Complementation of <i>erh1Δ</i> mutation and cross-species experiments.

<p>(A) Serial dilutions of cells were spotted on EMM2+ADE alone or EMM2+ADE supplemented with 2 M sorbitol, 10 mM hydroxyurea or 0.005% SDS agar plates and incubated for 5 days. Strains used: auxotrophic (ade⁻ leu⁻) <i>erh1⁺</i> FY7269 transformed with pREP1 (ZBM1023), FY7269 <i>erh1Δ</i> derivative ZBM1020 transformed with pREP1 (ZBM1024), ZBM1020 transformed with pREP1/SpErh1p (ZBM1025), ZBM1020 transformed pREP1/SjErh1p (ZBM1026) and ZBM1020 transformed with pREP1/HsERH (ZBM1027). (B) Intracellular localization of SpErh1p in human HeLa cells by confocal microscopy. Upper series of images, cells transfected with plasmid coding for EGFP-tagged human ERH (pEGFP-N1/ER) alone or cotransfected with plasmid coding for mCherry-tagged human Ciz1 (pmCherry-N1/Ciz1); lower series of images, cells transfected with plasmid coding for EGFP-tagged SpErh1p (pEGFP-N1/SpErh1p) alone or cotransfected with pmCherry-N1/Ciz1. Direct fluorescence of EGFP or mCherry was observed in live cells. (C) Yeast two-hybrid analysis with SpErh1p used as bait. The host <i>S. cerevisiae</i> L40 cells coexpressing human ERH and Ciz1, human ERH and PDIP46/SKAR (both pairs as positive controls), SpErh1p and Ciz1, and SpErh1p and PDIP46/SKAR were lysed and the activity of the <i>lacZ</i> reporter gene (conversion of X-gal to a blue precipitate represented here as a strong gray color of lysed cells) was determined.</p

Expression of <i>erh1⁺</i> and characterization of Erh1p in <i>S. pombe</i>.

<p>(A) Northern analysis of <i>erh1⁺</i> transcript. Total RNA from haploid strain FY12697 cultured to mid-log phase in YES, cultured to stationary phase in YES, subjected to nutritional stress in low-glucose EMM2, subjected to nutritional stress in EMM2-N or subjected to hyperosmotic stress in YES supplemented with 2 M sorbitol and a diploid constructed freshly by crossing strains FY12697 and FY7519, cultured to mid-log phase in YES or subjected to nutritional stress in EMM2-N, separated by 1.2% formaldehyde/agarose gel electrophoresis and stained with ethidium bromide (upper panel) followed by transfer to membrane and hybridization with radiolabeled <i>erh1⁺</i> cDNA (lower panel). (B) Intracellular localization of Erh1p. Upper series of images, cells expressing yEGFP-tagged Erh1p from pREP1 (strain ZBM1021) visualized with Nomarski Interference Contrast, nuclei stained with DAPI and localization of yEGFP-tagged Erh1p determined by direct fluorescence; lower series of images, cells expressing yEGFP-tagged Erh1p from <i>erh1⁺</i> chromosomal locus (strain ZBM1028), nuclei stained with DAPI and localization of yEGFP-tagged Erh1p determined by direct fluorescence. (C) Identification of two forms of Erh1p. Immunoprecipitates with anti-HA monoclonal antibody from lysed cells expressing 3HA-tagged Erh1p (strain ZBM1022) or negative control (strain ZBM1023) separated by 15% SDS/polyacrylamide gel electrophoresis and stained with silver. Protein molecular mass standard of 21.5 kDa is shown to the left. Both protein bands (p22 and p23) were identified by tandem mass spectrometry as Erh1p-3HA.</p

<i>ERH</i> genes and ERH proteins from four <i>Schizosaccharomyces</i> species.

<p>(A) Intron-exon organization of <i>ERH</i> genes from <i>S. pombe</i> (<i>Sperh1⁺</i>), <i>S. octosporus</i> (<i>Soerh1⁺</i>), <i>S. cryophilus</i> (<i>Scerh1⁺</i>) and <i>S. japonicus</i> (<i>Sjerh1⁺</i>). Number in parentheses gives total length of CDS plus introns. Black blocks represent exons and are drawn to scale; numbers on top give their lengths in bp. Incisions with numbers indicate intron positions and their length in bp. Consecutive introns are labeled with Roman numerals. (B) Alignment of ERH amino acid sequences from <i>S. pombe</i> (SpErh1p), <i>S. octosporus</i> (SoErh1p), <i>S. cryophilus</i> (ScErh1p) and <i>S. japonicus</i> (SjErh1p). Human ERH is shown as a reference sequence. Numbering according to SpErh1p. Number in parentheses indicates the length of the protein. Dots indicate identical residues and blanks denote missing amino acids. Table shows percent identity of sequences. (C) Predicted three-dimensional structure of Sperh1p generated by SWISS-MODEL using coordinates for human ERH from Protein Data Bank (PDB identifier: 2nmlA). Protein images produced with UCSF Chimera. Helices α1 and α2 and loop α1-α2 in both proteins and the first (Q46) and last (D55) amino acid residues of loop α1-α2 in SpErh1p are indicated. (D) Intron IV-exon V junctions in <i>Sperh1⁺</i> and <i>Sjerh1⁺</i> and intron I-exon II junction in <i>Sperh1⁺</i>. Sequences of introns are italicized. The AG sequence of the 3′ splice site is underlined and the neighboring AG is denoted by lower case. For details see text.</p

Effects of <i>erh1⁺</i> disruption in <i>S. pombe</i> on growth rate and cell morphology.

<p>(A) Growth curves for three <i>erh1⁺</i> strains (solid lines), prototrophic ZBM1004, auxotrophic (ade⁻ leu⁻) FY7269 and auxotrophic (ade⁻ leu⁻ ura⁻) FY7266 and three corresponding <i>erh1Δ</i> strains (dashed lines), ZBM1004 derivative ZBM1005, FY7269 derivative ZBM1020 and FY7266 derivative ZBM1030 in EMM2S minimal medium (left) or in YES rich medium (right). Cultures were inoculated to OD₆₀₀ of 0.1 from stationary cultures in the same medium. (B) Cell morphology by light microscopy with Hoffman Modulation Contrast. Images of strains as in (A) cultured to stationary phase in YES.</p

Effects of <i>erh1⁺</i> disruption on <i>S. pombe</i> sensitivity to stresses and on cell cycle progression.

<p>(A) Serial dilutions of cells were spotted on YES alone or YES supplemented with 2 M sorbitol, 10 mM hydroxyurea or 0.01% SDS agar plates and incubated for 5 days. Strains used: prototrophic <i>erh1⁺</i> ZBM1004 and its <i>erh1Δ</i> derivative ZBM1005, auxotrophic (ade⁻ leu⁻) <i>erh1⁺</i> FY7269 and its <i>erh1Δ</i> derivative ZBM1020, and auxotrophic (ade⁻ leu⁻ ura⁻) <i>erh1⁺</i> FY7266 and its <i>erh1Δ</i> derivative ZBM1030. (B) Cell cycle profiles following nutritional stress in nitrogen-free EMM2 minimal medium. Cells of strains as in (A) were stained with PI and sorted by flow cytometry. Upper series of profiles, cells before nutritional stress (in mid-log phase in YES); lower series of profiles, cells after 48-hour nutritional stress in EMM2-N. Peaks representing 1C and 2C DNA content are indicated. Numbers indicate the percentage of cells with a given DNA content.</p

Additional file 1: of Arabidopsis thaliana population analysis reveals high plasticity of the genomic region spanning MSH2, AT3G18530 and AT3G18535 genes and provides evidence for NAHR-driven recurrent CNV events occurring in this location

Figure S1. Distribution of DNA copy number in regions covered by CNV_610 and CNV_611 in 80 natural accessions of Arabidopsis (MPICao2010 set). Figure S2. A schematic map of Arabidopsis genes covered by AthMSH2-MLPA assay. Figure S3. Exemplar electropherograms of AthMSH2-MLPA assay results. Figure S4. Pairwise correlation of MLPA signals obtained with probes mlpaB-mlpaG in AthMSH2-MLPA genotyping assay of Arabidopsis populations. Figure S5. Nonhierarchical phylogenetic network of a subset of 154 accessions based on 20-kb regions flanking the CNVs and its relation to the genetic groups defined by 1001 Genomes Consortium. Figure S6. Linkage disequilibrium (LD) at genomic regions surrounding the investigated CNVs. Figure S7. Rate of missing calls at AT3G18530-AT3G18535 loci in pseudogenome sequences of 1135 Arabidopsis accessions. Figure S8. The sequence composition of the left and right breakpoints in accessions with “del-2” and “dupl-2” genotypes. Figure S9. Sequence alignment of CNV breakpoints in accessions with “del-2” genotype. Figure S10. Sequence alignment of CNV breakpoints in accessions with simple “dupl-2” genotype. Figure S11. Sequence alignment of CNV breakpoints in accessions with “dupl-2” genotype harboring extended duplication, that involves also the 3’ flank of the right LCR. Figure S12. Optimization of genomic DNA template input for ddPCR. Figure S13. Optimization of primer annealing temperatures for ddPCR. Table S2. Sequences of MLPA probes. Table S3. Gene specific primers used for ddPCR assays. (PDF 1563 kb

Additional file 2: of Arabidopsis thaliana population analysis reveals high plasticity of the genomic region spanning MSH2, AT3G18530 and AT3G18535 genes and provides evidence for NAHR-driven recurrent CNV events occurring in this location

Table S1. List of Arabidopsis accessions and gene copy numbers determined in MLPA- and ddPCR-based study. (XLSX 81 kb

Selection of Reference Genes for qPCR- and ddPCR-Based Analyses of Gene Expression in Senescing Barley Leaves

<div><p>Leaf senescence is a tightly regulated developmental or stress-induced process. It is accompanied by dramatic changes in cell metabolism and structure, eventually leading to the disintegration of chloroplasts, the breakdown of leaf proteins, internucleosomal fragmentation of nuclear DNA and ultimately cell death. In light of the global and intense reorganization of the senescing leaf transcriptome, measuring time-course gene expression patterns in this model is challenging due to the evident problems associated with selecting stable reference genes. We have used oligonucleotide microarray data to identify 181 genes with stable expression in the course of dark-induced senescence of barley leaf. From those genes, we selected 5 candidates and confirmed their invariant expression by both reverse transcription quantitative PCR and droplet digital PCR (ddPCR). We used the selected reference genes to normalize the level of the expression of the following senescence-responsive genes in ddPCR assays: <i>SAG12, ICL, AGXT, CS</i> and <i>RbcS</i>. We were thereby able to achieve a substantial reduction in the data variability. Although the use of reference genes is not considered mandatory in ddPCR assays, our results show that it is advisable in special cases, specifically those that involve the following conditions: i) a low number of repeats, ii) the detection of low-fold changes in gene expression or iii) series data comparisons (such as time-course experiments) in which large sample variation greatly affects the overall gene expression profile and biological interpretation of the data.</p></div

GST pull-down assay with substituted forms of human ERH.

<p>Indicated FLAG-tagged ERH forms incubated with either GST-tagged fragment L7 of human PDIP46/SKAR (GST-PDIP46/SKAR[L7]) or GST-tagged fragment B of human Ciz1 (GST-Ciz1[B]) and detected by western blotting with anti-FLAG antibody followed by enhanced chemiluminescence reaction. PDIP46/SKAR does not interact with ERH H3A Q9A or ERH H3A Q9A E37A T51A, and Ciz1 does not interact with ERH E37A T51A or ERH H3A Q9A E37A T51A.</p

Expression stability of candidate reference genes calculated with geNorm.

<p>Genes with M value ≤ 1.5 are considered highly stable across analyzed samples [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118226#pone.0118226.ref042" target="_blank">42</a>]</p><p>Expression stability of candidate reference genes calculated with geNorm.</p