9 research outputs found

    Methylated nucleotides display higher mutation rates than non-methylated positions.

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    <p>Positional co-occurrences of (in total 6031) SNPs at positions within (methylation) target motifs. Methylated positions highlighted in red within five target motifs (bold), as detected in the present study, with, each compared to two similar control sequences. Black bars in histogram represent nucleotides in methylated motifs, gray shades represent sequences not known as DNA-methylation targets. For each motif, counts of overlapping SNPs (for 5m-cytosine motifs: C/G in reference →N; or for 6m-adenosine: A/T→N) at each position are normalized by the genome-wide motif occurrences (numbers for methylated motifs in inset box). The dashed lines indicate the corresponding number of SNPs expected from random occurrence (G/C or A/T) across the genome and over-representation was tested with the χ2 statistics (*p value < 10–5).</p

    Variability at DNA methyltransferase loci.

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    <p>101 N. meningitidis isolates clustered according to SNP distance, yielding in two sequence type (ST) groups. Each column represents an isolate and rows specify the ORF status of 13 DNA methyltransferases (Rebase geneIDs of Z2491 reference strain). Bars in grey at the bottom represent the number of repeat units determining ON/OFF status of the phase-variable modA12 (M.NmeAORF1589P)</p

    Two N. meningitidis serogroup A strains Z2491 and NM1264 display divergent DNA modifications.

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    <p>DNA modification scores are plotted against the coverage in SMRT sequencing of Tet1 converted samples. Each dot represents a position on either strand with a modification score larger than 20, the color specifying the nucleotide base, on which the modification was detected. Modified adenosines (red dots) are predominantly detected in strain NM1264. The horizontal line indicates the threshold score 50 applied for subsequent motif finding.</p

    Depletion of cytosine methylated motifs in the immediate upstream region of ORFs.

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    <p>Occurrence counts of five (most frequent) methylation target motifs are plotted against their position (in bp) relative to 1997 oriented ORFs (genome annotation N. meningitidis strain Z2491). Motif counts are presented as sum over all ORFs within 50 bp windows, centered at position zero. Red lines in each panel compare to the GC content percentage (y-axis label to the right), averaged over all ORF regions. Dashed horizontal lines represent the averaged motif occurrences corresponding to statistically significant (p value 0.05) depletion of the corresponding motif, derived from a comparison to equally sized sets of random loci. The lower right panel represents occurrence counts of a set of six non-methylated control motifs with similar base composition (identical positions in bold), and similar occurrence frequencies as the non-palindromic target motifs T5mCTGG and AC6mACC. None of the control motifs display significant depletions at ORFs comparable to that of methylated motifs.</p

    Methylation-sensitive restriction assays to validate DNA methylation derived from SMRT sequencing (A) DNA samples separated via gel electrophoresis, lane labels indicating (M) 1 kb Plus DNA Ladder (Invitrogen), (gDNA) whole genomic DNA preparations, and (NlaIV) restriction digest products targeting sites 'GGNNCC' in a methylation-sensitive manner. Samples from 2 different strains (Z2491 and NM1264, genome size: 2.18 Mb) display differential resistance consistent with DNA methylation profiles.(B) single position resolution of modification signal averaged over ~1800 'GGNNCC' sites in the respective genome assemblies. The fractions of sites exhibiting a modification score above 50 are displayed for each position and strand. (C) For comparison, adenosine methylation featuring enhanced sensitivity and positional resolution averaged over ~4000 'ACACC' sites.

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    <p>Methylation-sensitive restriction assays to validate DNA methylation derived from SMRT sequencing (A) DNA samples separated via gel electrophoresis, lane labels indicating (M) 1 kb Plus DNA Ladder (Invitrogen), (gDNA) whole genomic DNA preparations, and (NlaIV) restriction digest products targeting sites 'GGNNCC' in a methylation-sensitive manner. Samples from 2 different strains (Z2491 and NM1264, genome size: 2.18 Mb) display differential resistance consistent with DNA methylation profiles.(B) single position resolution of modification signal averaged over ~1800 'GGNNCC' sites in the respective genome assemblies. The fractions of sites exhibiting a modification score above 50 are displayed for each position and strand. (C) For comparison, adenosine methylation featuring enhanced sensitivity and positional resolution averaged over ~4000 'ACACC' sites.</p

    Phase variability at modA12 locus: Alignment of representative read sequences derived from genome sequencing (strains NM1264 or Z2491).

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    <p>The number of repeat units is indicated in the second column, with blue cell background indicating a resulting ON status of the ORF, and yellow for an OFF status. The third column indicates the total number of reads matching the corresponding repeat configuration.</p

    Differential expression of novel TARs in G85R motor neurons.

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    <p>A. RNA-Seq signal plots in reads per million mapped reads at the Limk1 gene from wild-type (black) and G85R (red) motor neurons. The canonical Limk1 gene map is shown at the top. The enlarged region shows a clear 3′-UTR extension in G85R motor neuron RNA. Similar data were observed for Gak (not shown). B. Validation of novel 3′-UTRs of Gak and Limk1 by qRT-PCR. Values are relative expression for each 3′-UTR in G85R versus wild-type motor neuron RNA.</p

    RNA-Seq of laser capture microdissected spinal cord motor neurons from wtSOD1-YFP and G85R SOD1-YFP transgenic mice.

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    <p>A. Survival curve of G85R SOD1-YFP mice with copy number greater than 200. 80% of the mice were paralyzed (and euthanized) between ∼115 days and 205 days (red bar). N = 226. For the present study of motor neuron RNA, presymptomatic mice at ∼90 days of age were used. B.-D. Spinal cord from a 3 month old G85R SOD1-YFP mouse. Frozen section of right ventral horn region is shown, stained with Azure B dye, panel B (see Methods); incubated with anti-ChAT antibodies, panel C; or directly examined for YFP fluorescence, panel D. The large blue-stained cell bodies in panel B are motor neurons as indicated by anti-ChAT staining in panel C. Note that the same cells have YFP fluorescence in panel D. E. Large Azure B-stained cell bodies were laser captured directly into a guanidine thiocyanate solution (see Methods) and subsequent steps carried out as diagrammed (see Methods).</p

    Differential expression in G85R motor neurons.

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    <p>A. Heatmap of selected genes from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053575#pone.0053575.s007" target="_blank">Table S2</a> significantly differentially expressed (raw p-value <0.005) between wild-type and G85R SOD1-YFP mice. For each gene listed, the ratio of RPKM values (G85R/WT) for individual pairs of biological replicates (Rep1, Rep2) is plotted according to the color code below. B. Validation of differentially expressed genes in G85R by qRT-PCR. Shown are box plots representing relative expression values of each gene in G85R versus wild-type motor neurons. Upper whisker represents top 25% of values, box represents the middle 50% of values, and lower whisker represents bottom 25% of values. Median value is indicated by horizontal dashed line. Statistical significance calculated by REST 2009 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053575#pone.0053575-Pfaffl1" target="_blank">[37]</a> is indicated by *  =  p<0.05, **  =  p<0.005, ***  =  p<0.0005. RNAs from at least three different mouse pairs were compared for each gene. Note that the Hsp110 and B2m validations used one exon-junction-spanning and one non-spanning primer set; minus reverse transcriptase controls for these samples were negative for DNA contamination. The expression changes in the left nine genes were validated with 0.19 ng of total RNA, while the remaining six were validated with 1.5 ng of total RNA.</p
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