6 research outputs found
Expression of various BER proteins in different mouse organs.
<p>Total protein was extracted from different organs of 3 different FXD mice as described in the Materials and Methods. Since in our experience proteins used as ānormalizing controlsā including Ī²-actin and Ī±-tubulin and GAPDH differ significantly in different organs, we took care to analyze equal amounts of protein as assessed by the Bradford Assay. Ten micrograms of protein from the organs of each animal were pooled and loaded onto 3ā8% Tris-Acetate gels, resolved by gel electrophoresis and subjected to Western blotting as described in the Materials and Methods.</p
The effect of heterozygosity for the <i>PolBC</i> mutation on the number of expansions, contractions and unchanged alleles seen in the gametes of 3-month-old male mice.
<p>Small pool PCR was carried out on sperm DNA isolated from two 3-month-old <i>PolB+/+</i> and two 3-month-old <i>PolB+/C</i> male mice as described in the Materials and Methods. These animals all had ~140 repeats. The difference between the number of expansions, contractions and unchanged alleles within each genotype and between the two genotypes was evaluated by Fisherās exact test. The error bars represent the 95% confidence intervals. There were no significant within genotype differences in the frequency of expansions, contractions or unchanged alleles. Allele classes that are significantly different in <i>PolB+/C</i> mice are marked with asterisks. Expansions were significantly reduced in <i>PolB+/C</i> gametes (p = 0.0001) and contractions significantly increased (p = 0.0001) by Fisherās exact test.</p
PolB+/C mice show a reduced somatic instability index in testis and tail.
<p>The somatic instability index of different organs of three 16 month old <i>PolB+/+</i> and three 16 month old <i>PolB+/C</i> mice with ~140 repeats was determined as previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005181#pgen.1005181.ref046" target="_blank">46</a>]. Tail 1 and tail 2 refer to tail samples taken at 3 weeks of age and tail samples taken at 16 months respectively. The error bars represent the standard deviations. The significance of the differences in the SII for different genotypes was determined using Studentās t-test. The tissues in which the SII was significantly lower in <i>PolB+/C</i> mice are indicated by asterisks. The SII for <i>PolB+/C</i> testis was significantly lower at p = 0.001 and the SII for the <i>PolB+/C</i> tail 2 sample was significantly lower at p = 0.013.</p
The effect of heterozygosity for the <i>PolBC</i> mutation on the distribution of repeat number changes seen in the gametes of 3-month-old male mice.
<p>The percentage of alleles with the indicated gains or losses in repeat number for <i>PolB+/+</i> and <i>PolB+/C</i> mice was plotted. The mean gain of repeats was 3.17 (SD = 2.52) for <i>PolB+/+</i> and 5.43 (SD = 4.48) for <i>PolB+/C</i>. This resulted in a distribution of expanded alleles that was significantly different in the two genotypes (p = 0.0001; <i>t</i> test). The mean loss of repeats was 10.83 (SD = 11.05) for <i>PolB+/+</i> and 19.68 (SD = 31.58) for <i>PolB+/C</i>. The very high standard deviations due to the presence of some very large contractions particularly in the <i>PolB+/C</i> mice resulted in a distribution of contracted alleles that was not significantly different in the two genotypes. Inset: <i>PolB+/C</i> mice have fewer small expansions and more large expansions than <i>PolB+/+</i> mice. The error bars represent the 95% confidence interval. Repeat size classes that are significantly different in <i>PolB+/C</i> mice are marked with an asterisk. The decrease in the number of alleles with 1ā5 repeats was significant at p = 0.0001, and the increase in the number of alleles with >10 repeats was significant at p = 0.005.</p
The effect of heterozygosity for the <i>PolBC</i> mutation on the distribution of repeat number changes seen in the gametes of 11-month-old male mice.
<p>The percentage of alleles with the indicated change in repeat number that were seen in the gametes of three 11-month-old mice <i>PolB+/+</i> and three 11-month old <i>PolB+/C</i> mice. The grey arrowheads indicate the local maxima seen in the distribution of <i>PolB+/C</i> alleles. The mean gain of repeats was 8.75 (SD = 5.96) for <i>PolB+/+</i> and 14.04 (SD = 8.70) for <i>PolB+/C</i>. This resulted in a distribution of expanded alleles that was significantly different in the two genotypes (p = 0.0001; <i>t</i> test). Too few contractions were seen to carry out any statistical analysis. Inset: <i>PolB+/C</i> mice have fewer small expansions and more large expansions than <i>PolB+/+</i> mice. The error bars represent the 95% confidence interval. Repeat size classes that are significantly different in <i>PolB+/C</i> mice are marked with an asterisk. The decrease in the number of alleles with 1ā5 repeats was significant at p = 0.0001, and the increase in the number of alleles with >15 repeats was also significant at p = 0.0001.</p
Model for BER-mediated repeat expansion in the FX PM mouse.
<p>Nicks that do not get repaired by short patch BER may be channeled into one of two branches of the LP BER pathway. (<i>i) The PolĪ²-dependent</i>, <i>PolĪ“/PolĪµ-independent branch</i>. Nick processing is carried out by PolĪ², a poorly processive polymerase with weak strand-displacement activity. The resultant small flaps are processed by FEN1 to generate a ligatable 5ā end that still contains a few additional flap bases (shown in orange). (ii) The <i>PolĪ²/PolĪ“/PolĪµ-dependent branch</i>. Since both PolĪ“ and PolĪµ are more processive than PolĪ², more strand slippage and more extensive strand displacement may result. Repriming of DNA synthesis on the slipped-strand using PolĪ² would not remove looped out bases (shown in red) since PolĪ² lacks a suitable proofreading activity. Limited strand displacement by PolĪ² or more extensive strand displacement by PolĪ“/PolĪµ, followed by FEN1 cleavage could also result a ligatable end that still contains some flap bases (shown in orange). In either case, repair synthesis initiated on the complementary strand would fix the supernumerary bases into the derivative allele thus generating either a small (i) or large (ii) expansion. MMR proteins may facilitate this process by stabilizing the hairpins. These proteins may also directly generate expansions by channelling the hairpins formed during BER into the MMR pathway.</p