Histone H3K9me3 modification in the <i>disiRNA</i> loci is dependent on DIM-2.

<p>The distribution of the H3K9me3 modification in wild type, <i>dim-2^KO</i> and <i>dim-5^KO</i> mutants at the <i>disi-47</i> locus was determined by ChIP using an H3K9me3 antibody. Relative enrichment of DNA was calculated by normalizing with the relative DNA binding levels of the <i>dim-5^KO</i> mutant. The insert panel shows H3K9me3 ChIP assay results at the <i>ζ-η</i> region in the indicated strains.</p

<i>disiRNA</i> loci are methylated.

<p>(A) Methylation-specific PCR (MSP) analyses using <i>Dpn</i>II or <i>Bfu</i>CI showing that <i>disiRNA</i> loci are methylated. CD and CB are samples that were only treated with either <i>Dpn</i>II or <i>Bfu</i>CI restriction digestion buffer, respectively. <i>ζ-η</i> and <i>ncu06312</i> gene regions were used as positive (methylated) and negative (unmethylated) controls, respectively. Primer pair 113–114 covers a region without DpnII/BfuCI recognition sites. Agarose gel images show the results of semi-quantitaive PCR analyses. The estimated percentages of methylation (right) for these loci were determined by qPCR. (B) Correlation between DNA methylation level (upper panel) and disiRNA expression profile (low panel) spanning the <i>disi-47</i> locus as determined by MSP using qPCR. The <i>am</i> locus served as a negative control. (C) Correlation between DNA methylation level and disiRNA expression profile at the <i>disi-6</i> locus determined by MeDIP. Arrows indicate the locations of primer pairs (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003761#pgen.1003761.s001" target="_blank">Figure S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003761#pgen.1003761.s009" target="_blank">Table S1</a> for primer information).</p

Youth, Gender and Pornography - A Qualitative Study in Sweden

Introduction and objectives: The visibility and accessibility of pornography in public space has increased dramatically over the last decade. In many Western societies, among them Sweden, there is a wide-spread concern about the implications and consequences of this development, especially for young people. However, seldom are young people’s own voices being heard in this debate. Our research tries to remedy this by asking teenagers about their experiences, views and relationships to pornography. Methods: Data were collected in 2006 through qualitative research interviews and focus groups with young people; 73 informants between 14 and 20 years of age are included in the study, 36 girls and 37 boys. Results: The increasing accessibility of pornography has contributed to a process of normalization with regard to young people’s attitudes and behaviours in relation to pornography. This change, however, is related to both age and gender, which allows us to talk about gender specific pornography careers. Our study also confirms the influence and growing importance of the pornographic script as a frame of reference or behavioural code that more or less explicitly prescribes how to look and what to do. However, it seems that most of our interviewees have acquired the necessary skills in how to navigate in the pornographic landscape in a sensible and reflective manner. Most of them seem to have the ability to distinguish between pornographic fantasies and narratives on the one hand, and real life sexual interaction and relationships on the other. Conclusions: Growing up in a society with an easily accessible pornography both lead to a defused view on sexuality and to a critical and reflective outlook. The impact of the so-called pornographic script is clear. However, at the same time the script brings to the fore an ambivalence towards sexuality, and to pornography specifically. It contains both pleasure and harmfulness in a way that seems to be both tempting and frightening

DNA methylation is induced in the promoter region of an artificial convergent transcription construct upon induction of transcription.

<p>(A and B) MeDIP results showing the DNA methylation status of the <i>ccg-1</i> promoter, <i>luc</i> gene body, and the <i>qa-2</i> promoter of the indicated construct. The artificial construct <i>Pqa-2:cul:1-gccP</i> (A) or <i>Pqa-2:cul</i> (B) resides at the <i>his-3</i> locus. The top cartoon of each panel depicts the architecture of the construct and black bars indicate the approximate location of primer sets. Three independent repeats were performed. Values are mean ± s.d. (C) Distribution of DNA methylation at the endogenous location of the <i>qa-2</i> promoter of the <i>Pqa-2:cul:1-gccP</i> strain. (D) Distribution of DNA methylation around the <i>Pqa-2:cul:1-gccP</i> construct at the <i>his-3</i> locus with/without the activation of the <i>qa-2</i> promoter. In panels A-D, QA+ and QA- indicate presence or absence of quinic acid (QA), respectively. DNA methylation was measured with MeDIP. (E) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003761#s2" target="_blank">Results</a> of bisulfite PCR in the region upstream of the <i>qa-2</i> promoter of the <i>Pqa-2:cul:1-gccP</i> construct. Two aliquots of genomic DNA from <i>Pqa-2:cul:1-gccP</i> strain, one digested with <i>Bfu</i>CI and one untreated, were subject to bisulfite treatment and sequencing, respectively (strategy 2 of bisulfite sequencing, primer sequences in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003761#pgen.1003761.s012" target="_blank">table S4</a>). Each row of circles represents the order and number of cytosines in the subcloned sequence. Opened and filled circles indicate unmethylated and methylated cytosine, respectively.</p

DNA methylation and disiRNA in <i>disi-47</i> locus requires transcription.

<p>(A) The disiRNA distribution at <i>disi-47</i> locus in wild-type and <i>wc-2^KO</i> strain. Arrows indicate the transcription start sites. dLRE and pLRE are the two WC complex binding sites. The red bars indicate approximate primer set locations (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003761#pgen.1003761.s009" target="_blank">Table S1</a> for primer sequences). (B) RT-qPCR analyses of <i>frq</i> mRNA. (C) RT-qPCR analyses of the transcripts in the <i>frq</i> promoter region from a wild-type strain grown in constant light (LL) or constant darkness (DD) conditions and the <i>wc-2^KO</i> strain grown in LL. (D) MeDIP results of the dLRE region in the wild-type (LL and DD conditions) and the <i>wc-2^KO</i> strains (LL). In (B), (C) and (D), three independent repeats were performed. Values are mean ± s.d.</p

<i>disiRNA</i> loci have an on/off pattern of DNA methylation.

<p>(A) Bisulfite sequencing result of the <i>ζ-η</i> locus reveals significant methylation in all DNA clones examined. Each row of circles represents the number of cytosines in one subcloned <i>ζ-η</i> fragment. Opened and filled circles indicate unmethylated and methylated cytosines, respectively. (B) Strategy for detecting methylation in <i>disi-6</i> locus (strategy 1, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003761#s4" target="_blank">Materials and Methods</a>). The lack of cleavage by <i>Dpn</i>II indicates no methylation at the GATC recognition site of the original sequence, whereas the cleavage by <i>Dpn</i>II indicates that the DNA sequence contained 5mC. (C) 9 clones from “cut” and “uncut” populations were subject to sequencing. DNA methylation profiles were shown. (D) Southern blot analyses of <i>disi-29</i> and <i>disi-47</i> loci. The <i>am</i> and <i>ζ-η</i> loci were as the negative and positive control, respectively.</p

Exon-intron organization of Blocks 5–10 in <i>H.</i><i>triquetra rpoT</i>.

<p>A. Schematic of genomic DNA sequence determined by RT-PCR showing location of the three primer pairs (f2550/r2750, f2750/r3100 and p2f2/p2r1; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065387#pone.0065387.s002" target="_blank">Table S2</a>). Boxes, exons; lines, introns. (not to the same scale). B. Schematic of the cDNA sequence. (C) Genomic DNA encoding RACE10 transcript has lost the first intron, suggesting it originated by reverse transcription of a mRNA followed by integration into the genome. Numbers in A (2508, 3407) are nucleotide numbers.</p

Mitochondrial Genes of Dinoflagellates Are Transcribed by a Nuclear-Encoded Single-Subunit RNA Polymerase

<div><p>Dinoflagellates are a large group of algae that contribute significantly to marine productivity and are essential photosynthetic symbionts of corals. Although these algae have fully-functioning mitochondria and chloroplasts, both their organelle genomes have been highly reduced and the genes fragmented and rearranged, with many aberrant transcripts. However, nothing is known about their RNA polymerases. We cloned and sequenced the gene for the nuclear-encoded mitochondrial polymerase (RpoTm) of the dinoflagellate <i>Heterocapsa triquetra</i> and showed that the protein presequence targeted a GFP construct into yeast mitochondria. The gene belongs to a small gene family, which includes a variety of 3′-truncated copies that may have originated by retroposition. The catalytic C-terminal domain of the protein shares nine conserved sequence blocks with other single-subunit polymerases and is predicted to have the same fold as the human enzyme. However, the N-terminal (promoter binding/transcription initiation) domain is not well-conserved. In conjunction with the degenerate nature of the mitochondrial genome, this suggests a requirement for novel accessory factors to ensure the accurate production of functional mRNAs.</p></div

<i>H.</i><i>triquetra</i> mitochondrial RNA polymerase.

<p>A. Schematic of the RpoT structure derived from multiple sequence alignments. Boxes indicate the conserved sequence blocks in the C-terminal domain (residues 502–1106), numbered following <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065387#pone.0065387-Li1" target="_blank">[37]</a>. No region comparable to Block 1 of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065387#pone.0065387-Miller1" target="_blank">[40]</a> could be identified in the N-terminal domain. Black blocks, 35–37% amino acid identity; gray blocks, 20–27% identity, dotted blocks, less than 20% identity but more than 25% similarity. Although block 10 has only three conserved residues, one of them is the penultimate Phe822 which is essential for nucleotide binding <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065387#pone.0065387-Sousa1" target="_blank">[21]</a>. Block 2 (striped) consists of several smaller blocks of sequence relatedness. Lines (not to scale) represent regions with variable lengths and little or no relatedness among the RpoTs of different organisms. Grey oval, mitochondrial targeting sequence. B. Maximum likelihood phylogenetic tree of mitochondrial RNA polymerases plus that of bacteriophage T7, based on alignment of Blocks 3–10. Block 2 was not included because the T7 sequence was too divergent to make a reliable alignment. Full names and relevant accession numbers are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065387#pone.0065387.s003" target="_blank">Table S3</a>. The <i>Lingulodinium</i> sequence was derived from two separate transcripts deposited in the Genbank TSA archive.</p

RpoT-GFP localises to the <i>S.</i><i>cerevisiae</i> mitochondrion. <i>A</i>.

<p>Single optical section of a live cell transformed with a construct consisting of the first 30 amino acids of <i>H. triquetra</i> RpoT followed by GFP, showing its location in the mitochondria. <i>B</i>. Chemically fixed GFP-expressing cellls showing co-localization of (i) RpoT-GFP (green) (ii) mitochondrial ATP synthase immunofluorescence (red) and (iii) the overlay of these two signals.</p