Myc overexpression increases mutation frequency.

<p>(A) Schematic representation of the <i>lacZ</i> reporter we used to quantitate mutation frequency, and potential mutation events that can occur in this transgene. Adapted from Garcia et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074641#pone.0074641-Garcia3" target="_blank">[29]</a> (B) Western analyses showing 9-fold overexpression of Myc compared to the loading control of γ-tubulin. Samples are wing imaginal discs of the genotypes hs-FLP; <i>lacZ</i> #2/+; act>CD2>Gal4, UAS-GFP/+ (shown as “+”) and hs-FLP; <i>lacZ</i> #2/+; act>CD2>Gal4, UAS-GFP/UAS-Myc larvae (shown as “Myc”). Larvae were heat shocked (37°C) for 45 min during the 3<sup>rd</sup> larval instar and allowed to develop for 12 hours. Eight discs were loaded per lane. (C) <i>lacZ</i> mutation frequency of hs-FLP; <i>lacZ</i> #2/+; act>CD2>Gal4, UAS-GFP/+ (shown as “+”) and hs-FLP; <i>lacZ</i> #2/+; act>CD2>Gal4, UAS-GFP/UAS-Myc larvae (shown as “Myc”). Larvae are collected 3 days after a 45 minute heat shock at 37°C during the 1<sup>st</sup> instar larval stage and separated by sex. Experiments shown are from at least four biological repeats (D) <i>lacZ</i> mutation frequency of hs-FLP; <i>lacZ</i> #9/+; act>CD2>Gal4, UAS-GFP/+ (shown as “+”) and hs-FLP; <i>lacZ</i> #9/+; act>CD2>Gal4, UAS-GFP/UAS-Myc (shown as “Myc”) larvae. (E) A selection (at least 48) plasmids were chosen to digest with AvaI to determine plasmid size per experiment for hs-FLP; <i>lacZ</i> #2/+; act>CD2>Gal4, UAS-GFP/+ (shown as “+”) and hs-FLP; <i>lacZ</i> #2/+; act>CD2>Gal4, UAS-GFP/UAS-Myc (shown as “Myc”) larvae. Male and female data are shown separately for these analyses and do not show any differences in mutation spectra. White filled area of bar represents frequency of size change mutation (genome rearrangement), where as black solid area shows frequency of mutant plasmids that do not show a size change (point mutations). *indicates statistical significance of p<0.001 (student’s t-test).</p>

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