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

Mutations in the Mitochondrial Methionyl-tRNA Synthetase Cause a Neurodegenerative Phenotype in Flies and a Recessive Ataxia (ARSAL) in Humans

The study of Drosophila neurodegenerative mutants combined with genetic and biochemical analyses lead to the identification of multiple complex mutations in 60 patients with a novel form of ataxia/leukoencephalopathy

Structural health monitoring of stainless-steel nuclear fuel storage canister using acoustic emission

Nuclear power generation constitutes a significant component of the energy infrastructure within the United States. The dry cask storage system canister, used for storing highly radioactive spent fuel, demands vigilant structural health monitoring. This paper explores the feasibility of monitoring of stress corrosion cracking in large-scale dry cask storage system canisters using acoustic emission sensors. In this paper, a 304H stainless steel plate, reflecting a typical canister, was subjected to conditions inducing stress corrosion cracking by exposure to a potassium tetrathionate solution and tensile stresses. Analysis of the captured acoustic emission signals showed that the sensors were able to detect this corrosion even at considerable distances from the cracks. Moreover, a finite element model was designed to simulate the acoustic emission signals, thus providing a preliminary understanding of the signal profiles at different sensor locations and potentially providing guidance on the optimal placement of the sensors during field monitoring

Protein Phosphatase 1ß Limits Ring Canal Constriction during <i>Drosophila</i> Germline Cyst Formation

<div><p>Germline cyst formation is essential for the propagation of many organisms including humans and flies. The cytoplasm of germline cyst cells communicate with each other directly via large intercellular bridges called ring canals. Ring canals are often derived from arrested contractile rings during incomplete cytokinesis. However how ring canal formation, maintenance and growth are regulated remains unclear. To better understand this process, we carried out an unbiased genetic screen in <i>Drosophila melanogaster</i> germ cells and identified multiple alleles of <i>flapwing</i> (<i>flw</i>), a conserved serine/threonine-specific protein phosphatase. Flw had previously been reported to be unnecessary for early <i>D. melanogaster</i> oogenesis using a hypomorphic allele. We found that loss of Flw leads to over-constricted nascent ring canals and subsequently tiny mature ring canals, through which cytoplasmic transfer from nurse cells to the oocyte is impaired, resulting in small, non-functional eggs. Flw is expressed in germ cells undergoing incomplete cytokinesis, completely colocalized with the <i>Drosophila</i> myosin binding subunit of myosin phosphatase (DMYPT). This colocalization, together with genetic interaction studies, suggests that Flw functions together with DMYPT to negatively regulate myosin activity during ring canal formation. The identification of two subunits of the tripartite myosin phosphatase as the first two main players required for ring canal constriction indicates that tight regulation of myosin activity is essential for germline cyst formation and reproduction in <i>D. melanogaster</i> and probably other species as well.</p></div

Genetic interaction of <i>flw</i>, <i>DMYPT</i>, and <i>sqh</i> during oogenesis.

<p>Data shown for each panel is from a single experiment, though all experiments were repeated. Numbers in the columns of the histograms are ring canals or eggs measured or counted. Error bars are standard error of the means. The values that are significantly different are marked with asterisks (*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.0001). (<b>A</b>) Average diameters of ring canals of heterozygous <i>flw^XE55A</i> with the balancer <i>TM3</i> (black) or a copy of <i>DMYPT⁰³⁸⁰²</i> (upward slash), or homozygous <i>flw^XE55A</i> germline clones with <i>TM3</i> (grey) or a copy of <i>DMYPT⁰³⁸⁰²</i> (downward slash). Stage II is on the left and stage III on the right. 24 and 28 germaria were analyzed for those with TM3 and those without, respectively. (<b>B</b>) Percentage of unmeasurable ring canals at early stage IVg (left) and stage 2b (right) of the same germaria analyzed in panel A. (<b>C–J</b>) Stage 14 eggs developed from germline cysts with various levels of Flw and DMYPT: (C) Heterozygous <i>flw^XE55A</i>, (D) Heterozygous <i>flw^XE55A</i> with heterozygous <i>DMYPT⁰³⁸⁰²</i>, (E) Homozygous <i>flw^XE55A</i>, (F–H) Homozygous <i>flw^XE55A</i> with heterozygous <i>DMYPT⁰³⁸⁰²</i>, (I) Heterozygous <i>flw^XE55A</i> with TM3, (J) Homozygous <i>flw^XE55A</i> with <i>TM3</i>. All homozygous <i>flw^XE55A</i> eggs were germline clones generated in the corresponding <i>flw^XE55A</i> heterozygous females. All images have the same magnification. Scale bar: 100 µm. Eggs in panels F and H are slightly younger than those in other panels and still contain undegraded nurse cells. Nurse cells and the oocyte in panel H were outlined with dashed and solid lines, respectively. (<b>K</b>) Average length of mature eggs with various levels of Flw and DMYPT. (<b>L</b>) Average diameters of ring canals of homozygous <i>DMYPT⁰³⁸⁰²</i>cysts with a copy of null mutation of <i>sqh</i> (<i>sqh^AX3</i>, black), or the <i>FM7</i> balancer (dark grey), or a copy of <i>flw^XE55A</i> (light grey). Stage II is on the left and early stage IV on the right. Insert: percentage of unmeasurable ring canals. Germaria analyzed: seven for sqhAX3-carrying flies, nine for FM7-carrying, and eight for <i>flw^XE55A</i> -carrying.</p

Homozygous XE55E germline clones, but not follicle clones, lead to formation of minute actin-ring canals and small eggs.

<p>(<b>A–C</b>) Stage 10 egg chambers with heterozygous <i>XE55E</i> (A), homozygous <i>XE55E</i> in germ cells (B), or homozygous <i>XE55E</i> in somatic follicle cells (C). Actin phalloidin-staining on the left and nuclear RFP images on the right. The genotypes of the germ cells and somatic cells of the egg chambers are as indicated in the figure. The heterozygous <i>XE55E</i> is <i>XE55E</i>/<i>P{ovoD1} y FRT19A hsflp</i>. The inserts are magnified views of the ring canals marked with arrows in each panel. Note that small ring canals formed only when the egg chamber contains homozygous <i>XE55E</i> in its germ cells. (<b>D</b>) Stage 14 eggs with homozygous <i>XE55E</i> (I) or heterozygous (II) <i>XE55E</i> in germ cells. White field image on the left and nuclear RFP image on the right. Scale bars: 10 µm for A–C, 100 µm for D.</p

Mapping of <i>XE55</i> mutations.

<p>(<b>A</b>) Molecular nature of various <i>flw</i> mutations. Genomic annotation of <i>flw</i> is shown on the top, with the exons boxed. <i>flw⁷</i> is a <i>P</i>-element insertion in the 5′ untranslated region. <i>flw-YFP-159</i> and <i>flw-YFP-284</i> are two intronic <i>PiggyBac</i> yellow fluorescence protein trap lines. All other mutations are EMS-induced coding mutations. The three non-coding mutations are shown in the genomic map, while the coding mutations are shown in the annotated proteins. Protein regions shared between Flw-pA and Flw-pB are in blue. The Flw-pA-specific region is in grey. Amino acids are named with single letters. Positions according to the short isoform Flw-pB are in parentheses. Nonsense mutations are in red. <i>flw¹</i>, <i>flw⁶</i>, <i>flw⁷</i>, <i>flw-YFP-159</i>, <i>flw-YFP-284</i>, and <i>flw^FP41</i> have been described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070502#pone.0070502-Raghavan1" target="_blank">[23]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070502#pone.0070502-Sun1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070502#pone.0070502-Rees1" target="_blank">[28]</a>. Flw translation start and stop codons are indicated on the genomic map with green and red lines, respectively. The enzyme active site and the DMYPT binding site of Flw predictions are based on the Conserved Domain Database, which consists of a collection of well-annotated multiple sequence alignment models for full-length proteins (<a href="http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml" target="_blank">http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml</a>). (<b>B</b>) Flw mutations alter conserved residues. Protein sequence alignment of the short peptide pB of <i>D. melanogaster</i> Flw with PP1ß from Zebrafish (<i>Danio rerio)</i>, Frog (<i>Xenopus tropicalis</i>), Mouse (<i>Mus musculus</i>), Dog (<i>Canis lupus familiaris)</i> and Human (<i>Homo sapiens</i>). The amino acids mutated in Flw are boxed. Amino acid substitutes are included below each corresponding mutation, with “*”s indicate stop codons. Amino acids common to the majority of organisms are shown above the position lines and represented with dots in the alignment. Note that PP1ß is highly conserved across phylogeny, with only one amino acid difference between human PP1ß and <i>Xenopus</i> PP1ß or mouse PP1ß. The alignment was done using DNAStar software.</p

Homozygous GLCs of <i>XE55</i> mutations cause over-constriction of Anillin-stained ring canals during IC.

<p>Confocal images of part of germaria co-immunostained with antibodies against anillin (green) and α-spectrin (red). All cysts are at stage IVg, the sixth stage of the 4^th mitotic division. Homozygous germline clones are outlined with dashed lines. The mitotic origins of ring canals are labeled with 1, 2, 3, or 4 for the 1^st, 2^nd, 3^rd, or 4^th mitotic division. The unmeasurable ring canals are marked with asterisks. (<b>A</b>) Heterozygous <i>XE55A</i>. (<b>B</b>) Wild-type (<i>OreR</i>). (<b>C–G</b>) Homozygous XE55s: <i>XE55A</i> (C), <i>XE55B</i> (D), <i>XE55C</i> (E), <i>XE55D</i> (F), or <i>XE55E</i> (G). (<b>H</b>) Homozygous <i>flw^FP41</i>. Note that homozygous <i>XE55s</i> (C–G) and <i>flw^FP41</i> (H) in germ cells caused formation of small ring canals. Two normal M4 ring canals from heterozygous <i>XE55B</i> (D, right) and one normal M2 ring canal from heterozygous <i>XE55D</i> (F, left) were also labeled for comparison. The genotypes for the heterozygous XE55s in this and all the following figures are <i>XE55^mutation</i>/<i>ubi-RFP^NLS hsFlp¹²² FRT19A</i>. All panels have the same magnification. Scale bar: 2 µm.</p

The <i>XE55</i> IC phenotype is more severe after all four mitotic divisions than during those divisions.

<p>(<b>A–F</b>) Confocal images of anillin (green) and α-spectrin (red) immunostained germline cysts at stages IId (A–B), IIId (C–D), and IVg (E–F). Genotypes of the germ cells housing the ring canals of interest are heterozygous <i>XE55A</i> for panels A, C, and E and homozygous <i>XE55A</i> for panels B, D, and F. Homozygous germline mosaic clones, marked by the absence of RFP (not shown), are outlined. The numbers mark the mitotic origins of ring canals. Two normal M4 ring canals from heterozygous <i>XE55A</i> (F, right) were also labeled for comparison; the top M4 ring canal appeared small because the current focal plane does not reveal its real diameter. All images have the same magnification. Scale bar: 2 µm. (<b>G</b>) Average diameters of ring canals of cysts with heterozygous (grey) or homozygous (doted) <i>XE55A</i>. Stage II is on the left and stage IVg on the right. (<b>H</b>) Percentage of ring canals too small to be measured. Error bars are standard error of the means. Numbers in panels G and H are ring canals measured or counted. The ring canal diameters that are significantly different (<i>P</i><0.05) are marked with asterisks. 14 germaria were analyzed. Data shown is from a single representative experiment.</p

Lethal phases of <i>flw</i> alleles and rescue data.

<p>Note: Lethal phase is defined as the maximum stage the animals can reach. Some animals die at earlier stages, and if the culture is too crowded, the lethal phase can be earlier. The lethal phases of <i>flw⁶</i>, <i>flw⁷</i>, and <i>flw^FP41</i> are listed as already published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070502#pone.0070502-Raghavan1" target="_blank">[23]</a>-<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070502#pone.0070502-Sun1" target="_blank">[25]</a>. In addition, since the eggs for lethal phase analysis were from heterozygous females, maternal contributions were included. Maternal contributions cannot be avoided because Flw is required for oogenesis. All alleles except <i>XE55C</i> were rescued to fertile adults with an <i>flw</i>-carrying duplication <i>Dp(1;3)DC224</i>.</p>*<p>: <i>XE55C</i> itself was not rescued, but <i>XE55C</i> females transheterozygous with <i>XE55D</i> or <i>XE55E</i> were rescued by <i>Dp(1;3)DC224</i> to fertile adults. This suggests that there is a second non-<i>flw</i> lethal hit on the <i>XE55C</i>-carrying chromosome.</p><p>For rescues with Pp1ß9C (<i>flw</i> cDNA under the control of UASt promoter, which expresses Flw-pB in somatic cells specified by Gal4 lines used <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070502#pone.0070502-Raghavan1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070502#pone.0070502-Vereshchagina1" target="_blank">[24]</a>), the following Gal4 lines were used: tub-Gal4 (<i>w; UAS-Pp1ß9C/CyO; TubGal4/TM6C</i>) expressing Gal4 under the control of the <i>α-tubulin84B</i> promoter, maternal Gal4 (<i>w*; P{w[+mC] = matalpha4-GAL-VP16}V37</i>) expressing GAL4-VP16 fusion protein under the control of the <i>α-tubulin67C</i> promoter, act Gal4-II (<i>y¹w*; P{w[+mC] = Act5C-GAL4}25FO1/CyO, y⁺</i>) and act Gal4-III (<i>y¹w*; P{w[+mC] = Act5C-GAL4}25FO1/CyO, y⁺</i>) expressing Gal4 under the control of <i>actin5C</i> promoter. For rescuing with tubGal4, males of <i>w; UAS-Pp1ß9C/CyO; TubGal4/TM6C</i> were crossed with females of <i>flw^mutations/FM7</i>. For other Gal4-UASt-Pp1ß9C mediated rescues, females of <i>flw^mutations/FM7; UASPp1ß9C/CyO</i> were crossed with males of corresponding Gal4 lines. For rescuing with duplication, males carrying <i>Dp(1;3)DC224</i> were crossed with females of <i>flw^mutations/FM7</i>. For female rescues, rescued <i>XE55</i> mutant males were crossed with females of <i>flw^mutations/FM7.</i> NA: not available.</p