Higher serum lipids and oxidative stress in patients with normal tension glaucoma, but not pseudoexfoliative glaucoma

This study entailed a cross-examination of oxidant/antioxidant balance, high-density lipoprotein (HDL)-linked paraoxonase 1 (PON1) phenotypes, and levels of serum routine lipids among patients with normal tension glaucoma (NTG) or pseudoexfoliative glaucoma (PEXG) compared with healthy control groups. We aimed to investigate the links between oxidative stress (OS), HDL-related antioxidant enzyme activities and dyslipidemia in distinct subtypes of glaucoma. The study included 32 patients with NTG, 31 patients with PEXG, and 40 control subjects. Levels of PON1 and arylesterase enzymatic activity, total oxidant status (TOS), and total antioxidant status were measured by spectrophotometry and OS indexes (OSI) were calculated. The phenotype distribution of PON1 was determined using the dual substrate method. Blood serum levels of HDL, low-density lipoprotein, total cholesterol (TC), and triglyceride (TG) were measured. The TOS and OSI values in the NTG group were significantly higher compared with the other groups (both p < 0.01). The phenotype distribution found in the glaucoma and control groups were NTG: QQ, 59.4%; QR, 37.5%; RR, 3.1%; PEXG: QQ, 45.1%; QR, 48.4%; RR, 6.5%; and in the control group: QQ, 42.5%; QR, 50.0%; RR, 7.5%. Serum TC levels were significantly higher than the control in both NTG and PEXG groups, whereas TG was significantly higher in NTG only (p < 0.01 and p < 0.02, respectively). Hyperlipidemia, OS and variations in phenotype distribution of PON1 may play a role in the pathogenesis of different types of glaucoma

Identification of a novel type of spacer element required for imprinting in fission yeast

Asymmetrical segregation of differentiated sister chromatids is thought to be important for cellular differentiation in higher eukaryotes. Similarly, in fission yeast, cellular differentiation involves the asymmetrical segregation of a chromosomal imprint. This imprint has been shown to consist of two ribonucleotides that are incorporated into the DNA during laggingstrand synthesis in response to a replication pause, but the underlying mechanism remains unknown. Here we present key novel discoveries important for unravelling this process. Our data show that cis-acting sequences within the mat1 cassette mediate pausing of replication forks at the proximity of the imprinting site, and the results suggest that this pause dictates specific priming at the position of imprinting in a sequence-independent manner. Also, we identify a novel type of cis-acting spacer region important for the imprinting process that affects where subsequent primers are put down after the replication fork is released from the pause. Thus, our data suggest that the imprint is formed by ligation of a not-fullyprocessed Okazaki fragment to the subsequent fragment. The presented work addresses how differentiated sister chromatids are established during DNA replication through the involvement of replication barriers

Substitutions in the region around and of the imprinted nucleotides.

<p>(A) The wild-type sequence is given on top. The <i>H1</i> domain sequences are highlighted with a grey shadow. The position of the inverted repeat that flank the imprint is shown with arrows. Below, the mutant sequences introduced are given. Substituted nucleotides are shown in italics and are underlined. (B) Southern analysis of the strains given in panel A. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001328#pgen-1001328-g002" target="_blank">Figure 2</a> legend for method. The position of the DSB products is shown. (C) High-resolution Southern blot of the wild-type and mutant strains carrying substitutions at the imprinted nucleotides. A strand-specific probe that hybridizes to the imprinted strand was used. For detailed explanation of the method, see the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001328#s4" target="_blank">Materials and Methods</a> section. The positions of the fragments generated by hydrolysis of the imprint are shown.</p

Working model for <i>MPS1</i> replication pausing and <i>mat1</i> imprinting.

<p>The <i>mat1</i> locus and the events that occur during replication of it are depicted with line drawings. Left, molecular events that take place in the wild-type strain. Right, events occurring when cis-acting mutations are introduced. Positions of the spacer and <i>abc</i> regions and the <i>H1</i> domain (grey boxes) are shown. The orientations of the DNA strands are indicated. The putative proteins mediating <i>MPS1</i> activity and maintenance of the imprint are displayed as a circle and a pentagon, respectively. The imprint is marked with a black spot. Leading- and lagging-strand replication is represented with black half arrowheads. Specific priming is depicted as wavy lines, while loss of it is indicated with grey half arrowheads. Left, positions of the cis-acting deletions are given. See main text for description of each step.</p

Mating-type switching in <i>S. pombe</i>.

<p>(A) Switching pedigree. The cells of minus (M) and plus (P) mating type are represented with light and dark brown colours, respectively. Division of an unswitchable (u) cell leads to the formation of an unswitchable and a switchable (s) cell of the same mating type. Division of the switchable cell gives rise to a switchable cell of the same mating type and an unswitchable cell of the opposite mating type. (B) The mating-type region on chromosome II. The transcriptionally active <i>mat1</i> locus and the silenced donor loci, <i>mat2P</i> and <i>mat3M</i>, are shown. The <i>M</i> and <i>P</i> information is indicated with light and dark brown colours, respectively. The homology domains <i>H1</i>, <i>H2</i> and <i>H3</i> flanking the loci are represented by grey boxes. The centromere (<i>cen2</i>) and the origin of replication located centromere-distal to <i>mat1</i> are shown with a hollow box and a circle, respectively. The replication termination element <i>RTS1</i> is indicated with grey triangles, and direction of fork movement through <i>mat1</i> is shown above with grey arrows, representing unidirectional replication. The imprint is indicated by a yellow star. (C) The replication of the <i>mat1</i> locus in unswitchable and switchable cells. The nascent leading and lagging strands are represented by red and blue lines, respectively. In the second generation, the inherited template strands are also shown in red or blue, depending on which strand they were replicated as during the S phase of the parent. The polarities of DNA strands are indicated with 3′ and 5′ symbols. The S phases of both cell types involves pausing of the replication fork at <i>mat1</i> and the introduction of the imprint in the newly synthesized upper strand. In addition, during the S phase of a switchable cell, the imprint on the upper template strand acts as a lesion to block leading-strand synthesis. The stalled leading-strand replication complex induces homologous recombination between <i>mat1</i> and the donor loci leading to mating-type switching.</p

Out-competition of the putative trans-acting factor(s) that interacts with the <i>M abc</i> and <i>P abc</i> regions.

<p>(A) The effect of plasmids that carry arrays of the <i>abc</i> region on sporulation efficiency. Two different plasmids (constructed using pREP3X or pREP4X parental vectors) that each carries an array of approximately 10 copies of either the <i>M abc</i> or <i>P abc</i> region were introduced in the <i>h⁹⁰</i> wild-type background (strain JZ1), such that two plasmid-borne arrays of either <i>M</i> or <i>P abc</i> were present. The sporulation efficiency relative to that observed for a strain carrying empty vectors (100%) are given as a graph. The deviation is shown as error bars. (B) Quantification of <i>mat1</i> imprinting in strains used in panel A. <i>Hind</i>III fragments were run and probed with the <i>mat1P Hind</i>III fragment. The intensity of the signal from the <i>P abc</i> array is higher due to greater sequence homology. Quantification of intensities of the DSB products relative to the strain that carries the empty vectors (100%) is given below. (C) Neither <i>P abc</i> nor <i>M abc</i> induces replication pausing at the transcriptionally silenced donor loci. Left, 2D-gel analyses of <i>mat2P</i> and <i>mat3M</i> replication are shown. Right, line drawings of the fragments analyzed are given. The direction of replication through each locus is indicated with grey arrows. The positions of the restriction enzyme sites and the probes used are shown. The sizes of the fragments are given.</p

Characterization of the cis-acting region required for replication pausing.

<p>(A) DNA sequence of the <i>abc</i> region, aligned to the corresponding sequence in the <i>P</i> cassette, is given. The borders of <i>a</i>, <i>b</i>, and <i>c</i> regions are indicated by double arrows above the sequence. The sequence was divided into 10 segments and each segment was substituted by a random DNA sequence and denominated as substitution 1 to 10 (<i>Sub1</i>-<i>10</i>). (B) 2D-gel analysis of plasmids that carry different substitutions within the <i>abc</i> region. Top left, line drawing of the parent plasmid. The analyzed fragment contains the <i>abc</i> region and the first 32 bp of the <i>H1</i> domain. The plasmids were digested with <i>Mps</i>A1I and <i>Afe</i>I, which produce a 2.5-kb fragment for the empty vector. The membranes were probed with same fragment of the parental plasmid. Relative intensities of the pause signals observed in plasmids carrying the substitutions are given as percentages of the wild-type value below each 2D gel. (C) 2D-gel analysis of replication of plasmids with different DNA fragments inserted.</p

Identification of a cis-acting “spacer” element in <i>mat1M</i>.

<p>(A) Left, line drawing representation of the strains used is given. The grey line segment indicates the fragment that is substituted by a random stretch of DNA. Right, the names of the strains and quantification of the DSB products (including standard error) and of the pause signals are given. (B) Southern analysis of the strains shown in panel A. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001328#pgen-1001328-g002" target="_blank">Figure 2</a> legend for method. (C) 2D-gel analysis of the strains shown in panel A. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001328#pgen-1001328-g002" target="_blank">Figure 2</a> legend for method. (D) Schematic representation of the mating-type region of the strain used for analyzing the spacer deletion <i>in vivo</i>. The largest spacer deletion (same as in SS13, see A) was introduced into the donor locus <i>mat3M</i> to yield a mutant form of the donor <i>M</i> information (denoted as <i>M*</i>). The <i>ura4</i>⁺ gene linked to the <i>mat3M</i> locus, which was used during strain construction, is marked by a triangle. (E) Sporulation phenotype caused by the spacer deletion. The <i>h⁹⁰</i> wild-type strain and the mutant described in panel D (SS56) were allowed to form single colonies on sporulation media (PMA+), which were subsequently stained by iodine vapours. Iodine reacts with the starch found in spores to yield dark-coloured colonies. (F) Southern analysis of the strain SS56. Genomic DNA from <i>h⁹⁰</i> wild type and SS56 strains was digested with <i>Hind</i>III and probed for the <i>mat1</i> locus. Because of the presence of homology, the donor loci are also visualized. The positions of the bands corresponding to <i>mat1</i>, <i>mat2P</i>, <i>mat3M</i>, DSB products, and <i>mat3M</i> linked to the <i>ura4</i>⁺ gene (<i>mat3</i>::<i>ura4</i>⁺) are indicated.</p

Characterization of a cis-acting region named <i>abc</i> that is required for replication pausing and imprinting.

<p>(A) Left, line drawing representation of the strains used. A gap represents a deletion and a gray line segment represents a substitution of a DNA sequence. The defined <i>abc</i> region is shown and the borders of the element are indicated by vertical lines. Right, names of the strains and quantification of DSB products and pause signals on 2D gels (including standard error) are given. (B) Southern analysis of the strains shown in panel A. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001328#pgen-1001328-g002" target="_blank">Figure 2</a> legend for method. (C) 2D-gel analysis of the strains shown in panel A. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001328#pgen-1001328-g002" target="_blank">Figure 2</a> legend for method.</p

Southern analysis of <i>mat1</i> replication intermediates.

<p>(A) Line drawing of the fragments generated for different strains for denaturing polyacrylamide gel electrophoresis (PAGE) analysis of replicating <i>mat1M</i> DNA shown in panels B and C. The positions of the restriction enzyme sites and the mapped priming site, and the lengths of the fragments are given. The site of the imprint is indicated with a circle. (B) Denaturing PAGE analysis of the <i>mat1M</i> lagging strand. Replicating DNA from log-phase cells was isolated as for 2D-gel analysis, digested with restriction enzymes given in panel A, then was subjected to denaturing PAGE analysis. After blotting, the membrane was probed with a strand-specific probe that hybridizes to the nascent lagging strand and the parental upper strand. Denatured and labelled DNA ladder is given to the left with size markers. The strains that exhibit imprinting (WT and SS25) produced two strong bands that correspond to the centromere-distal and proximal DSB products created by hydrolysis of the imprint (1d and 1p, respectively). Specific priming products at the site of the imprint (2) migrate to the same position as the distal DSB product observed for the WT and SS25 strains (1d). See panel D. Two fainter putative priming products that are shared between WT and SS25 are marked by arrowheads. (C) Denaturing PAGE analysis of the <i>mat1M</i> leading strand. The nascent leading strand and the parental lower strand were detected by the strand-specific probe. The co-migrating signals from the leading strand intermediates stalled at the imprint in WT and SS25 strains (3) and at <i>MPS1</i> in the strain SS13 (4) are indicated. See panel D. Denatured and labelled DNA ladder is given to the left with size markers. (D) Line drawings of the replication intermediates observed in panel B and C are given. Fragments that produce bands in PAGE analysis are marked. The <i>H1</i> domain is represented as a grey rectangle. The imprint is shown as a black circle. The positions of the restriction enzyme sites used are indicated by vertical lines. (E) 2D-gel analysis of the <i>mat1P</i> and <i>mat1M</i> imprint regions contained in <i>Dra</i>I fragments. Migration of size markers after the first-dimension electrophoresis step is shown below each 2D gel. A line drawing of the region is given on right. The positions of the mapped priming site and the probes are shown. The site of the imprint is indicated with a circle.</p