Large-scale Identification of Chemically Induced Mutations in Drosophila melanogaster.

Forward genetic screens using chemical mutagens have been successful in defining the function of thousands of genes in eukaryotic model organisms. The main drawback of this strategy is the time-consuming identification of the molecular lesions causative of the phenotypes of interest. With whole-genome sequencing (WGS), it is now possible to sequence hundreds of strains, but determining which mutations are causative among thousands of polymorphisms remains challenging. We have sequenced 394 mutant strains, generated in a chemical mutagenesis screen, for essential genes on the Drosophila X chromosome and describe strategies to reduce the number of candidate mutations from an average of -3500 to 35 single-nucleotide variants per chromosome. By combining WGS with a rough mapping method based on large duplications, we were able to map 274 (-70%) mutations. We show that these mutations are causative, using small 80-kb duplications that rescue lethality. Hence, our findings demonstrate that combining rough mapping with WGS dramatically expands the toolkit necessary for assigning function to genes

Correction: Impaired Mitochondrial Energy Production Causes Light-Induced Photoreceptor Degeneration Independent of Oxidative Stress.

[This corrects the article DOI: 10.1371/journal.pbio.1002197.]

<i>ppr</i> is required to maintain PR depolarization upon repetitive stimulation.

<p>(A) ERG traces during repetitive light stimuli (1 sec light and 1.5 sec dark) recorded from eye clones of control, <i>ppr</i>^<i>A</i> and <i>ppr</i>^<i>E</i> (left). Quantification of the change in ERG amplitude, relative to the amplitude of the response to the initial light stimulus, is shown on the right. Error bars represent ± SEM. (B) Recovery time of ERG amplitude (light response) in control, <i>ppr</i>^<i>A</i><i>and ppr</i>^<i>E</i> eye clones. Following 30 sec of light exposure (~1,700 Lux), ERG amplitudes were measured after 5, 30, 60, 90 or 120 sec of recovery in dark. ERG traces after 5 sec and 2 min are shown (middle), and quantification of the relative ERG amplitude is shown on the right. Error bars represent ± SEM; NS (two-tailed Student’s <i>t</i> test not significant). (C) Schematic presentation of the phototransduction cascade and the mechanism of Rh1 recycling and endocytosis. When exposed to blue light, Rh1 is converted to meta-Rh1 (MRh1). Through a G-protein cascade, MRh1 activates the TRP and TRPL channels, leading to a Ca²⁺ influx and PR depolarization. MRh1 is quickly phosphorylated by GPRK1 and bound by Arrestin2 (Arr2), leading to the inactivation of MRh1 [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002197#pbio.1002197.ref030" target="_blank">30</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002197#pbio.1002197.ref031" target="_blank">31</a>]. Subsequently, MRh1 is converted to Rh1 by orange light. Rh1 is recycled through a Ca²⁺-dependent pathway leading to Arr2 released from Rh1 [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002197#pbio.1002197.ref032" target="_blank">32</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002197#pbio.1002197.ref034" target="_blank">34</a>]. A fraction of Rh1 forms a stable complex with Arr2 when exposed to light. This complex is endocytosed and degraded by the endolysosomal system (Reviewed in [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002197#pbio.1002197.ref015" target="_blank">15</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002197#pbio.1002197.ref017" target="_blank">17</a>]).</p

Light-induced ATP synthesis is reduced in <i>ppr</i> mutant eyes.

<p>(A) Relative mtRNA levels, normalized to precursor RNA transcribed from the heavy strand (+) of the mitochondrial genome, in control and <i>ppr</i>^<i>A</i> third instar larvae. (B) Relative mtDNA content, normalized to nuclear DNA, in control and <i>ppr</i>^<i>A</i> third instar larvae. (C) Relative mtRNA levels, normalized to nuclear RNA RP49. (D) Activities of mitochondrial electron transport chain (ETC) protein complexes (CI, CII, CIII, CIV) and Citrate synthase (CS) from third instar larval extracts. Genotypes shown are control, <i>ppr</i>^<i>A</i> and <i>ppr</i>^<i>A</i>; genomic rescue (CH322-75O21). ETC complex activity was normalized to CS, and data are expressed as percentage of the activity detected in controls. (E) O_<b>2</b> consumption assayed by polarography. O_<b>2</b> consumption was measured from isolated third instar larvae-derived mitochondria in the presence of CI-specific substrates. State III is the ADP-stimulated O_<b>2</b> consumption rate; State IV represents the ADP-limited O_<b>2</b> consumption rate; RCR is the Respiratory Control Ratio (state III rate / state IV rate). (F–G) Relative ATP levels from control and <i>ppr</i>^<i>A</i> third instar larval extracts (F) and adult eyes (exposed to 1 h light, 1,800 Lux) (G). (H) Relative change in ATP levels in adult heads upon 1 h exposure to light (1,800 Lux). In A–H, error bars represent mean ± standard deviation (SD); statistical significance was determined using a two-tailed Student’s <i>t</i> test (<i>p</i>-values: *** < 0.001, ** < 0.01, * <0.05).</p

PR degeneration is not induced by oxidative stress in <i>ppr</i> mutants.

<p>(A–C) Detection of ROS levels by DHE (red) staining in <i>ppr</i> mutant clones in eye imaginal discs. Mutant clones, encircled by dotted lines, are marked by loss of GFP (green). (B, C) Detection of ROS levels by DHE staining (red) in control (B) and <i>ppr</i> mutant (C) eyes from adult flies exposed to 24 h light (1,800 Lux). (D) Aconitase activity, which is negatively correlated to ROS levels because of its sensitivity to oxidation, is measured in mitochondrial extracts from third instar larvae (Native) or upon treatment with a reducing agent (Reactivated) to control for variations in the total amount of enzyme. Error bars represent ± SD. (E) Relative ERG amplitude from retinas of control, <i>ppr</i>^<i>A</i> and <i>ppr</i>^<i>A</i> expressing hSOD1 in R1-R6 using Rh1-Gal4. All flies carried Rh1-GAL4 in this experiment. Flies were raised in constant light for seven days. Error bars represent ± SEM; NS (two-tailed Student’s <i>t</i> test not significant).</p

Rh1-dependent degeneration of <i>ppr</i> mutant PRs.

<p>(A) ERG traces from control (top) or <i>ppr</i>^<i>A</i> (bottom) eye clones. Flies were raised on normal food (blue, left) or low vitamin A food (red, right) and kept in the dark or constant light for seven days. (B) Quantification of relative ERG amplitude from the experiment shown in A. Error bars represent ± SEM; two-tailed Student's <i>t</i> test (<i>p</i>-value ***<0.001). (C, D) TEM of control and <i>ppr</i>^<i>A</i> ommatidia. Flies were raised on normal food (C) or low vitamin A food (D) and raised in the dark (middle, <i>ppr</i>^<i>A</i>) or 12 h light/dark cycle (left, control and right, <i>ppr</i>^<i>A</i>) for three weeks.</p

<i>ppr</i> and <i>bsf</i> are partially redundant genes.

<p>(A) In the <i>bsf</i>^{<i>SH1181</i>} allele, P{lacW} is inserted within the coding sequence of <i>bsf</i>. (B) No Bsf protein is detected on western blots of larval extract of <i>bsf</i>^<i>SH118</i>. (C) Colocalization of Bsf (red) with GFP-tagged Ppr (green) in adult testes. (D, E) Relative mtRNA levels from control (precise excision line) and <i>bsf</i>^{<i>SH1181</i>} third instar larvae normalized to mitochondrial precursor RNA (D) and nuclear RNA RP49 (E). (F) Activities of mitochondrial ETC protein complexes (CI, CII, CIII, CIV) and CS from <i>yw</i>, <i>bsf</i>^{<i>SH1181</i>} and precise excision (Pre-Ex). ETC complex activity was normalized to CS, and data are expressed as percentage of the activity detected in controls. In D–F, error bars represent mean ± SD; statistical significance was determined using a two-tailed Student’s <i>t</i> test (<i>p</i>-values: *** < 0.001, ** < 0.01, * <0.05). (G) Lethal stages of <i>bsf</i>^{<i>SH1181</i>}, <i>ppr</i>^<i>A</i>, and double mutants (<i>ppr</i>^<i>A</i>; <i>bsf</i>^{<i>SH1181</i>}).</p

Light induces Rh1 accumulation in ppr mutant PRs.

<p>(A) Western blots of proteins regulating phototransduction. Protein was extracted from heads of flies containing control or <i>ppr</i>^<i>A</i> mutant eyes. (B–C) TEM of a single PR from 2–3-d-old, dark-reared control or <i>ppr</i>^<i>A</i> eye clones. (D–G) Whole mount Rh1 (red) immunostaining in control (D, F) and <i>ppr</i>^<i>A</i> mutant PR (E, G). Rhabdomeres are marked by Phalloidin/Actin (green). Flies used in this experiment were 3–4 d old and raised in the dark (D, E) or exposed to ~30 h of light (F, G). (H–K) Arr2::GFP (green or grey) levels in rhabdomeres of <i>ppr</i>^<i>A</i> mosaic retina. RFP (red) marks wild-type PRs (-/+) and yellow dotted lines encircle <i>ppr</i>^<i>A</i> mutant PRs (-/-, lacking RFP). Rhabdomeres are costained with Phalloidin/Actin (blue). Flies were raised in constant dark and dissected and fixed under dim red light. Prior to fixation, flies were kept in dark (H) or blue light (I) for 1.5 min, allowing Arr2::GFP to translocate to rhabdomeres. Alternatively, flies were kept in blue light for 30 min and then shifted to orange light for 60 min to assess release of Arr2::GFP from rhabdomeres (J). (K) Quantification of the difference in green fluorescence intensity between mosaic ommatidia of <i>ppr</i> and wild-type rhabdomeres. Error bars represent ± SEM; statistical significance was determined using a two-tailed Student’s <i>t</i> test (<i>p</i>-values: * <0.05, ***<0.001).</p

PR degeneration due to <i>ppr</i> loss of function is light-dependent.

<p>(A) ERG traces from control and <i>ppr</i>^<i>A</i> mutant eye clones. Flies were raised either in constant dark for three days, constant dark for five weeks, or a 12 h light/dark cycle for five weeks. Dashed line indicates ERG amplitude. (B) Quantification of ERG amplitudes for experiment shown in (A). (C) Quantification of the ERG amplitude of control and <i>ppr</i>^<i>A</i> mutant eye clones of flies that were grown in dark or constant light for one week. Error bars represent mean ± standard error of the mean (SEM); statistical significance was determined using a two-tailed Student’s <i>t</i> test (<i>p</i>-value: *** < 0.001). (D–K) Transmission Electron Microscopy (TEM) of a single ommatidium from control (D, G), <i>ppr</i>^<i>A</i> (E, H), <i>ppr</i>^<i>E</i> (F, I), <i>ppr</i>^<i>A</i>; genomic rescue (J) and <i>ppr</i>^<i>E</i>; genomic rescue (K). Flies were raised in the dark (D–F) or in a 12 h light/dark cycle for three weeks (G–K). The dark structures indicated by arrows in (D) are rhabdomeres. The red arrow specifically indicates R7 or R8 rhabdomeres, which do not degenerate in mutants exposed to light (H, I). Light intensity during light periods was ~1,800 Lux.</p