNA molecule. In addition, if the activation of the initiation sites is not synchronous (as in the molecules described above), we will refer to the initiation events used to begin the duplication of the EBV genome as primary and any subsequent initiation event as secondary. The secondary events visible in molecules 17 and 43 are both located within the long transcription unit of the Epstein Barr nuclear antigen [EBNA] genes . In particular, molecule 43 shows a secondary initiation event that occurred when the duplication of the EBV episome was almost complete (red patch near the pSalF hybridization signal). Therefore, initiation events are not limited to the RRF of the Raji episomes. Initiation events located throughout the EBV genome, as well as multiple initiation events, were also identified in a much larger fraction of Mutu I EBV episomes (see below). We conclude that the entire EBV genome constitutes a large initiation zone, although the frequency of the initiation events is reduced throughout RRL (see below). | 15208711_p17 | 15208711 | Active Initiation Sites Are Not Limited to the RRF | 4.450789 | biomedical | Study | [
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] | [
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0.000346414337400347,
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] | en | 0.999997 |
In the previous sections we showed that different portions of the EBV genome are not equivalent with respect to when and where DNA replication begins and how DNA replication progresses. Here we wanted to determine the quantitative aspects of DNA replication in different portions of the EBV genome. The data obtained by SMARD can be analyzed quantitatively and used to determine the average time required to duplicate any portion of the EBV genome . By knowing Td and the length of the segment analyzed, the corresponding duplication speed (Sd) can also be calculated . Importantly, these measurements are based on all the images collected during each SMARD experiment, including the molecules entirely stained in red or in green (several hundred). Therefore, the conclusions reached by this analysis are not solely dependent on the appearance of the immunostaining patterns in a small fraction of the DNA molecules. In addition, the quantitative analysis is performed on relatively large genomic segments; therefore, it is not significantly affected by the resolution at which the positions of the replication forks are determined. | 15208711_p18 | 15208711 | DNA Replication Proceeds at Different Speeds throughout Different Portions of the Raji EBV Genome | 4.136368 | biomedical | Study | [
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] | [
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0.00022571840963792056,
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] | en | 0.999997 |
We calculated the value of Sd for each portion of the Raji EBV genome, depicted with double-headed arrows in Figure 5 (segments A–K; Td is reported in Table 1 ). The results obtained from the PacI and the SwaI experiments were analyzed independently but show remarkable similarities . Therefore, the quantitative analysis is highly reproducible. | 15208711_p19 | 15208711 | DNA Replication Proceeds at Different Speeds throughout Different Portions of the Raji EBV Genome | 3.821609 | biomedical | Study | [
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] | [
0.9986724853515625,
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] | en | 0.999997 |
We found that different portions of the EBV genome replicate at different speeds, with values that range from a minimum of 0.3 kb/min to a maximum of 3.5–4.7 kb/min (segment I ). More details are provided later in the text. However, it is important to note that the highest Sd values were detected within the central portion of the Raji RRF (segments A, I, and G ). This result can be explained in two ways. First, replication forks may move faster throughout the RRF. Alternatively, the RRF could contain a significant level of multiple initiation events. For the reasons mentioned below we favor the second possibility. | 15208711_p20 | 15208711 | DNA Replication Proceeds at Different Speeds throughout Different Portions of the Raji EBV Genome | 4.089117 | biomedical | Study | [
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] | [
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] | en | 0.999998 |
Three lines of evidence indicate that multiple initiation events take place within the Raji RRF. Two lines of evidence are discussed in this section (the presence of multiple red patches in the immunostaining patterns of some EBV episomes and the detection of termination events by 2D gel analysis); the third is discussed in the last section of Results (differences in duplication speed across segments of the RRF of different sizes). | 15208711_p21 | 15208711 | Multiple Initiation Events Can Take Place on the Same Raji EBV Episome within the RRF | 3.843614 | biomedical | Study | [
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] | [
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0.0075904252007603645,
0.003142311703413725,
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] | en | 0.999999 |
The first line of evidence is provided by the immunostaining pattern of the EBV molecules. Although discontinuities in the immunostaining make it difficult to detect multiple initiation events when the distance between converging forks is 5 kb or less, the replication patterns of some of the molecules are compatible with the presence of multiple initiation events within RRF . For example, in molecule 9 three regions stained in red (divergent arrows) are separated by two regions stained in green about 4 kb in size (convergent arrows); shorter patches are also visible (asterisks) and might indicate the presence of additional initiation events. In these molecules, the genomic regions stained in red are very close to each other and approach the resolution limits of SMARD. If these signals were produced by multiple initiation events, we should conclude that they took place at about the same time and with a short interorigin distance. Alternatively, secondary initiation events might have taken place in proximity to an incoming replication fork (generated by a primary initiation event). In both cases, the different replication bubbles would rapidly fuse into a larger bubble, making the detection of these events extremely difficult. Since these patterns are too short to be unequivocally ascribed to DNA replication, the presence of multiple initiation events within the RRF was confirmed using a replication mapping approach independent of SMARD. | 15208711_p22 | 15208711 | Multiple Initiation Events Can Take Place on the Same Raji EBV Episome within the RRF | 4.369236 | biomedical | Study | [
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] | [
0.9991496801376343,
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A second line of evidence is provided by the structure of the replication intermediates examined by 2D gel electrophoresis in exponentially growing Raji cells ( see Materials and Methods ). We analyzed nine restriction fragments, indicated in Figure 3 A as gray bars (a–i). We also reexamined the hybridization patterns of ten different fragments analyzed in a previous study . In total, we considered 19 restriction fragments spanning approximately 65% of the Raji EBV genome. The summary of these 2D gel analyses is shown in Figure 3 E. Replication intermediates indicative of initiation events were found in several restriction fragments . The restriction fragments containing replication bubbles are contiguous and span the genomic regions underlined by the red dashed line at the bottom of Figure 3 E (approximately corresponding to the RRF). As expected, termination events were detected in many of the fragments located within the RRL. Importantly, random termination events were also detected in most of the fragments in which we detected bubble arcs. However, the source of these events could not be identified in our previous studies. As discussed earlier, SMARD shows unequivocally that the RRF completes its duplication before forks originating elsewhere have the time to reach its central portion. We conclude that the termination events detected by 2D gel electrophoresis derive from the collision of replication forks generated by multiple initiation events taking place within the RRF. An estimate of the frequency of these multiple initiation events is reported later in the text. | 15208711_p23 | 15208711 | Multiple Initiation Events Can Take Place on the Same Raji EBV Episome within the RRF | 4.292602 | biomedical | Study | [
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] | [
0.9993256330490112,
0.00022084653028286994,
0.0003699262742884457,
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] | en | 0.999999 |
Previous observations have suggested that some initiation sites (such as oriP) are used at a different frequency in different EBV strains . However, it was not known whether these changes were the result of modifications in the activity of individual initiation sites or involved multiple initiation sites throughout the EBV genome. In order to determine the extent of these differences, we performed SMARD in another Burkitt's lymphoma cell line called Mutu I . A brief description of this cell line and of the conditions used for SMARD is reported in Materials and Methods . From this experiment we recovered the images of 271 PacI-linearized EBV episomes substituted along their entire length with halogenated nucleotides (122 red, 107 green, 42 red and green). The results of this analysis are shown in Figure 6 . | 15208711_p24 | 15208711 | In Mutu I Cells, the Order of Activation of the Initiation Sites Varies Throughout the Viral Genome | 4.060429 | biomedical | Study | [
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] | [
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0.0002116139221470803,
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] | en | 0.999996 |
The most striking differences in the replication of the two EBV strains were found in the order of activation of the initiation sites. In Raji episomes, primary initiation events are restricted to an 80-kb region approximately corresponding to the RRF . In contrast, in Mutu I episomes primary initiation events occur at many locations throughout the viral genome . Multiple initiation events (mostly largely spaced), firing either synchronously (molecules 2 and 5) or asynchronously (molecules 11, 15, 31 and 34), are also frequent in Mutu I. This explains the heterogeneity detected in the positions of the termination events (green patches in molecules 32–40), as well as in the replication patterns of the episomes at intermediate stages of duplication. Hence, DNA replication initiates, progresses, and terminates differently in different Mutu I episomes. Even if the order of activation of the initiation sites varies from molecule to molecule, more than 80% of the Mutu I episomes are stained in red between intervals 6 and 9 . This indicates that this EBV strain also contains an RRF, although its genomic location differs from that found in Raji . In summary, these results confirm that initiation sites are not confined to a specific portion of the EBV genome and allow us to conclude that their utilization in different viral strains can change throughout the EBV genome. | 15208711_p25 | 15208711 | In Mutu I Cells, the Order of Activation of the Initiation Sites Varies Throughout the Viral Genome | 4.393945 | biomedical | Study | [
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] | [
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] | en | 0.999997 |
Modifications in the activity of individual initiation sites (such as oriP) could potentially explain the different location of the RRF in Raji and Mutu I episomes. However, the following considerations indicate that this is not the case. Initiation events occurring at oriP take place in the vicinity of the dyad symmetry (DS) element and produce replication forks that stall in the leftward orientation at the family of repeats (FR) element . Initiation events taking place to the left of oriP (such as in Raji episomes) instead produce replication forks that pause in the rightward orientation . In the Mutu I genome, oriP is at the center of the RRF, but replication forks accumulate in both orientations within interval 8 . This indicates that primary initiation events occur on both sides of oriP. Examples of initiation events that took place near the DS element, or to the left of oriP, are visible, respectively, in molecule 5 and in molecules 1 and 3 . The presence of initiation events on both sides of oriP is also supported by the replication pattern of molecule 2, in which two initiation events spaced approximately 10 kb apart are visible on the same episome. Therefore, in the Mutu I EBV genome, the RRF (∼20 kb in size) contains multiple active initiation sites that have a shared tendency to be activated at the beginning of the duplication of each episome. | 15208711_p26 | 15208711 | The RRFs Are Produced by Clusters of Initiation Sites Frequently Activated at the Beginning of the Duplication of the Episomes | 4.488549 | biomedical | Study | [
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] | [
0.9984851479530334,
0.0008581013535149395,
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] | en | 0.999998 |
Similar conclusions apply to the RRF of the Raji EBV genome (∼80 kb in size), in which primary initiation events were detected at various locations . This could explain why only weak bubble arcs were detected by 2D gel electrophoresis throughout the RRF, even though the duplication of the episomes usually begins within this genomic region . We conclude that the RRFs in the Raji and Mutu I EBV genomes are similar in that they contain various initiation sites that have a shared tendency to be activated at the beginning of the duplication of each episome. | 15208711_p27 | 15208711 | The RRFs Are Produced by Clusters of Initiation Sites Frequently Activated at the Beginning of the Duplication of the Episomes | 4.279161 | biomedical | Study | [
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] | [
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] | en | 0.999997 |
In the previous sections we have shown that the order of activation of the initiation sites in Raji and Mutu I EBV episomes is not conserved. Here, we wanted to determine whether the quantitative aspects of DNA replication were also different. SMARD was used to calculate Sd for each portion of the Mutu I EBV genome, depicted as double-headed arrows in Figure 6 F (segments A′ – M′; see also Table 1 ). We found that DNA replication proceeds at different speeds throughout different portions of the Mutu I EBV genome (from a minimum of 0.5 kb/min in segment K′, to a maximum of 3.8 kb/min in segment G′ ). This is very similar to the range of speeds found in the Raji episomes (0.3–4.7 kb/min). Therefore, DNA replication appears to progress with similar kinetics in the two EBV strains. | 15208711_p28 | 15208711 | The Duplication Speed of Various Portions of the EBV Genome Is Different in Raji and Mutu I EBV Episomes | 4.1578 | biomedical | Study | [
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] | [
0.9994563460350037,
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0.00006437687261495739
] | en | 0.999998 |
We also noticed that similar portions of the EBV genome have different Sd values in the two viral strains. Segments A′–G′ divide the Mutu I EBV genome in seven parts of identical size . These segments encompass portions of the EBV genome similar to segments A–G in the Raji genome . However, the values of Sd differ significantly in almost every segment. Interestingly, in Mutu I episomes, the highest Sd values were not detected within the RRF (segment G′ ). Instead, the RRF contained some of the lowest Sd values (segment B′ ). This is probably due to the presence of strong pausing sites within this portion of the Mutu I EBV genome. Nevertheless, segment B′ replicates faster than the corresponding portion of the Raji EBV genome , a phenomenon that could be explained by the presence of multiple initiation events on both sides of oriP in a fraction of the EBV episomes. In any case, these results indicate that there is no simple correlation between the Sd of a genomic segment and its location within the RRF or the RRL. | 15208711_p29 | 15208711 | The Duplication Speed of Various Portions of the EBV Genome Is Different in Raji and Mutu I EBV Episomes | 4.259351 | biomedical | Study | [
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] | [
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Previous observations have suggested that in mammalian cells the speed of the replication forks can vary . Here, we wanted to determine if some of the differences detected in the replication of Raji and Mutu I episomes could be ascribed to modifications in the rate of progression of the replication forks as proposed for other systems . For a genomic segment replicated by forks moving in a single direction, Sd corresponds to the average speed of the replication forks (provided pausing sites are absent). This allowed us to measure the average speed of the replication forks in various portions of the EBV genomes in which these conditions are satisfied. | 15208711_p30 | 15208711 | Replication Forks Progress at Similar Rates Across Different Portions of the EBV Genome and in Different EBV Strains | 4.095655 | biomedical | Study | [
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] | en | 0.999998 |
In Raji episomes, we found that the average speed of the replication forks was about 1.0 kb/min throughout both segment H and segment J ; these segments are replicated by forks moving predominantly in one direction . Interestingly, a similar value (1.1 kb/min) was found for two different portions of the Mutu I EBV genome in which replication forks also move predominantly in one direction . Therefore, in both EBV strains, we found that the average speed of the replication forks is approximately 1.0 kb/min within every segment that could be analyzed. | 15208711_p31 | 15208711 | Replication Forks Progress at Similar Rates Across Different Portions of the EBV Genome and in Different EBV Strains | 4.182025 | biomedical | Study | [
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] | en | 0.999999 |
Studies performed in different systems have suggested that transcription could interfere with the progression of the replication forks . In the Raji EBV genome, segments J and H are located within the long transcription unit of the EBNA genes . Throughout segment J, replication forks progress in the same direction of transcription, whereas in segment H their orientation is reversed . Nevertheless, as demonstrated above, replication forks move at the same speed in both segments. Replication forks also move at a similar speed across two nontranscribed regions in the Mutu I EBV genome . We conclude that in our systems the progression of the replication forks is not significantly influenced by transcription. This could be so either because the level of transcription is not sufficiently high or because, as suggested by others, transcription and DNA replication do not occur at the same time in mammalian cells . | 15208711_p32 | 15208711 | Replication Forks Progress at Similar Rates Across Different Portions of the EBV Genome and in Different EBV Strains | 4.220762 | biomedical | Study | [
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Variation in the utilization of the initiation sites and similarity in the speed of the replication forks suggest that the former should have a stronger influence on the duplication speed of a genomic segment. If we assume that the speed of the replication forks is constant throughout the EBV genome (except for the regions containing pausing sites), Sd becomes a function of the number of replication forks actively synthesizing DNA. High Sd values would indicate that a large number of replication forks participate in the replication of a genomic segment. If we apply this assumption to the central portion of the Raji RRF, we can see that the values of Sd for segments A and G are compatible with the presence 2–3 active forks/segment (corresponding to about one initiation event per duplication cycle within each of these 25-kb segments). Importantly, a larger segment spanning the same portion of the EBV genome (segment I; 35 kb in size) replicates even faster . This increase could not be explained if the changes in duplication speed were caused by modifications in the speed of the replication forks. However, it is precisely what would be expected if an average of two initiation events take place within the 35-kb segment of the RRF (as suggested by the immunostaining pattern of the episomes and supported by the 2D gel analysis). Therefore, the observed duplication speeds support a model in which, within the RRF, initiation sites spaced 25 kb apart or less can become licensed on the same EBV episome. We also noticed that the EBV genome duplicates faster in Mutu I than in Raji cells ( Table 1 ). This is in agreement with the higher level of widely spaced multiple initiation events detected in Mutu I . We conclude that the differences in Sd across the EBV genome and between the two EBV strains reflect different frequencies of initiation and termination events. | 15208711_p33 | 15208711 | The Duplication Speed of a Genomic Segment Reflects the Average Number of Replication Forks Involved in Its Replication | 4.540548 | biomedical | Study | [
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] | [
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] | en | 0.999996 |
In this study, we determined how DNA replication initiates and progresses in EBV episomes latently replicating in two human Burkitt's lymphoma cell lines (Raji and Mutu I). Previous experiments had suggested that some variability in the utilization of oriP might exist among different EBV strains . Here, however, we found that the replication dynamics vary across the entire EBV genome to an extent that could have not been predicted from previous studies. As exemplified by the replication profiles, the immunostaining patterns of the episomes is strikingly different in the Raji and Mutu I strains . This indicates that the order of activation of the initiation sites is not conserved. Differences were also found in the direction of movement of the replication forks and in the duplication speed of different portions of the EBV genome . The last, in particular, indicates that the frequency of initiation and termination events varies across the EBV genome and between the two viral strains. We did not find a simple correlation between the Sd of a genomic segment and its location within the RRFs. For example, the high Sd value for segment G′ indicates the presence of active initiation sites outside the Mutu I RRF. Therefore the factors that influence the order of activation of the initiation sites are at least partially distinct from the factors that control their frequency of activation. | 15208711_p34 | 15208711 | Conserved and Nonconserved Features in the Latent Replication of Different EBV Strains | 4.306999 | biomedical | Study | [
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] | [
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The EBV episomes replicating in these two cell lines have a similar size and genomic organization. However, the number of internal repeats 1 (also called Bam HI W) is reduced by one unit in the Mutu I strain (not shown), while the Raji EBV genome contains two short deletions . In principle, these differences could affect some initiation sites. On the other hand, initiation events were detected throughout the EBV genomes. It is unlikely that localized modifications of the DNA sequence (affecting one or few initiation sites) could account for all the differences in the replication of Raji and Mutu I episomes. Primary or secondary events were detected within almost every 25-kb segment of the Mutu I EBV genome , such as segment A′ , segment B′ (molecules 1, 2, 3, and 5), segment C′ (molecule 34), segment D′ (molecule 8), segment F′ (molecules 5 and 15), and segment G′ (molecules 4 and 31). Similarly, in Raji episomes, replication bubbles were detected within every restriction fragment analyzed by 2D gel electrophoresis throughout a region of about 80 kb (the sizes of these fragments ranged from 3 to 12 kb; Little and Schildkraut and this study). Using SMARD, low frequencies of secondary initiation events were also detected in the remaining portion of the Raji EBV genome. Therefore, even if SMARD and the 2D gel analysis do not have the resolution to pinpoint the locations of the initiation events at the nucleotide level, our results clearly indicate that the average distance between the initiation sites is below 25 kb. | 15208711_p35 | 15208711 | Conserved and Nonconserved Features in the Latent Replication of Different EBV Strains | 4.417957 | biomedical | Study | [
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] | [
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] | en | 0.999998 |
This study also revealed modifications in the pausing of the replication forks in the oriP region. Accumulation of replication forks is clearly present in both EBV strains within this genomic region. However, only 25% of replicating Mutu I episomes contain replication forks at this location , compared with 43% of Raji episomes . Quantitative estimates of the average pausing of the replication forks suggested values of about 30 min in Raji episomes and 10 min in Mutu I episomes (data not shown). The decreased pausing could reflect the presence of active initiation sites on both sides of oriP in the Mutu I strain (as suggested by the immunostaining pattern of the episomes). Alternatively, a decreased efficiency of the pausing sites could also contribute to the significant reduction in pausing detected in Mutu I. | 15208711_p36 | 15208711 | Conserved and Nonconserved Features in the Latent Replication of Different EBV Strains | 4.186725 | biomedical | Study | [
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] | [
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] | en | 0.999998 |
We also found that some features of the episomal duplication do not vary. In both Raji and Mutu I cells, replication forks move freely throughout the EBV genome , and their progression rate appears to be constant. This indicates that modifications in the speed of the replication forks do not contribute significantly to the differences in DNA replication described above. This contrasts with results obtained by another laboratory for an amplified genomic locus , in which the slower progression of the replication forks—caused by a reduction in nucleotide pools—was presented as the cause for a more frequent activation of initiation sites. Instead, the changes in DNA replication detected in our experiments appear to be caused by differences in the order and frequency of activation of groups of initiation sites encompassing large genomic regions (see next section). | 15208711_p37 | 15208711 | Conserved and Nonconserved Features in the Latent Replication of Different EBV Strains | 4.160759 | biomedical | Study | [
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] | [
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] | en | 0.999998 |
Another common feature between Raji and Mutu I cells is the presence of a genomic region that usually replicates first during the duplication of each episome. The position of this RRF differs in the two EBV strains. However, the direction of movement of the replication forks throughout the RRFs is similar. For example, within the central portion of each RRF, replication forks move in both directions, while along its distal portions, replication forks move predominantly outward . We conclude that the direction of movement of the replication forks throughout the EBV genome is mainly a consequence of the dynamics of the initiation of DNA replication. | 15208711_p38 | 15208711 | Conserved and Nonconserved Features in the Latent Replication of Different EBV Strains | 4.260644 | biomedical | Study | [
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] | [
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] | en | 0.999997 |
Even if the EBV episomes utilize the same replication machinery (provided by the host cell), several aspects of their duplication are not conserved between Raji and Mutu I. In mammalian cells, prereplication complexes are believed to form at the end of mitosis, when general transcription is shut off . However, the selection of specific initiation sites occurs only later in G1, at the origin decision point . This suggests that there must be a mechanism that, during G1, restricts the utilization of initiation sites to specific regions of the mammalian genomes. In this study, we have shown that initiation sites are present throughout the EBV genome and that their utilization differs dramatically in different EBV strains. It is therefore reasonable to assume that the utilization of the initiation sites in the EBV episomes is restricted by the same mechanisms responsible for the selection of the initiation sites in mammalian chromosomes. | 15208711_p39 | 15208711 | Utilization of Initiation Sites is Regulated at the Level of Genomic Regions Rather Than at the Level of Individual Initiation Sites | 4.214394 | biomedical | Study | [
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] | [
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One of the questions we tried to answer is whether initiation of DNA replication is regulated at the level of individual initiation sites. Clues to a possible regulatory mechanism can be found in the replication profiles of the EBV episomes. The RRFs are localized in specific portions of the EBV genome that differ in the two EBV strains. These regions are tens of kilobases in size (about 80 kb in Raji episomes and 20 kb in Mutu I episomes) and encompass multiple initiation sites. The early activation of an individual initiation site could be sufficient to generate a RRF. However, our results have demonstrated that within each RRF various initiation sites have a similar tendency to be activated at the beginning of the duplication of each episome. Therefore, the order of activation of the initiation sites varies at the level of genomic regions rather than at the level of individual initiation sites and might reflect the presence of a particular chromatin organization. | 15208711_p40 | 15208711 | Utilization of Initiation Sites is Regulated at the Level of Genomic Regions Rather Than at the Level of Individual Initiation Sites | 4.226023 | biomedical | Study | [
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] | [
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Recent findings have shown that histone acetylation can influence the timing of replication origin firing in yeast . In this study we found that even if initiation events were detected at many locations within the MutuI episomes, primary initiation events occurred predominantly within the RRFs. Modifications in chromatin structure could be used in mammalian cells to regulate the order of activation of the initiation sites across genomic regions that encompass multiple initiation sites. The early activation of the initiation sites located in these regions would increase the chance of passively replicating the neighboring initiation sites contributing, at least in part, to the process of selection of the initiation sites. | 15208711_p41 | 15208711 | Utilization of Initiation Sites is Regulated at the Level of Genomic Regions Rather Than at the Level of Individual Initiation Sites | 4.164083 | biomedical | Study | [
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In addition to changes in the order of activation of the initiation sites, other mechanisms could influence their utilization by affecting their frequency of activation. We noticed that in Raji episomes the frequency of initiation events across the RRL appears to be reduced compared to that of the RRF. This difference is reflected in the higher levels of Sd detected within the RRF compared to the RRL and in the absence of bubble arcs outside the RRF . The replication profile of the EBV episomes also indicates that the RRL (the genomic region stained in red in less than 40% of the EBV episomes) is larger in Raji than in Mutu I and that it mirrors the positions of the longest transcription units active in each strain . This suggests that the presence of a long transcription unit could delay the duplication of the corresponding genomic region. This delay is unlikely to be caused by an impaired progression of the replications forks, since we have shown that the average speed of the replication forks is not significantly influenced by transcription . An alternative possibility could be that transcription decreases the frequency of initiation events across the genomic regions traversed by RNA polymerases, as previously suggested by others . Perhaps the passage of RNA polymerase removes, or inactivates, prereplication complexes deposited on the DNA at the end of mitosis (see next section). The observation that initiation events appear more diffusely across the Mutu I EBV genome than in Raji is consistent with this hypothesis and might reflect the presence of larger nontranscribed regions in Mutu I . Further experiments will be required to shed light on this phenomenon. However, the detection of some initiation events within the transcribed regions suggests that the relationship between transcription and replication could be more complex. | 15208711_p42 | 15208711 | Utilization of Initiation Sites is Regulated at the Level of Genomic Regions Rather Than at the Level of Individual Initiation Sites | 4.519415 | biomedical | Study | [
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] | [
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In this study, we have shown that initiation events are not confined to a specific portion of the episomes, suggesting that DNA sequences capable of functioning as initiation sites must be rather common. This can explain why, under various experimental conditions, individual initiation sites do not appear to play an essential role in the replication of EBV episomes. For example, a hundred-nucleotide deletion encompassing the DS element of oriP is sufficient to abrogate both initiation of DNA replication and the binding of ORC and MCM proteins at this genomic location . This deletion, however, has no apparent effect on the stable replication of the episomes in established cell lines . Therefore, other efficiently licensed initiation sites are present in different portions of the EBV genome. | 15208711_p43 | 15208711 | Initiation Sites Are Redundant Elements of the EBV Genome | 4.183215 | biomedical | Study | [
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] | [
0.99937504529953,
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] | en | 0.999998 |
Large deletions are also well tolerated , even when they encompass portions of the EBV genome known to contain multiple initiation sites (such as the Raji RRF). One of the deleted EBV genomes shown in Figure 7 was recently analyzed to detect binding sites for ORC and MCM proteins. Significant binding of these proteins was detected only at oriP . However, we found that in this mini-EBV genome, oriP is used at a frequency that approaches 100% (B. Chaudhuri and C. L. Schildkraut, unpublished data). Therefore, the absence/reduction of replication complexes at other locations correlates with an increased usage of the licensed initiation site oriP. Interestingly, these short versions of the EBV genome were specifically engineered to preserve the latency genes by removing most of the untranscribed regions. Therefore, a possible effect of transcription could be to reduce the number of replication complexes present throughout the EBV genome. Reductions in initiation events throughout transcribed regions could be relevant in the maintenance of genomic stability. In fact, it has been reported that at least three extremely large transcription units ( FHIT, WWOX, and Parkin; each ∼1 Mb in size) encompass known common fragile sites in mammalian genomes . We conclude that initiation sites are redundant elements of the EBV genome and that the deletion of some of them can be compensated for by an increased usage of the remaining sites. | 15208711_p44 | 15208711 | Initiation Sites Are Redundant Elements of the EBV Genome | 4.41186 | biomedical | Study | [
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] | [
0.999052107334137,
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] | en | 0.999999 |
Initiation site oriP is the best characterized initiation site of the EBV genome. Initiation of DNA replication has been detected at this site in every EBV strain analyzed to date by 2D gel electrophoresis. However, the frequency of the initiation events at oriP varies in different EBV strains, and it is particularly low in Raji . The infrequent use of this initiation site in Raji does not appear to be caused by an inability to assemble a prereplication complex. In fact, in this cell line, both ORC and MCM proteins efficiently bind oriP . Changes in the DNA sequence of oriP are also an unlikely cause for this difference since only single nucleotide polymorphisms between the Raji and Mutu I strains have been described at this location . | 15208711_p45 | 15208711 | Role of oriP in the Replication of EBV Episomes | 4.365325 | biomedical | Study | [
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] | [
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] | en | 0.999998 |
In our current study we show that primary initiation events are frequently detected by SMARD near oriP in Mutu I but not in Raji episomes. This could be explained, in part, by the decreased frequency of utilization of this site in the Raji strains. However, this is unlikely to be the only reason. Even infrequent primary initiation events occurring at oriP would produce replication forks that pause in the leftward orientation within interval 8 (as seen in Mutu I), but none of the Raji EBV episomes showed forks paused in this orientation . Therefore, an alternative explanation is that in Raji episomes the activation of oriP is delayed compared to initiation sites located in the RRF. This would cause an increase in the passive replication of this genomic region, explaining the reduced frequency of activation detected by 2D gel electrophoresis. In this context, the residual activity of oriP in Raji could represent secondary initiation events that take place in proximity to replication forks that originated in the RRF and paused near oriP. These events would produce small red patches, such as the one marked by an asterisk in molecule 35 of Figure 3 , that are too close to the paused forks to be unequivocally identified by SMARD as separate initiation events. We conclude that oriP is one of the initiation sites preferentially utilized to begin the duplication of the Mutu I episomes, while in Raji only secondary initiation events usually occur at this site. | 15208711_p46 | 15208711 | Role of oriP in the Replication of EBV Episomes | 4.361882 | biomedical | Study | [
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] | [
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] | en | 0.999997 |
Various groups have suggested that different cellular proteins could participate in regulating the activity of oriP . However, it is currently not clear why, in Raji episomes, oriP is not among the preferred initiation sites. Interestingly, it has been reported that oriP is more extensively methylated in Raji than in other EBV strains . It is therefore tempting to speculate that epigenetic modifications of the DNA template, or modifications of the chromatin structure, could be responsible for the differences in the order of activation detected in these EBV strains. The epigenetic regulation of oriP activity could be particularly important during the establishment of latent replication, since it has been demonstrated that an epigenetic event is required for the establishment of oriP-dependent replication . | 15208711_p47 | 15208711 | Role of oriP in the Replication of EBV Episomes | 4.255044 | biomedical | Study | [
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] | [
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] | en | 0.999999 |
In this study we have shown that, while the basic features of DNA replication are conserved (i.e., the progression of the replication forks), the activity of the initiation sites (order and frequency of activation) varies significantly in different EBV strains and across different portions of the EBV genome. Importantly, using SMARD we are now beginning to detect similar modifications in the utilization of initiation sites across transcriptionally active chromosomal loci of the mouse genome (data not shown). Additional mechanisms could regulate DNA replication at transcriptionally silent loci, as suggested by the complete absence of initiation events throughout an approximately 450-kb portion of the mouse IgH locus in non–B cell lines . These results are compatible with the flexible utilization of initiation sites also suggested by other laboratories . It is therefore likely that the large redundancy in initiation site usage and the regulation of initiation site activity at the level of genomic regions represent common features of DNA replication in mammalian cells. In particular, our results suggest that long-range changes in chromatin structure or chromosomal organization could be far more important than local modifications at individual initiation sites in regulating DNA replication. This could represent an efficient way for eukaryotic cells to control the replication of their very large genomes, and could have broad implications for the maintenance of genomic stability. By using SMARD on primary cells, we will soon be able to determine if similar dynamics are also present in nontransformed mammalian cells. | 15208711_p48 | 15208711 | Conclusions—Flexible Utilization of Initiation Sites in Higher Eukaryotes | 4.355237 | biomedical | Study | [
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] | [
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Raji cells were grown in exponential phase , keeping the cell density between 3 × 10 5 and 8 × 10 5 cells/ml. The experiments presented in this manuscript were performed at approximately 5 × 10 5 Raji cells/ml, using two labeling periods (240 min each) with 25 μM IdU (first label) and 25 μM CldU (second label). IdU was added directly to the growing culture, followed by low-speed centrifugation of the cells at the end of the first labeling period and resuspension in warm medium containing CldU. | 15208711_p49 | 15208711 | Cell cultures, EBV strains, and double-labeling of replicating DNA | 4.085213 | biomedical | Study | [
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] | [
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] | en | 0.999994 |
Early passages of the Mutu I cells were provided by Alan B. Rickinson and grown for only seven additional passages (keeping the cell density between 4 × 10 5 and 8 × 10 5 cells/ml) before the replicating DNA was labeled. The conditions used for growth and labeling were the same as those used for Raji cells, with the exception that the labeling periods were only 210 min each. This Mutu I cell line is characterized by the presence of a small fraction of cells in which EBV replicates lytically, producing molecules linearized at the terminal repeats. However, this did not affect our analysis of the latently replicating episomes because only the DNA molecules that were circular before the digestion with PacI were recovered from the agarose gels and analyzed by SMARD. | 15208711_p50 | 15208711 | Cell cultures, EBV strains, and double-labeling of replicating DNA | 4.143893 | biomedical | Study | [
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0.00028159661451354623
] | [
0.9988898634910583,
0.0007830847753211856,
0.0002509618934709579,
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] | en | 0.999997 |
Only ten EBV genes (out of about 100) can be expressed during latency . In EBV-associated diseases, where the viral genome is maintained as a circular episome, the phenotype of the infected cell influences the viral patterns of expression . Three different latent transcription patterns have been described : type I (only EBNA1 and EBERs expressed), type II (only EBNA1, the LMPs, and the EBERs expressed), and type III (all the EBNAs, the LMPs, and the EBERs expressed). Although both Raji and Mutu I are human Burkitt's lymphoma cell lines, their transcription profiles are different. The Mutu I cell line used in this study was an early passage of a type I clone isolated in the Alan B. Rickinson laboratory. Raji cells, instead, have a type III -like transcription pattern and they also carry a deletion of the EBNA3C gene . | 15208711_p51 | 15208711 | Cell cultures, EBV strains, and double-labeling of replicating DNA | 4.26985 | biomedical | Study | [
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] | [
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0.00006151866546133533
] | en | 0.999996 |
In order to collect a sufficient number of images of the EBV genome, the population of replicated episomes needed to be enriched by a partial purification using pulsed field gel electrophoresis. However, starting from the limited amount of DNA that can be purified from a pulsed field gel, we could not stretch a sufficient number of DNA molecules on microscope slides by molecular combing . As a consequence, the collection of several hundred images of EBV episomes would have required the analysis of a very large number of microscope slides and the use of large amounts of pulsed field–purified DNA. In order to solve this problem, we decided to stretch the DNA molecules using a modification of the method originally introduced for the optical mapping of restriction sites on individual DNA molecules as well as for other applications . The stretching was achieved by the movement of a DNA solution (a few microliters) gently deposited at the interface between a silanized microscope slide and a nonsilanized coverslip. In this way it was possible to complete our analysis using just few microscope slides and a fraction of the pulsed field–purified DNA derived from the digestion of 10 6 cells. The molecules stretched by capillary action vary in their orientation and in their size . Nevertheless, the EBV molecules were clearly identified by the two hybridization signals. These images were aligned with the map of the EBV genome by computer adjustment of the image size for the entire DNA molecule , as we did previously when EBV episomes were stretched by molecular combing . | 15208711_p52 | 15208711 | Improved method to stretch a large number of EBV molecules on individual slides | 4.19122 | biomedical | Study | [
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] | [
0.9993126392364502,
0.00035803928039968014,
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] | en | 0.999998 |
Hybridization was performed as previously described using probes prepared by nick translation in the presence of biotin-16-dUTP (Roche, Basel, Switzerland). The probes used in this study, pSalF, p107.5, and pWW (provided by John L. Yates), were detected using a modification of the DIRVISH procedure . Briefly, five layers of Alexa Fluor 350 conjugated NeutrAvidin (Molecular Probes, Eugene, Oregon, United States) and biotinylated anti-avidin antibodies (Vector Laboratories, Burlingame, California, United States) were deposited on the microscope slide, washing with PBS, 0.03% Igepal CA-630 (Sigma, St. Louis, Missouri, United States) after each step. The purpose of the hybridization signals is to identify and orient the EBV episomes. Since the DNA molecules studied by SMARD are substituted along their entire length with halogenated nucleotides, they are very easy to detect even in presence of substantial hybridization background . This hybridization background does not affect SMARD, therefore it was digitally removed from the images of the molecules shown in Figures 3 B, 4 B, and 6 B . | 15208711_p53 | 15208711 | Hybridization, probe detection, and immunostaining of the individual DNA molecules stretched on microscope slides | 4.139997 | biomedical | Study | [
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] | [
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Immunostaining to detect IdU and CldU was performed simultaneously with the detection of the biotinylated DNA probes. Mouse anti-IdU (Becton-Dickinson, Palo Alto, California, United States) and rat anti-CldU (Accurate Chemical, Westbury, New York, United States) were used as primary antibodies (monoclonal), while Alexa Fluor 568-conjugated goat anti-mouse (Molecular Probes) and Alexa Fluor 488-conjugated goat anti-rat (Molecular Probes) were used as secondary antibodies. The immunostaining has almost no background. As described previously , the specificity of the immunostaining was tested on DNA fully substituted with IdU or with CldU. No cross-reaction of the antibodies was detected using the detection procedure utilized in this study, and both antibodies were unable to recognize the unlabeled DNA. In practice, the background visible in the red and green channels is mainly represented by other DNA molecules containing halogenated nucleotides . These molecules can be fully or partially stretched (sometimes collapsed or broken in pieces), but are usually clearly distinguishable from the unbroken, fully substituted EBV molecules. By using appropriate dilutions of the DNA sample during stretching, we minimized the overlap of different molecules. | 15208711_p54 | 15208711 | Hybridization, probe detection, and immunostaining of the individual DNA molecules stretched on microscope slides | 4.151271 | biomedical | Study | [
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] | [
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Studies performed by fiber autoradiography have previously shown that the results obtained using DNA fibers (such as the average size of the replicons) are significantly affected by the length of the labeling period utilized to label the replicating DNA . In these studies bias could also be introduced during the collection of the data as a result of the criteria utilized by the experimenter in the choice of the images analyzed. In addition, if synchronized cells are considered, the length of the labeling period also defines the potential resolution at which initiation sites can be mapped, and the estimate of the replication fork speed. Replacing the radioactive detection of the labels with fluorescence microscopy does not solve any of these problems, nor does the statistical analysis of the data. | 15208711_p55 | 15208711 | Advantages in the labeling scheme utilized for SMARD and internal controls | 4.105308 | biomedical | Study | [
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] | [
0.9910171031951904,
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] | en | 0.999998 |
These problems are completely eliminated by the labeling scheme that characterizes SMARD . For our experiments we utilized exponentially growing cells and labeling periods that are longer than the time required to fully replicate the genomic region of interest. In practice, since the replication of a specific genomic region can proceed differently in various DNA molecules, we utilize labeling periods that are sufficiently long to insure the duplication of even the slowest replicating molecules. In addition, only the molecules completely replicated during these labeling periods are examined. By studying this particular population of molecules, we introduce an objective criterion in the collection of the data, eliminating possible biases. Therefore, the molecules replicated during these labeling periods will faithfully represent the distribution of the replication forks in the steady-state population of replicating molecules . | 15208711_p56 | 15208711 | Advantages in the labeling scheme utilized for SMARD and internal controls | 4.108617 | biomedical | Study | [
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] | en | 0.999996 |
Using long labeling periods, and limiting our analysis to the molecules entirely substituted with the halogenated nucleotides, also provides multiple internal controls. These controls cannot be performed when short labeling periods are used. Since the molecules are immunostained throughout their length, their images can be easily aligned to the map of the genomic region analyzed. This allows us to detect the presence of unevenly stretched molecules that can therefore be discarded. In addition, since the immunostaining is visible along the entire length of the molecules, the loss of signal caused by the breakage of some molecules is immediately revealed. The complete substitution of the DNA molecule with halogenated nucleotides also allowed us to easily detect overlaps between different DNA molecules. These overlaps can occur relatively frequently during the stretching of the molecules and their frequency increases as the density and the size of the DNA molecules increases. It is also worth noting that the presence of hybridization probes decreases the intensity of the immunostaining along the corresponding portion of the DNA molecules . This causes a significant loss of information along the hybridized regions, but it also represents an additional control indicating that the immunostaining is indeed present on the DNA molecules that we intend to study (rather than on adventitiously overlapping molecules). Finally, our labeling scheme allows us to insure that the replication proceeded normally during the labeling of the replicated DNA and that no bias was introduced during the collection of the images. In fact, when these conditions are satisfied, the number of molecules fully substituted with IdU is expected to be very similar to the number of molecules fully substituted with CldU. These controls represent a strong proof that the images of the molecules are representative of a steady-state population of replicating molecules. | 15208711_p57 | 15208711 | Advantages in the labeling scheme utilized for SMARD and internal controls | 4.27791 | biomedical | Study | [
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] | [
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] | en | 0.999998 |
The procedures for the enrichment of replication intermediates, 2D gel electrophoresis, and Southern analysis were essentially as described previously . Preparations of replication intermediates from Raji cells were digested with different restriction enzymes depending on the fragment analyzed: EcoRI/DraI for fragments a–e, EcoRI/EcoRV for fragments f and I, EcoRI/HindIII for fragment g, and EcoRI/XbaI for fragment h . The positions (EBV strain B95–8 coordinates) of the restriction fragments analyzed by 2D gel electrophoresis were as follows: fragment a, DraI –EcoRI ; fragment b, EcoRI –DraI ; fragment c, DraI –EcoRI ; fragment d, EcoRI –EcoRI ; fragment e, EcoRI –DraI ; fragment f, EcoRV –EcoRV ; fragment g, HindIII –EcoRI ; fragment h, XbaI –EcoRI (1); and fragment I, EcoRV –EcoRI . The probes used to detect the restriction fragments were as follows: pHindLHI for fragments a–c, pHindE for fragments d–e, pSalF for fragment f, the pHindC fragment XbaI –BglII for fragment g, the p107.5 fragment XhoI –XhoI for fragment h, and the pHindC fragment HpaI –XbaI for fragment i . The plasmids pHindLHI, pHindE, pSalF, pHindC, and p107.5 were kindly provided by John L. Yates. Two different preparations of replication intermediates were used to study the replication patterns of fragments a–c. | 15208711_p58 | 15208711 | Analysis of the replication intermediates by 2D gel electrophoresis at neutral pH | 4.124649 | biomedical | Study | [
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Genetic variation in pathogen virulence (harm to the host) has been found whenever it has been looked for. A considerable body of theory, based on the transmission consequences of virulence, has been developed to predict how natural selection will act on this genetic variation and how it will shape virulence levels in natural populations of disease-causing organisms . For instance, natural or vaccine-acquired host immunity protects hosts from dying, thereby relieving the parasite of the potential fitness costs of prematurely shortened infections. Thus, host populations with high levels of immunity can maintain more virulent pathogens than can naïve host populations . To date, the best example of virulence evolving upwards in response to enhanced levels of host defense comes from an uncontrolled “experiment” in the field: upon release into a highly susceptible host population, the myxomatosis virus evolved lower virulence but then later increased in virulence once the host population had evolved resistance . | 15221031_p0 | 15221031 | Introduction | 4.234485 | biomedical | Study | [
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As well as altering between-host selection pressures on virulence, host immunity can alter the nature of inhost selection. Different directions of virulence evolution are expected depending on the details of inhost competition among parasites . Unfortunately, these details are not well understood for any pathogen . The only generality is that serial passage of pathogens almost always increases virulence , implying that virulent variants have a fitness advantage within hosts. However, all serial passage experiments of which we are aware were conducted in immunologically naïve hosts, so the effects of immunity on virulence evolution are unknown. In theory, immunity could impose selection in several ways. For instance, lower parasite loads should reduce resource competition (e.g., for red blood cells) among parasites occupying the same host, but increase the competition for enemy-free space (e.g., by immune evasion). This could lead to more aggressive parasites racing to stay ahead of proliferating immune responses ; it could also lead to the evolution of novel antigenic variants that have a selective advantage only in immunized hosts. Immunization will also alter the timing of immune selection, thus potentially selecting for changes in parasite life history parameters that affect virulence, such as an earlier or higher rate of production of transmission stages . Finally, the rate at which virulence evolution occurs may be limited by the size of the parasite population inside the host, and therefore may be retarded by host immunity. Thus, at least in theory, there are many potential consequences for virulence evolution of prior host immunity, both long-term and short-term in nature. | 15221031_p1 | 15221031 | Introduction | 4.372326 | biomedical | Study | [
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] | [
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One barrier to testing theoretical models of virulence evolution is that the models typically predict the outcome at evolutionary and epidemiological equilibrium. New equi-libria may or may not take a long time to reach, but will in any case depend on the dynamics of the host population and the environmental conditions under which transmission occurs: this means that experimental evolution to new equilibria will be hard to study in the laboratory for medically relevant pathogens. However, the short-term consequences for virulence evolution, which are at least as important to public health policy as the long-term consequences, may be more tractable. This is especially true for diseases for which animal models are available. | 15221031_p2 | 15221031 | Introduction | 3.983006 | biomedical | Study | [
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] | en | 0.999998 |
In this study, we begin the empirical effort to determine the likely direction of immune-mediated virulence evolution by performing experimental evolution of the rodent malaria parasite, Plasmodium chabaudi , in laboratory mice. We evolved multiple lines of P. chabaudi in immunized and naïve mice by repeated serial passage of blood-stage parasites (i.e., bypassing the normally obligate mosquito vector) starting from two different starting populations. After 20 passages, the lines had evolved sufficiently to make comparisons between the immune-selected lines (I-lines) and naïve-selected lines (N-lines) for virulence and transmissibility. | 15221031_p3 | 15221031 | Introduction | 4.111251 | biomedical | Study | [
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We found that both the I-lines and N-lines evolved to become more virulent than their ancestral populations, but the I-lines became even more virulent than the N-lines . This higher virulence was manifest in both naïve and immunized mice. When the lines were transmitted through mosquitoes, there was generally a reduction in virulence across all the lines, but the I-lines remained more virulent than the N-lines to naïve mice, though not to immunized mice . We discuss these two principal findings separately below. | 15221031_p4 | 15221031 | Results/Discussion | 3.983609 | biomedical | Study | [
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] | [
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The results suggest that immune selection on blood-stage parasites is more efficient at selecting virulent variants than is selection in naïve mice. Response to selection is a function of the amount of variation in the population and the proportion of the population that survives to produce offspring, i.e., the selection intensity. The higher selection response in the I-lines is unlikely to be due to greater variation on which selection could act because the parasite population size on the day of transfer in immunized mice was on average 2-fold smaller than in naïve mice . It is also unlikely to be due to lower host death in the I-lines as there were no line differences in mortality in naïve mice over the entire course of the experiment (10/223 naïve mice infected with N-lines versus 2/40 naïve mice infected with I-lines, p > 0.10 by 2-tailed Fisher's Exact test, zero mortality in immunized mice), and all but one of the deaths occurred after the day of transfer. The most likely explanation is that immunity generated more intense selection by killing a greater proportion of the parasite population up until the point of transfer . Winners of the race into the syringe on day 7 were those parasite variants that survived immune selection, and these parasites proceeded to cause more damage to their host later in the infection. | 15221031_p5 | 15221031 | Immunity Selects for Higher Virulence | 4.193689 | biomedical | Study | [
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] | [
0.9992055296897888,
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But why would selection favor more virulent parasites? Our previous studies have consistently shown that peak parasite densities in the acute phase are positively correlated to the level of virulence that they generate . We therefore expected to find that the higher virulence in I-lines was accompanied by higher parasite densities, in which case we would deduce that immune selection had favored variants that were better able to outgrow immune defenses. While we found positive relationships between asexual multiplication and virulence across all the lines including the ancestral ones , the I-lines and N-lines were statistically indistinguishable ( p > 0.05) for (i) parasitemia on day 4, (ii) parasitemia on day 6 or 7, (iii) the increase in parasitemia from day 4 to day 6 or 7, and (iv) maximum parasitemia, with one exception: maximum parasitemia was significantly higher in I-lines than N-lines derived from unadapted ancestors when measured in immunized mice, and this only in one of the two replicate experiments (23% versus 6.9% parasitemia, p < 0.001). Thus, there is little evidence to suggest that the increased virulence was due to a higher asexual multiplication rate (or a lower death rate of asexuals) in those parasites that successfully made it into the syringe. Our data demonstrate that immunity acts as a powerful and upward inhost selective force on virulence, but the precise mechanism awaits further study. | 15221031_p6 | 15221031 | Immunity Selects for Higher Virulence | 4.273593 | biomedical | Study | [
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] | [
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There were positive relationships between virulence and lifetime transmission potential across all the lines , consistent with our previous studies , but the differences between the I-lines and N-lines were not statistically significant ( p > 0.05). Gametocyte densities are a good predictor of transmission probability in P. chabaudi and other Plasmodium species , so these results demonstrate that the more virulent parasites evolved in semi-immune mice would transmit as successfully as the less virulent parasites evolved in naïve hosts. Thus, in the absence of a cost, virulent variants favored by within-host immune selection are expected to spread throughout an immunized host population. | 15221031_p7 | 15221031 | Immunity Selects for Higher Virulence | 4.105534 | biomedical | Study | [
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] | [
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] | en | 0.999993 |
Malaria parasites, like many microbes , are remarkable in their ability to rapidly adapt to changes in their host environment, and some of this is known to be due to phenotypic switching mechanisms in virulence-related phenotypes such as binding to host cells , red cell surface antigen expression , and red cell invasion pathways . Some of these phenotype-based changes are transient, while others appear to be stable, i.e., maintained over sequential blood-stage passages. In our experiment, it is possible that the increases in virulence we observed following serial passage were at least partly due to altered gene expression rather than changes at the genome level. The public health consequences of this sort of change depend on whether the higher virulence is maintained during mosquito transmission, and upon transfer to hosts with different levels of immunity from those in which selection took place. We found that the I-lines were more virulent than the N-lines in both naïve and immunized hosts . However, after mosquito transmission, the I-lines remained more virulent than the N-lines, only in naïve hosts: the difference in immune hosts was negated by mosquito transmission . Possible reasons for this are discussed further below. For now, we note that the data are consistent with (though do not directly test) the prediction that enhancement of host immunity by anti-blood-stage vaccination will render malaria populations more dangerous to naïve hosts, at least in the short- to medium-term. Whether or not our long-term prediction that immunized populations will drive virulence to a higher level at evolutionary equilibrium proves true can be established only by monitoring vaccine-covered parasite populations in the field. | 15221031_p8 | 15221031 | The Effects of Mosquito Transmission | 4.24454 | biomedical | Study | [
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] | [
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We observed a general reduction in virulence across all lines following mosquito transmission , particularly when measured in immunized mice, and particularly in lines that had been selected under immune pressure, i.e., the I-lines, and in the CW-A ancestral line, which had been serially passaged on day 12 postinfection (PI). Many laboratory studies in malaria have shown that high or low virulence phenotypes accrued through serial passage can be maintained upon transmission through mosquitoes , although occasional major losses (or gains) of virulence do occur . Mosquito transmission could play a significant role in virulence evolution that is driven by inhost selective processes (as distinct from the between-host selective processes underlying the vaccination hypothesis in Gandon et al. ). | 15221031_p9 | 15221031 | The Effects of Mosquito Transmission | 4.164851 | biomedical | Study | [
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] | [
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The mechanistic basis for the reduction in virulence following mosquito transmission remains to be determined. We offer the following speculations. It may be that the virulence reductions we and others have observed are due to stochastic loss of virulent variants during the population bottlenecking that occurs during mosquito transmission (the variability between lines in virulence loss during mosquito transmission favors this hypothesis). Alternatively, virulence reduction may be due to the deterministic forces of selection against virulent variants that have lost or reduced the ability to transmit through mosquitoes : the potential trade-off between virulence in the vertebrate host and production and infectivity of sporozoites in the mosquito has not yet been explored. A further possibility is that the virulence reductions observed following mosquito transmission are due to the systematic resetting during meiosis of the expression of genes that have been switched on or up-regulated during asexual serial passage. For example, it is known that mosquito transmission induces the expression of a different set of the clonally variant (i.e., phenotypically switching) surface antigens from those expressed at the time of ingestion by the mosquito . It is possible that the variants that appear early in the infection, either because of some genetically programmed ordering of expression or because of higher intrinsic switching rates, are recognized by the immune system in a preimmunized host, thus giving the late-appearing variants a selective advantage. Our data are consistent with this idea, since mosquito transmission eliminated the difference between the I-lines and N-lines in immunized mice but not in naïve mice, suggesting that part of the virulence advantage in immunized hosts was due to novelty in the clonally variant surface antigens. Finally, an interesting possibility is that it is loss of diversity per se during mosquito transmission (either at the genetic level or at the phenotypic expression level) that causes a reduction in virulence by limiting the invading parasites' ability to evade immune defenses: our data are also consistent with this hypothesis. | 15221031_p10 | 15221031 | The Effects of Mosquito Transmission | 4.587007 | biomedical | Study | [
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] | [
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Any of these mechanisms could explain the loss of virulence during mosquito transmission, but none are sufficient to explain why the I-lines were more virulent than the N-lines in naïve mice both before and after mosquito transmission. Thus, more than one distinct underlying mechanism probably explains the virulence differences observed here, such as differences in intrinsic virulence properties and differences in levels of antigenic diversity within the lines. Identifying the mechanisms, any links between them, and their relative roles in determining parasite survival in naïve versus immunized hosts are of key importance in understanding virulence evolution and immunoepidemiology of malaria in the field. | 15221031_p11 | 15221031 | The Effects of Mosquito Transmission | 4.153528 | biomedical | Study | [
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] | [
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] | en | 0.999998 |
To what extent do our observations accord with previous work on serial passage of malaria in immune-modified environments? Results from other studies are difficult to interpret as none maintained control lines for selection (i.e., lines that were passaged in the nonmanipulated immune environment), most had no replication of lines within selection treatment, and some used just a single selection step. Nevertheless, some tentative conclusions may be drawn. Comparisons of selected and ancestral parasites have been made after three different forms of immune manipulation: (i) down-regulation of immunity by removal of the spleen prior to infection, (ii) up-regulation of immunity by transfer of immune serum at the beginning of infection, and (iii) up-regulation of immunity by infection, sometimes with subcurative drug treatment in order to establish a chronic infection. In the first two, parasites were selected from the primary wave of parasitemia, as in our experiment, whereas in the third, selected parasites were isolated from relapses much later in the infection (40–150 d PI). Parasite lines passaged through splenectomized hosts often lose the ability to bind to host endothelial cells (cytoadherence) in the microvasculature of the deep tissues and therefore the ability to avoid being passaged through the spleen , the primary site of immune-mediated clearance . This loss of binding is often accompanied by a loss of ability to express —or a major alteration in the level of expression of —the highly variable and clonally variant switching parasite antigens on the surface of the red cell known to be important for the maintenance of long-term chronic infections . In P. falciparum at least , this coincident change in the two properties is because both phenotypes are mediated by the same parasite molecule, denoted PfEMP1 . Importantly, in two of three studies, the line of parasites that lost cytoadherence and/or surface antigen expression had much-reduced virulence to spleen-intact naïve hosts compared to their ancestral lines . If our immunization procedure was priming the spleen for effective parasite clearance, our results are consistent with these findings. | 15221031_p12 | 15221031 | Other Serial Passage Studies in Malaria | 4.360569 | biomedical | Study | [
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] | [
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However, the second form of immune selection—passage of acute-phase parasites from hosts injected with antiserum at the beginning of the infection—yielded parasites with lower virulence to naïve mice than their ancestors in one study , although it had no impact on virulence in two other studies . The third type of immune selection—isolation of parasites from relapses late in the infection—has generated parasites with virulence to naïve hosts that is lower than , higher than , or similar to that of their ancestors. In all these studies, which involved only single passages, selected parasites were more virulent than their ancestors to immunized hosts, suggesting that the selected parasites were predominantly of a novel antigenic type . Whether antigenic novelty is traded off against multiplication rate or virulence among the repertoire of variants expressed during a single infection—as has also been suggested from field population studies —is an interesting question that deserves more attention. However, in our study, in which we focused on the longer-term and more natural environment of hosts preimmunized with a heterogeneous parasite population, the higher virulence of the I-lines compared to the N-lines in both naïve and immunized mice leads us to deduce that selection associated with virulence overrides selection for immune evasion alone. | 15221031_p13 | 15221031 | Other Serial Passage Studies in Malaria | 4.251507 | biomedical | Study | [
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] | en | 0.999997 |
Our data demonstrate that host immunity can increase the potency of inhost selection for higher virulence in malaria. Whether our results generalize to other immunization protocols, parasite clones, parasite species, host genotypes, repeated mosquito passage, and so on requires extensive further experimentation. But, coupled with the malaria parasite's famous ability to rapidly adapt to novel conditions in the laboratory (see above) and to variant-specific vaccine pressure and drugs in the field, these results urge the continuous monitoring of virulence of parasite populations if asexual-stage malaria vaccines become widely used. And for other microparasites (bacteria, viruses, and protozoa) that rely on rapid multiplication within the host for successful transmission, similar concerns might apply. | 15221031_p14 | 15221031 | Conclusion | 4.138462 | biomedical | Study | [
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] | en | 0.999997 |
Starting from two separate ancestral lines derived from clone CW (see below), five parasite lines (“sublines”) from each ancestral line were repeatedly passaged in mice (female C57Bl/6J, 7–10 wk old) that were naïve to malaria infection (N-lines), and five from each ancestral line were passaged in immunized mice (I-lines, see below), forming 20 lines (“sublines”) in total. Passages involved the syringe transfer to a fresh mouse of 0.1 ml of diluted blood containing 5 × 10 5 parasites from a donor mouse that had been infected 7 d previously. Day 7 PI is during the period of rapid population growth, and is about 2 d prior to peak parasitemia, after which population size rapidly declines . Parasite lines under the same selection regime (i.e., passage in immune versus naïve mice) were not mixed at each transfer, thus yielding five independent replicate sublines in each of the four selection treatment–ancestral line groups. | 15221031_p15 | 15221031 | Selection phase. | 4.113912 | biomedical | Study | [
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] | [
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] | en | 0.999998 |
Immunization was by infection with 10 4 parasites of a different clone (denoted ER), followed by drug cure with 10 mg/kg of mefloquine for 4 d starting on day 5 PI. Naïve mice were injected with parasite-free media but were not drug treated. Re-infection took place on average 3 wk after the end of drug treatment (range 1.5–5 wk): as the half-life of mefloquine in mice is reported to be 18 h , the residual amount in the blood by this stage was expected to be very low. The same deep-frozen stock of ER was used each generation. ER is genetically distinct from CW at marker loci (data not shown) and was originally isolated from different hosts. Before use in this experiment, ER had undergone two passages since mosquito transmission and more than 20 passages prior to that. No recrudescent infections in immunized mice were detected prior to challenge. In generations 10 and 11, all lines were passaged through naïve mice. | 15221031_p16 | 15221031 | Selection phase. | 4.115658 | biomedical | Study | [
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] | [
0.9993413090705872,
0.0003526747168507427,
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0.00006961810140637681
] | en | 0.999995 |
The serial passage experiments in this study were replicated using two different starting populations (ancestral lines)—one avirulent (CW-0) and one virulent (CW-A). CW-0 had been cloned by serial dilution from an isolate obtained from its natural host, the thicket rat, Thamnomys rutilans, and then blood passaged every 12 d for a total of 12 passages to produce the CW-A line. During these passages, CW-A was subjected to selection for low virulence on the basis of how much weight loss it caused to mice. Despite this selection, however, CW-A increased in virulence relative to CW-0 during these passages . Prior to use in the current experiments, both CW-0 and CW-A underwent four further serial passages in naïve mice, and were not recloned. | 15221031_p17 | 15221031 | Selection phase. | 4.091252 | biomedical | Study | [
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] | [
0.9995542168617249,
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0.0001516038755653426,
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] | en | 0.999995 |
All the lines, including the ancestral lines, were transmitted once through Anopheles stephensi mosquitoes by allowing 50–100 mosquitoes aged 2–5 d to take a blood meal for 20–30 min on an anaesthetized gametocytemic mouse that had been inoculated 6–10 d previously, i.e., prior to the peak of infection. Then, 11–12 d later these mosquitoes—typically 10–20 of them infected as assessed by random surveys of oocyst prevalence—were allowed to feed back onto anaesthetized naïve mice. After 7–10 d, the blood from these sporozoite-infected mice was harvested and stored in liquid nitrogen. These aliquots were used to initiate blood infections in naïve mice that were then used as donors of asexual parasites to mice involved in the posttransmission experiments. As the lines were transmitted through mosquitoes noncontemporaneously, and involved typically one mouse per subline, comparisons among the lines for infectivity to mosquitoes were not made during these transmission exercises. | 15221031_p18 | 15221031 | Selection phase. | 4.14347 | biomedical | Study | [
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] | [
0.9992491602897644,
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] | en | 0.999997 |
After 18 passages, the pretransmission lines were evaluated in two replicate experimental blocks in naïve (generations 19 and 21) and immunized mice (generations 20 and 22). Ancestral lines were only evaluated in generations 21 and 22. This set of trials was denoted the “pretransmission experiments.” In a separate set of experiments, the “posttransmission experiments,” the mosquito-transmitted lines were compared with each other, as well as with the nontransmitted ancestral lines in two replicate experimental blocks in both naïve (generations 23 and 24) and immunized mice (generations 25 and 26). In both these experiments, across both blocks, ten mice were used for each of the four selection groups (two per subline), and five mice were used per ancestral line. Red blood cell density was measured every 1 or 2 d until day 18 PI by flow cytometry (Coulter Electronics, Luton, United Kingdom), and the minimum density reached was taken as a measure of virulence. Liveweight of the mouse was also recorded every 1–2 d. During the pretransmission experiments (generations 19–22), parasitemia and gametocytemia (proportions of red blood cells infected with asexual parasites and gametocytes, respectively) were evaluated from Giemsa-stained thin blood smears every 2 d from day 4 PI until day 18 PI, and then four more times until day 43 PI. Total lifetime transmission potential was measured as the average gametocytemia throughout the infection from day 4 to day 18 PI. | 15221031_p19 | 15221031 | Evaluation phase. | 4.123895 | biomedical | Study | [
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] | [
0.9994131326675415,
0.0002084485604427755,
0.00031170915463007987,
0.00006677982310066
] | en | 0.999997 |
Statistical analyses were performed separately for the pretransmission and posttransmission experiments as these were carried out at different times. The virulence measure used for the final analysis was minimum red blood cell density, though other measures of virulence were also analyzed (unpublished data). Since selection treatment was replicated on sublines, thus making subline the independent experimental unit, the means of mice within sublines were first calculated. These were then analyzed for the effects of immune environment on selection response by fitting a linear model to these data with factors for selection line (with three levels for nontransmitted ancestral lines, N-lines, and I-lines in the case of the pretransmission experiments, and four levels for the transmitted versions of these three lines plus the nontransmitted ancestral lines in the case of the posttransmission experiments), ancestral population (CW-0, CW-A), and an interaction between these two factors. Thus, statistical tests of differences between the selection lines and other factors in the model were made using t-tests, with the variance for subline means as the residual. An alternative model fitted to data on individual mice (rather than means of sublines) that incorporated subline as a random effect was found to be unsatisfactory because in some treatment groups, the model did not converge and estimates of the subline variance were highly variable between groups. To determine the effects of mosquito transmission on the line differences in virulence, a further analysis was performed on the combined data from the pretransmission and posttransmission experiments fitting a fixed effect factor of line-within-experiment in the statistical model (seven levels—three lines for the pretransmission experiment and four for the posttransmission experiment). These analyses were carried out separately for each of the four immune-treatment-by-ancestral-line groups. Since the pretransmission ancestral line was included in both the pretransmission and posttransmission experiments, the effect of mosquito transmission (and its standard error) on the N-lines and I-lines, which was not measured directly (i.e., in a single experiment), could be estimated by reference to this line. For example, the effect of mosquito transmission in the N-lines was estimated from the difference between the N-lines and their pretransmission ancestral line in the pretransmission experiment minus the analogous contrast in the posttransmission experiment. This was done using the method of linear contrasts provided for in the SAS GLM procedure . The effect of mosquito transmission on the difference between the I-lines and N-lines was similarly calculated but without reference to the pretransmission ancestral line. The effect of mosquito transmission on the ancestral lines was estimated from the direct comparison available from only the posttransmission experiment data. | 15221031_p20 | 15221031 | Analysis. | 4.184043 | biomedical | Study | [
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] | [
0.999406099319458,
0.00021698129421565682,
0.0003162022039759904,
0.00006068286165827885
] | en | 0.999998 |
There is now compelling evidence that a subset of T-cells with regulatory activity suppresses T-cell activation in both mice and humans . Regulatory T-cells (Treg cells) have been shown to inhibit various autoimmune and allergic diseases , mediate transplantation and self-tolerance , and block the activation and proliferation of T-cells both in vitro and in vivo . These findings strongly suggest that Treg cells play a key role in immune regulation. | 15252446_p0 | 15252446 | Introduction | 4.02304 | biomedical | Review | [
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] | [
0.16890233755111694,
0.006899511441588402,
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] | en | 0.999996 |
Subsets and Splits