Quantitative investigation of two linguistic variables in Hohhot, China: exploring attitudes-language correlation

Abstract of presentation for ALS201

Effect of <i>rad51</i>Δ and <i>rad52</i>Δ on the Extent of RPA Binding to an Unrepairable DSB

<p>An unrepairable DSB was created in wild-type (yXW1), <i>rad51</i>Δ (ySL306), and <i>rad52</i>Δ (ySL177) strains and RPA-bound chromatin was immunoprecipitated using anti-Rfa1 antibody. PCR-amplified DNA from the <i>MAT</i> locus was run on ethidium bromide-stained gels (reverse images are shown). DNA signals were quantitated and graphed as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020021#pbio-0020021-g001" target="_blank">Figure 1</a>. Error bars show one standard deviation.</p

<i>rfa1-t11</i> Mutation Does Not Affect the Recruitment of Itself or Rad51 to an Unrepairable DSB

<div><p>(A) An unrepairable DSB was created in wild-type (yXW1), <i>rfa1-t11</i> (ySL31), <i>rad51</i>Δ (ySL306), and <i>rfa1-t11 rad51</i>Δ (ySL351) strains, and half of the DNA sample was immunoprecipitated with anti-Rfa1 antibody to extract <i>rfa1-t11</i>-bound chromatin.</p> <p>(B) For wild-type (yXW1) and <i>rfa1-t11</i> (ySL31) strains, the other half of the DNA sample was applied with anti-Rad51 antibody to extract Rad51-associated chromatin. PCR-amplified DNA from the <i>MAT</i> locus was run on ethidium bromide-stained gels (reverse images are shown). DNA signals were quantitated and graphed as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020021#pbio-0020021-g002" target="_blank">Figure 2</a>.</p></div

Timing of Recruitment of RPA versus Rad51 to the DSB

<p>An unrepairable DSB was created in the wild-type strain (yXW1), and closer timepoints were harvested at 20 min and 30 min after the HO cut. DNA samples extracted at each timepoint were split. One half was applied with antibody against Rfa1 to immunoprecipitate RPA-associated DNA, while the other half was applied with anti-Rad51 antibody to immunoprecipitate Rad51-bound chromatin. RPA- or Rad51-associated <i>MAT</i> DNA was PCR-amplified and run on ethidium bromide-stained gels (reverse images are shown). DNA signals were quantitated and graphed as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020021#pbio-0020021-g001" target="_blank">Figure 1</a> for RPA ChIP. PCR-amplified <i>ARG5,6</i> signals from the input DNA were used as controls for quantitation and graphing for Rad51 ChIP (see Materials and Methods).</p

<i>rfa1-t11</i> Mutants Are Defective in the Strand Invasion Step of Gene Conversion

<div><p>(A) One half of the DNA extract collected from a typical timecourse experiment as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020021#pbio-0020021-g006" target="_blank">Figure 6</a> was applied with anti-Rad51 antibody to immunoprecipitate Rad51-bound chromatin. Primers P1 and P2 and P1 and P3 were used to PCR-amplify Rad51-bound <i>MAT</i> and <i>HML</i> DNA, respectively (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020021#pbio-0020021-g004" target="_blank">Figure 4</a>A). Samples were run on ethidium bromide-stained gels (reverse images are shown). DNA signals were quantitated and graphed as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020021#pbio-0020021-g002" target="_blank">Figure 2</a>.</p> <p>(B) Input DNA was used to PCR-amplify strand invasion product using a unique primer distal to <i>MAT</i> (pB) and a primer within the Yα sequence from <i>HML</i> (pA) (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020021#pbio-0020021-White1" target="_blank">White and Haber 1990</a>). PCR-amplified <i>ARG5,6</i> signals from the input DNA were used as loading control.</p></div

Recruitment of RPA to a DSB in the Absence of DNA Repair

<div><p>A strain deleted for donors (yXW1), thus incapable of repairing a DSB by gene conversion, was pregrown in YP–lactate medium, and 2% galactose was added to the culture to induce a DSB at <i>MAT</i>. DNA was extracted at intervals after HO cutting, to which polyclonal antibody against Rfa1 was applied to immunoprecipitate RPA-bound chromatin. Another set of DNA samples were taken at the same time for Southern blot analysis.</p> <p>(A) Map of <i>MAT</i> showing the locations of the HO-cut site as well as the StyI restriction sites and the primers (P1 and P2), 189 bp to 483 bp distal to the DSB, used to PCR-amplify RPA-associated <i>MAT</i> DNA from the immunoprecipitated extract. Purified genomic DNA was digested with StyI, separated on a 1.4% native gel, and probed with a ³²P-labeled <i>MAT</i> distal fragment to monitor the appearance of the HO-cut fragment (see Materials and Methods). The 1-h timepoint represents 1 h after galactose induction of the HO endonuclease.</p> <p>(B) PCR-amplified RPA-bound <i>MAT</i> DNA in a wild-type strain (yXW1). As controls, primers to an independent locus, <i>ARG5,6</i> (see Materials and Methods), were used to amplify DNA from the immunoprecipitated chromatin. PCR samples were run on ethidium bromide-stained gels (reverse images are shown). Quantitated signals were graphed for the wild-type strain. IP represents ratio of the <i>MAT</i> IP signal to <i>ARG5,6</i> IP signal. Error bars show one standard deviation.</p> <p>(C) RPA-bound chromatin was PCR-amplified from sites located proximal and distal to the DSB and then quantitated and graphed as described in (B). The DSB is shown at 0 bp.</p> <p>(D) Effect of formaldehyde cross-linking on RPA binding to ssDNA. In both the noncross-linked samples and the cross-linked samples, 4 ng of single-stranded heterologous β-lactamase (<i>AMP</i>) gene DNA was added during the extract preparation step of ChIP. The amount of input genomic and heterologous DNA was measured by PCR primers specific to the <i>ARG5,6</i> locus and to the <i>AMP</i> sequence, respectively. RPA-associated <i>ARG5,6</i> and <i>AMP</i> DNA were analyzed from the IP samples. PCR samples were run on ethidium bromide-stained gels (reverse images are shown).</p></div

<i>rfa1-t11</i> Was Not Able to Associate with the Donor Sequence during Gene Conversion

<div><p>The wild-type strain carrying the <i>HML</i>α donor (yXW2) and an isogenic strain carrying the <i>rfa1-t11</i> mutation (yXW3) were treated with 2% galactose to induce HO endonuclease and then with 2% glucose after 1 h to repress further HO expression. DNA extracted at intervals after HO cutting was split. One half was applied with antibody against Rfa1 to immunoprecipitate RPA-associated DNA, while the other half was applied with anti-Rad51 antibody to immunoprecipitate Rad51-bound chromatin. Another set of DNA samples was taken at the same time for Southern blot analysis.</p> <p>(A) Purified genomic DNA was digested with StyI, separated on a 1.4% native gel, and probed with a ³²P-labeled <i>MAT</i> distal fragment to monitor the appearance of the HO-cut fragment and the repaired product Yα (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020021#pbio-0020021-g001" target="_blank">Figure 1</a>A; see Materials and Methods). Arrowheads indicate the switched product Yα.</p> <p>(B) RPA-bound <i>MAT</i> and <i>HML</i> DNA was PCR-amplified with primers P1 and P2 and with P1 and P3, respectively (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020021#pbio-0020021-g004" target="_blank">Figure 4</a>A). Samples were run on ethidium bromide-stained gels (reverse images are shown). DNA signals were quantitated and graphed as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020021#pbio-0020021-g001" target="_blank">Figure 1</a>.</p></div

Biological function and KEGG pathway analysis of target genes.

<p>The overlapping target genes were predicted using TargetScan and miRDB online analysis tools. (A) the enriched GO biological processes of target genes (B). the enriched KEGG pathways of target genes.</p

Kaplan-Meier survival curves for 10 miRNAs associated with disease free survival identification of three-miRNA signature in KIRC diagnosis and prognosis.

<p>Kaplan-Meier survival curves for 10 miRNAs associated with disease free survival identification of three-miRNA signature in KIRC diagnosis and prognosis.</p

Association between differentially expressed miRNAs and clinical characteristics.

<p>Association between differentially expressed miRNAs and clinical characteristics.</p