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 G3: Genes|Genomes|Genetics publishes high-quality, valuable findings, regardless of perceived impact. G3 publishes foundational research that generates useful genetic and genomic information such as genome maps, single gene studies, QTL studies, mutant screens and advances in methods and technology, novel mutant collections, genome-wide association studies (GWAS)  including gene expression, SNP, and CNV studies; exome sequences related to a specific disease but lacking functional follow-up, personal exome and genome sequencing case, disease, and population reports, and more.   Conceived by the Genetics Society of America, with its first issue published June 2011, G3 is fully open access. G3 uses a Creative Commons license that allows the most free use of the data, which anyone can download, analyze, mine, and reuse, provided that the authors of the article receive credit. GSA believes that rapid dissemination of useful data is the necessary foundation for analysis that leads to mechanistic insights. It is our hope is that this strategy will spawn new discovery.   Like GENETICS, G3 is fast—with a 31-day turnaround time from submission to first decision—and rapid time-to-publication. And like GENETICS, G3 manuscripts are thoroughly peer-reviewed, with careful decisions made by practicing scientists. Before publication, G3 articles receive a thorough copy-edit, ensuring that articles enjoy maximum clarity and impact. Thompson Reuters JCR Impact Factor (2014): 3.198 EigenFactor (2014): 0.00978 Cited Half-life (2014): 2.1 years       What's Inside the Current Issue of G3    Thursday, October 13 2016 09:06:55 AM Multiparental Populations: A Call for Papers Thursday, October 13 2016 09:06:55 AM A Drosophila LexA Enhancer-Trap Resource for Developmental Biology and Neuroendocrine Research Kockel, L., Huq, L. M., Ayyar, A., Herold, E., MacAlpine, E., Logan, M., Savvides, C., Kim, G. E. S., Chen, J., Clark, T., Duong, T., Fazel-Rezai, V., Havey, D., Han, S., Jagadeesan, R., Kim, E. S. J., Lee, D., Lombardo, K., Piyale, I., Shi, H., Stahr, L., Tung, D., Tayvah, U., Wang, F., Wang, J.-H., Xiao, S., Topper, S. M., Park, S., Rotondo, C., Rankin, A. E., Chisholm, T. W., Kim, S. K. Novel binary gene expression tools like the LexA-LexAop system could powerfully enhance studies of metabolism, development, and neurobiology in Drosophila. However, specific LexA drivers for neuroendocrine cells and many other developmentally relevant systems remain limited. In a unique high school biology course, we generated a LexA-based enhancer trap collection by transposon mobilization. The initial collection provides a source of novel LexA-based elements that permit targeted gene expression in the corpora cardiaca, cells central for metabolic homeostasis, and other neuroendocrine cell types. The collection further contains specific LexA drivers for stem cells and other enteric cells in the gut, and other developmentally relevant tissue types. We provide detailed analysis of nearly 100 new LexA lines, including molecular mapping of insertions, description of enhancer-driven reporter expression in larval tissues, and adult neuroendocrine cells, comparison with established enhancer trap collections and tissue specific RNAseq. Generation of this open-resource LexA collection facilitates neuroendocrine and developmental biology investigations, and shows how empowering secondary school science can achieve research and educational goals. Thursday, October 13 2016 09:06:55 AM Tandem Duplication Events in the Expansion of the Small Heat Shock Protein Gene Family in Solanum lycopersicum (cv. Heinz 1706) Krsticevic, F. J., Arce, D. P., Ezpeleta, J., Tapia, E. In plants, fruit maturation and oxidative stress can induce small heat shock protein (sHSP) synthesis to maintain cellular homeostasis. Although the tomato reference genome was published in 2012, the actual number and functionality of sHSP genes remain unknown. Using a transcriptomic (RNA-seq) and evolutionary genomic approach, putative sHSP genes in the Solanum lycopersicum (cv. Heinz 1706) genome were investigated. A sHSP gene family of 33 members was established. Remarkably, roughly half of the members of this family can be explained by nine independent tandem duplication events that determined, evolutionarily, their functional fates. Within a mitochondrial class subfamily, only one duplicated member, Solyc08g078700, retained its ancestral chaperone function, while the others, Solyc08g078710 and Solyc08g078720, likely degenerated under neutrality and lack ancestral chaperone function. Functional conservation occurred within a cytosolic class I subfamily, whose four members, Solyc06g076570, Solyc06g076560, Solyc06g076540, and Solyc06g076520, support ~57% of the total sHSP RNAm in the red ripe fruit. Subfunctionalization occurred within a new subfamily, whose two members, Solyc04g082720 and Solyc04g082740, show heterogeneous differential expression profiles during fruit ripening. These findings, involving the birth/death of some genes or the preferential/plastic expression of some others during fruit ripening, highlight the importance of tandem duplication events in the expansion of the sHSP gene family in the tomato genome. Despite its evolutionary diversity, the sHSP gene family in the tomato genome seems to be endowed with a core set of four homeostasis genes: Solyc05g014280, Solyc03g082420, Solyc11g020330, and Solyc06g076560, which appear to provide a baseline protection during both fruit ripening and heat shock stress in different tomato tissues. Thursday, October 13 2016 09:06:55 AM Genome-Wide Analysis of Polyadenylation Events in Schmidtea mediterranea Lakshmanan, V., Bansal, D., Kulkarni, J., Poduval, D., Krishna, S., Sasidharan, V., Anand, P., Seshasayee, A., Palakodeti, D. In eukaryotes, 3' untranslated regions (UTRs) play important roles in regulating posttranscriptional gene expression. The 3'UTR is defined by regulated cleavage/polyadenylation of the pre-mRNA. The advent of next-generation sequencing technology has now enabled us to identify these events on a genome-wide scale. In this study, we used poly(A)-position profiling by sequencing (3P-Seq) to capture all poly(A) sites across the genome of the freshwater planarian, Schmidtea mediterranea, an ideal model system for exploring the process of regeneration and stem cell function. We identified the 3'UTRs for ~14,000 transcripts and thus improved the existing gene annotations. We found 97 transcripts, which are polyadenylated within an internal exon, resulting in the shrinking of the ORF and loss of a predicted protein domain. Around 40% of the transcripts in planaria were alternatively polyadenylated (ApA), resulting either in an altered 3'UTR or a change in coding sequence. We identified specific ApA transcript isoforms that were subjected to miRNA mediated gene regulation using degradome sequencing. In this study, we also confirmed a tissue-specific expression pattern for alternate polyadenylated transcripts. The insights from this study highlight the potential role of ApA in regulating the gene expression essential for planarian regeneration. Thursday, October 13 2016 09:06:55 AM Characterization of a Novel MMS-Sensitive Allele of Schizosaccharomyces pombe mcm4+ Ranatunga, N. S., Forsburg, S. L. The minichromosome maintenance (MCM) complex is the conserved helicase motor of the eukaryotic replication fork. Mutations in the Mcm4 subunit are associated with replication stress and double strand breaks in multiple systems. In this work, we characterize a new temperature-sensitive allele of Schizosaccharomyces pombe mcm4+. Uniquely among known mcm4 alleles, this mutation causes sensitivity to the alkylation damaging agent methyl methanesulfonate (MMS). Even in the absence of treatment or temperature shift, mcm4-c106 cells show increased repair foci of RPA and Rad52, and require the damage checkpoint for viability, indicating genome stress. The mcm4-c106 mutant is synthetically lethal with mutations disrupting fork protection complex (FPC) proteins Swi1 and Swi3. Surprisingly, we found that the deletion of rif1+ suppressed the MMS-sensitive phenotype without affecting temperature sensitivity. Together, these data suggest that mcm4-c106 destabilizes replisome structure. Thursday, October 13 2016 09:06:55 AM The Evolution of the FT/TFL1 Genes in Amaranthaceae and Their Expression Patterns in the Course of Vegetative Growth and Flowering in Chenopodium rubrum Drabešova, J., Černa, L., Mašterova, H., Kolouškova, P., Potocky, M., Štorchova, H. The FT/TFL1 gene family controls important aspects of plant development: MFT-like genes affect germination, TFL1-like genes act as floral inhibitors, and FT-like genes are floral activators. Gene duplications produced paralogs with modified functions required by the specific lifestyles of various angiosperm species. We constructed the transcriptome of the weedy annual plant Chenopodium rubrum and used it for the comprehensive search for the FT/TFL1 genes. We analyzed their phylogenetic relationships across Amaranthaceae and all angiosperms. We discovered a very ancient phylogenetic clade of FT genes represented by the CrFTL3 gene of C. rubrum. Another paralog CrFTL2 showed an unusual structural rearrangement which might have contributed to the functional shift. We examined the transcription patterns of the FT/TFL1 genes during the vegetative growth and floral transition in C. rubrum to get clues about their possible functions. All the genes except for the constitutively expressed CrFTL2 gene, and the CrFTL3 gene, which was transcribed only in seeds, exhibited organ-specific expression influenced by the specific light regime. The CrFTL1 gene was confirmed as a single floral activator from the FT/TFL1 family in C. rubrum. Its floral promoting activity may be counteracted by CrTFL1. C. rubrum emerges as an easily manipulated model for the study of floral induction in weedy fast-cycling plants lacking a juvenile phase. Thursday, October 13 2016 09:06:55 AM Php4 Is a Key Player for Iron Economy in Meiotic and Sporulating Cells Brault, A., Rallis, C., Normant, V., Garant, J.-M., Bahler, J., Labbe, S. Meiosis is essential for sexually reproducing organisms, including the fission yeast Schizosaccharomyces pombe. In meiosis, chromosomes replicate once in a diploid precursor cell (zygote), and then segregate twice to generate four haploid meiotic products, named spores in yeast. In S. pombe, Php4 is responsible for the transcriptional repression capability of the heteromeric CCAAT-binding factor to negatively regulate genes encoding iron-using proteins under low-iron conditions. Here, we show that the CCAAT-regulatory subunit Php4 is required for normal progression of meiosis under iron-limiting conditions. Cells lacking Php4 exhibit a meiotic arrest at metaphase I. Microscopic analyses of cells expressing functional GFP-Php4 show that it colocalizes with chromosomal material at every stage of meiosis under low concentrations of iron. In contrast, GFP-Php4 fluorescence signal is lost when cells undergo meiosis under iron-replete conditions. Global gene expression analysis of meiotic cells using DNA microarrays identified 137 genes that are regulated in an iron- and Php4-dependent manner. Among them, 18 genes are expressed exclusively during meiosis and constitute new putative Php4 target genes, which include hry1+ and mug14+. Further analysis validates that Php4 is required for maximal and timely repression of hry1+ and mug14+ genes. Using a chromatin immunoprecipitation approach, we show that Php4 specifically associates with hry1+ and mug14+ promoters in vivo. Taken together, the results reveal that in iron-starved meiotic cells, Php4 is essential for completion of the meiotic program since it participates in global gene expression reprogramming to optimize the use of limited available iron. Thursday, October 13 2016 09:06:55 AM Genome Evolution in Three Species of Cactophilic Drosophila Sanchez-Flores, A., Penaloza, F., Carpinteyro-Ponce, J., Nazario-Yepiz, N., Abreu-Goodger, C., Machado, C. A., Markow, T. A. We report genomes of two species of cactophilic Drosophila: Drosophila arizonae and D. navojoa. These two are the closest relatives of D. mojavensis, forming the D. mojavensis cluster. D. mojavensis and D. arizonae diverged from D. navojoa ~5.8 Mya, while the split between D. arizonae and D. mojavensis is more recent, at 1.5 Mya. Together the three genomes provide opportunities to examine genomic changes associated with speciation and host shifts in this ecologically defined group of flies. The three species are also separated by fixed inversion differences in three of their six chromosomes. While the levels of nucleotide divergence in the colinear chromosomes are significantly lower than in the inverted chromosomes, consistent with a past role of the inversions in preventing gene flow, the patterns differ among the inverted chromosomes when the locations of nucleotides inside or outside of the inversions are considered. For Muller element E, there is greater divergence external to the inversion breakpoints. For Muller A, the divergence is slightly higher inside the inversions, while for Muller B, the breakpoints and hence the difference in substitutions in relation to the inversions could not be determined. The differences among the inverted chromosomes, especially once the breakpoints are clearly established, could aid in dating the origins of the inversions. Thursday, October 13 2016 09:06:55 AM Cross-Validation Without Doing Cross-Validation in Genome-Enabled Prediction Gianola, D., Schon, C.-C. Cross-validation of methods is an essential component of genome-enabled prediction of complex traits. We develop formulae for computing the predictions that would be obtained when one or several cases are removed in the training process, to become members of testing sets, but by running the model using all observations only once. Prediction methods to which the developments apply include least squares, best linear unbiased prediction (BLUP) of markers, or genomic BLUP, reproducing kernels Hilbert spaces regression with single or multiple kernel matrices, and any member of a suite of linear regression methods known as "Bayesian alphabet." The approach used for Bayesian models is based on importance sampling of posterior draws. Proof of concept is provided by applying the formulae to a wheat data set representing 599 inbred lines genotyped for 1279 markers, and the target trait was grain yield. The data set was used to evaluate predictive mean-squared error, impact of alternative layouts on maximum likelihood estimates of regularization parameters, model complexity, and residual degrees of freedom stemming from various strengths of regularization, as well as two forms of importance sampling. Our results will facilitate carrying out extensive cross-validation without model retraining for most machines employed in genome-assisted prediction of quantitative traits. Thursday, October 13 2016 09:06:55 AM Cryptic Genetic Variation for Arabidopsis thaliana Seed Germination Speed in a Novel Salt Stress Environment Yuan, W., Flowers, J. M., Sahraie, D. J., Purugganan, M. D. The expansion of species ranges frequently necessitates responses to novel environments. In plants, the ability of seeds to disperse to marginal areas relies in part to its ability to germinate under stressful conditions. Here we examine the genetic architecture of Arabidopsis thaliana germination speed under a novel, saline environment, using an Extreme QTL (X-QTL) mapping platform we previously developed. We find that early germination in normal and salt conditions both rely on a QTL on the distal arm of chromosome 4, but we also find unique QTL on chromosomes 1, 2, 4, and 5 that are specific to salt stress environments. Moreover, different QTLs are responsible for early vs. late germination, suggesting a temporal component to the expression of life history under these stress conditions. Our results indicate that cryptic genetic variation exists for responses to a novel abiotic stress, which may suggest a role of such variation in adaptation to new climactic conditions or growth environments. Thursday, October 13 2016 09:06:55 AM Interallelic Transcriptional Enhancement as an in Vivo Measure of Transvection in Drosophila melanogaster Noble, G. P., Dolph, P. J., Supattapone, S. Transvection—pairing-dependent interallelic regulation resulting from enhancer action in trans—occurs throughout the Drosophila melanogaster genome, likely as a result of the extensive somatic homolog pairing seen in Dipteran species. Recent studies of transvection in Drosophila have demonstrated important qualitative differences between enhancer action in cis vs. in trans, as well as a modest synergistic effect of cis- and trans-acting enhancers on total tissue transcript levels at a given locus. In the present study, we identify a system in which cis- and trans-acting GAL4-UAS enhancer synergism has an unexpectedly large quantitative influence on gene expression, boosting total tissue transcript levels at least fourfold relative to those seen in the absence of transvection. We exploit this strong quantitative effect by using publicly available UAS-shRNA constructs from the TRiP library to assay candidate genes for transvection activity in vivo. The results of the present study, which demonstrate that in trans activation by simple UAS enhancers can have large quantitative effects on gene expression in Drosophila, have important new implications for experimental design utilizing the GAL4-UAS system. Thursday, October 13 2016 09:06:55 AM Glucose or Altered Ceramide Biosynthesis Mediate Oxygen Deprivation Sensitivity Through Novel Pathways Revealed by Transcriptome Analysis in Caenorhabditis elegans Ladage, M. L., King, S. D., Burks, D. J., Quan, D. L., Garcia, A. M., Azad, R. K., Padilla, P. A. Individuals with type 2 diabetes display metabolic abnormalities, such as hyperglycemia, increased free fatty acids, insulin resistance, and altered ceramide levels, that contribute to vascular dysfunctions and compromised oxygen delivery. Caenorhabditis elegans fed a glucose-supplemented diet or with altered ceramide metabolism, due to a hyl-2 mutation, are sensitive to oxygen deprivation (anoxia). Our experiments showed that the combination of these factors further decreased the anoxia survival. RNA-sequencing analysis was performed to assess how a glucose-supplemented diet and/or a hyl-2 mutation altered the transcriptome. Comparison analysis of transcripts associated with anoxia-sensitive animals [hyl-2(tm2031) mutation or a glucose diet] revealed 199 common transcripts encoded by genes with known or predicted functions involving innate immunity, cuticle function (collagens), or xenobiotic and endobiotic phase I and II detoxification system. Use of RNA interference (RNAi) to target gene products of the xenobiotic and endobiotic phase I and II detoxification system (UDP-glycosyltransferase and Cytochrome p450 genes; ugt-15, ugt-18, ugt-19, ugt-41, ugt-63, cyp-13A12, cyp-25A1, and cyp-33C8) increased anoxia survival in wild-type animals fed a standard diet. Anoxia sensitivity of the hyl-2(tm2031) animals was suppressed by RNAi of cyp-25A1 or cyp-33C8 genes. A glucose diet fed to the P0 hermaphrodite decreased the anoxia survival of its F1 embryos; however, the RNAi of ugt-63 and cyp-33C8 suppressed anoxia sensitivity. These studies provide evidence that the detoxification system impacts oxygen deprivation responses and that C. elegans can be used to model the conserved detoxification system. Thursday, October 13 2016 09:06:55 AM Efficient CRISPR-Mediated Post-Transcriptional Gene Silencing in a Hyperthermophilic Archaeon Using Multiplexed crRNA Expression Zebec, Z., Zink, I. A., Kerou, M., Schleper, C. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-mediated RNA degradation is catalyzed by a type III system in the hyperthermophilic archaeon Sulfolobus solfataricus. Earlier work demonstrated that the system can be engineered to target specifically mRNA of an endogenous host reporter gene, namely the β-galactosidase in S. solfataricus. Here, we investigated the effect of single and multiple spacers targeting the mRNA of a second reporter gene, α-amylase, at the same, and at different, locations respectively, using a minimal CRISPR (miniCR) locus supplied on a viral shuttle vector. The use of increasing numbers of spacers reduced mRNA levels at progressively higher levels, with three crRNAs (CRISPR RNAs) leading to ~ 70–80% reduction, and five spacers resulting in an α-amylase gene knockdown of > 90% measured on both mRNA and protein activity levels. Our results indicate that this technology can be used to increase or modulate gene knockdown for efficient post-transcriptional gene silencing in hyperthermophilic archaea, and potentially also in other organisms. Thursday, October 13 2016 09:06:55 AM A New Advanced Backcross Tomato Population Enables High Resolution Leaf QTL Mapping and Gene Identification Fulop, D., Ranjan, A., Ofner, I., Covington, M. F., Chitwood, D. H., West, D., Ichihashi, Y., Headland, L., Zamir, D., Maloof, J. N., Sinha, N. R. Quantitative Trait Loci (QTL) mapping is a powerful technique for dissecting the genetic basis of traits and species differences. Established tomato mapping populations between domesticated tomato (Solanum lycopersicum) and its more distant interfertile relatives typically follow a near isogenic line (NIL) design, such as the S. pennellii Introgression Line (IL) population, with a single wild introgression per line in an otherwise domesticated genetic background. Here, we report on a new advanced backcross QTL mapping resource for tomato, derived from a cross between the M82 tomato cultivar and S. pennellii. This so-called Backcrossed Inbred Line (BIL) population is comprised of a mix of BC2 and BC3 lines, with domesticated tomato as the recurrent parent. The BIL population is complementary to the existing S. pennellii IL population, with which it shares parents. Using the BILs, we mapped traits for leaf complexity, leaflet shape, and flowering time. We demonstrate the utility of the BILs for fine-mapping QTL, particularly QTL initially mapped in the ILs, by fine-mapping several QTL to single or few candidate genes. Moreover, we confirm the value of a backcrossed population with multiple introgressions per line, such as the BILs, for epistatic QTL mapping. Our work was further enabled by the development of our own statistical inference and visualization tools, namely a heterogeneous hidden Markov model for genotyping the lines, and by using state-of-the-art sparse regression techniques for QTL mapping. Thursday, October 13 2016 09:06:55 AM An Eye on Trafficking Genes: Identification of Four Eye Color Mutations in Drosophila Grant, P., Maga, T., Loshakov, A., Singhal, R., Wali, A., Nwankwo, J., Baron, K., Johnson, D. Genes that code for proteins involved in organelle biogenesis and intracellular trafficking produce products that are critical in normal cell function . Conserved orthologs of these are present in most or all eukaryotes, including Drosophila melanogaster. Some of these genes were originally identified as eye color mutants with decreases in both types of pigments found in the fly eye. These criteria were used for identification of such genes, four eye color mutations that are not annotated in the genome sequence: chocolate, maroon, mahogany, and red Malpighian tubules were molecularly mapped and their genome sequences have been evaluated. Mapping was performed using deletion analysis and complementation tests. chocolate is an allele of the VhaAC39-1 gene, which is an ortholog of the Vacuolar H+ ATPase AC39 subunit 1. maroon corresponds to the Vps16A gene and its product is part of the HOPS complex, which participates in transport and organelle fusion. red Malpighian tubule is the CG12207 gene, which encodes a protein of unknown function that includes a LysM domain. mahogany is the CG13646 gene, which is predicted to be an amino acid transporter. The strategy of identifying eye color genes based on perturbations in quantities of both types of eye color pigments has proven useful in identifying proteins involved in trafficking and biogenesis of lysosome-related organelles. Mutants of these genes can form the basis of valuable in vivo models to understand these processes. Thursday, October 13 2016 09:06:55 AM Rapid Screening for CRISPR-Directed Editing of the Drosophila Genome Using white Coconversion Ge, D. T., Tipping, C., Brodsky, M. H., Zamore, P. D. Adoption of a streamlined version of the bacterial clustered regular interspersed short palindromic repeat (CRISPR)/Cas9 defense system has accelerated targeted genome engineering. The Streptococcus pyogenes Cas9 protein, directed by a simplified, CRISPR-like single-guide RNA, catalyzes a double-stranded DNA break at a specific genomic site; subsequent repair by end joining can introduce mutagenic insertions or deletions, while repair by homologous recombination using an exogenous DNA template can incorporate new sequences at the target locus. However, the efficiency of Cas9-directed mutagenesis is low in Drosophila melanogaster. Here, we describe a strategy that reduces the time and effort required to identify flies with targeted genomic changes. The strategy uses editing of the white gene, evidenced by altered eye color, to predict successful editing of an unrelated gene-of-interest. The red eyes of wild-type flies are readily distinguished from white-eyed (end-joining-mediated loss of White function) or brown-eyed (recombination-mediated conversion to the whitecoffee allele) mutant flies. When single injected G0 flies produce individual G1 broods, flies carrying edits at a gene-of-interest were readily found in broods in which all G1 offspring carried white mutations. Thus, visual assessment of eye color substitutes for wholesale PCR screening of large numbers of G1 offspring. We find that end-joining-mediated mutations often show signatures of microhomology-mediated repair and that recombination-based mutations frequently involve donor plasmid integration at the target locus. Finally, we show that gap repair induced by two guide RNAs more reliably converts the intervening target sequence, whereas the use of Lig4169 mutants to suppress end joining does not improve recombination efficacy. Thursday, October 13 2016 09:06:55 AM Whole-Genome Sequencing and iPLEX MassARRAY Genotyping Map an EMS-Induced Mutation Affecting Cell Competition in Drosophila melanogaster Lee, C.-H., Rimesso, G., Reynolds, D. M., Cai, J., Baker, N. E. Cell competition, the conditional loss of viable genotypes only when surrounded by other cells, is a phenomenon observed in certain genetic mosaic conditions. We conducted a chemical mutagenesis and screen to recover new mutations that affect cell competition between wild-type and RpS3 heterozygous cells. Mutations were identified by whole-genome sequencing, making use of software tools that greatly facilitate the distinction between newly induced mutations and other sources of apparent sequence polymorphism, thereby reducing false-positive and false-negative identification rates. In addition, we utilized iPLEX MassARRAY for genotyping recombinant chromosomes. These approaches permitted the mapping of a new mutation affecting cell competition when only a single allele existed, with a phenotype assessed only in genetic mosaics, without the benefit of complementation with existing mutations, deletions, or duplications. These techniques expand the utility of chemical mutagenesis and whole-genome sequencing for mutant identification. We discuss mutations in the Atm and Xrp1 genes identified in this screen. Thursday, October 13 2016 09:06:55 AM The Genetic Architecture of Noise-Induced Hearing Loss: Evidence for a Gene-by-Environment Interaction Lavinsky, J., Ge, M., Crow, A. L., Pan, C., Wang, J., Salehi, P., Myint, A., Eskin, E., Allayee, H., Lusis, A. J., Friedman, R. A. The discovery of environmentally specific genetic effects is crucial to the understanding of complex traits, such as susceptibility to noise-induced hearing loss (NIHL). We describe the first genome-wide association study (GWAS) for NIHL in a large and well-characterized population of inbred mouse strains, known as the Hybrid Mouse Diversity Panel (HMDP). We recorded auditory brainstem response (ABR) thresholds both pre and post 2-hr exposure to 10-kHz octave band noise at 108 dB sound pressure level in 5–6-wk-old female mice from the HMDP (4–5 mice/strain). From the observation that NIHL susceptibility varied among the strains, we performed a GWAS with correction for population structure and mapped a locus on chromosome 6 that was statistically significantly associated with two adjacent frequencies. We then used a "genetical genomics" approach that included the analysis of cochlear eQTLs to identify candidate genes within the GWAS QTL. In order to validate the gene-by-environment interaction, we compared the effects of the postnoise exposure locus with that from the same unexposed strains. The most significant SNP at chromosome 6 (rs37517079) was associated with noise susceptibility, but was not significant at the same frequencies in our unexposed study. These findings demonstrate that the genetic architecture of NIHL is distinct from that of unexposed hearing levels and provide strong evidence for gene-by-environment interactions in NIHL. Thursday, October 13 2016 09:06:55 AM Ctr9, a Key Component of the Paf1 Complex, Affects Proliferation and Terminal Differentiation in the Developing Drosophila Nervous System Bahrampour, S., Thor, S. The Paf1 protein complex (Paf1C) is increasingly recognized as a highly conserved and broadly utilized regulator of a variety of transcriptional processes. These include the promotion of H3K4 and H3K36 trimethylation, H2BK123 ubiquitination, RNA Pol II transcriptional termination, and also RNA-mediated gene silencing. Paf1C contains five canonical protein components, including Paf1 and Ctr9, which are critical for overall complex integrity, as well as Rtf1, Leo1, and Cdc73/Parafibromin(Hrpt2)/Hyrax. In spite of a growing appreciation for the importance of Paf1C from yeast and mammalian studies, there has only been limited work in Drosophila. Here, we provide the first detailed phenotypic study of Ctr9 function in Drosophila. We found that Ctr9 mutants die at late embryogenesis or early larval life, but can be partly rescued by nervous system reexpression of Ctr9. We observed a number of phenotypes in Ctr9 mutants, including increased neuroblast numbers, increased nervous system proliferation, as well as downregulation of many neuropeptide genes. Analysis of cell cycle and regulatory gene expression revealed upregulation of the E2f1 cell cycle factor, as well as changes in Antennapedia and Grainy head expression. We also found reduction of H3K4me3 modification in the embryonic nervous system. Genome-wide transcriptome analysis points to additional downstream genes that may underlie these Ctr9 phenotypes, revealing gene expression changes in Notch pathway target genes, cell cycle genes, and neuropeptide genes. In addition, we find significant effects on the gene expression of metabolic genes. These findings reveal that Ctr9 is an essential gene that is necessary at multiple stages of nervous system development, and provides a starting point for future studies of the Paf1C in Drosophila. Thursday, October 13 2016 09:06:55 AM Genome-Wide Association Studies with a Genomic Relationship Matrix: A Case Study with Wheat and Arabidopsis Gianola, D., Fariello, M. I., Naya, H., Schon, C.-C. Standard genome-wide association studies (GWAS) scan for relationships between each of p molecular markers and a continuously distributed target trait. Typically, a marker-based matrix of genomic similarities among individuals (G) is constructed, to account more properly for the covariance structure in the linear regression model used. We show that the generalized least-squares estimator of the regression of phenotype on one or on m markers is invariant with respect to whether or not the marker(s) tested is(are) used for building G, provided variance components are unaffected by exclusion of such marker(s) from G. The result is arrived at by using a matrix expression such that one can find many inverses of genomic relationship, or of phenotypic covariance matrices, stemming from removing markers tested as fixed, but carrying out a single inversion. When eigenvectors of the genomic relationship matrix are used as regressors with fixed regression coefficients, e.g., to account for population stratification, their removal from G does matter. Removal of eigenvectors from G can have a noticeable effect on estimates of genomic and residual variances, so caution is needed. Concepts were illustrated using genomic data on 599 wheat inbred lines, with grain yield as target trait, and on close to 200 Arabidopsis thaliana accessions. Thursday, October 13 2016 09:06:55 AM Regulation of the MEI-1/MEI-2 Microtubule-Severing Katanin Complex in Early Caenorhabditis elegans Development Beard, S. M., Smit, R. B., Chan, B. G., Mains, P. E. Thursday, October 13 2016 09:06:55 AM Characterization of the Far Transcription Factor Family in Aspergillus flavus Luo, X., Affeldt, K. J., Keller, N. P. Metabolism of fatty acids is a critical requirement for the pathogenesis of oil seed pathogens including the fungus Aspergillus flavus. Previous studies have correlated decreased ability to grow on fatty acids with reduced virulence of this fungus on host seed. Two fatty acid metabolism regulatory transcription factors, FarA and FarB, have been described in other filamentous fungi. Unexpectedly, we find A. flavus possesses three Far homologs, FarA, FarB, and FarC, with FarA and FarC showing a greater protein similarity to each other than FarB. farA and farB are located in regions of colinearity in all Aspergillus spp. sequenced to date, whereas farC is limited to a subset of species where it is inserted in an otherwise colinear region in Aspergillus genomes. Deletion and overexpression (OE) of farA and farB, but not farC, yielded mutants with aberrant growth patterns on specific fatty acids as well as altered expression of genes involved in fatty acid metabolism. Marked differences included significant growth defects of both farA and farB on medium-chain fatty acids and decreased growth of OE::farA on unsaturated fatty acids. Loss of farA diminished expression of mitochondrial β-oxidation genes whereas OE::farA inhibited expression of genes involved in unsaturated fatty acid catabolism. FarA also positively regulated the desaturase genes required to generate polyunsaturated fatty acids. Aflatoxin production on toxin-inducing media was significantly decreased in the farB mutant and increased in the OE::farB mutant, with gene expression data supporting a role for FarB in tying β-oxidation processes with aflatoxin accumulation. Thursday, October 13 2016 09:06:55 AM Head Transcriptomes of Two Closely Related Species of Fruit Flies of the Anastrepha fraterculus Group Reveals Divergent Genes in Species with Extensive Gene Flow Rezende, V. B., Congrains, C., Lima, A. L. A., Campanini, E. B., Nakamura, A. M., Oliveira, J. L. d., Chahad-Ehlers, S., Junior, I. S., Alves de Brito, R. Several fruit flies species of the Anastrepha fraterculus group are of great economic importance for the damage they cause to a variety of fleshy fruits. Some species in this group have diverged recently, with evidence of introgression, showing similar morphological attributes that render their identification difficult, reinforcing the relevance of identifying new molecular markers that may differentiate species. We investigated genes expressed in head tissues from two closely related species: A. obliqua and A. fraterculus, aiming to identify fixed single nucleotide polymorphisms (SNPs) and highly differentiated transcripts, which, considering that these species still experience some level of gene flow, could indicate potential candidate genes involved in their differentiation process. We generated multiple libraries from head tissues of these two species, at different reproductive stages, for both sexes. Our analyses indicate that the de novo transcriptome assemblies are fairly complete. We also produced a hybrid assembly to map each species’ reads, and identified 67,470 SNPs in A. fraterculus, 39,252 in A. obliqua, and 6386 that were common to both species. We identified 164 highly differentiated unigenes that had a mean interspecific index ($$\overline{D}$$) of at least 0.94. We selected unigenes that had Ka/Ks higher than 0.5, or had at least three or more highly differentiated SNPs as potential candidate genes for species differentiation. Among these candidates, we identified proteases, regulators of redox homeostasis, and an odorant-binding protein (Obp99c), among other genes. The head transcriptomes described here enabled the identification of thousands of genes hitherto unavailable for these species, and generated a set of candidate genes that are potentially important to genetically identify species and understand the speciation process in the presence of gene flow of A. obliqua and A. fraterculus. Thursday, October 13 2016 09:06:55 AM Sporadic Gene Loss After Duplication Is Associated with Functional Divergence of Sirtuin Deacetylases Among Candida Yeast Species Rupert, C. B., Heltzel, J. M. H., Taylor, D. J., Rusche, L. N. Gene duplication promotes the diversification of protein functions in several ways. Ancestral functions can be partitioned between the paralogs, or a new function can arise in one paralog. These processes are generally viewed as unidirectional. However, paralogous proteins often retain related functions and can substitute for one another. Moreover, in the event of gene loss, the remaining paralog might regain ancestral functions that had been shed. To explore this possibility, we focused on the sirtuin deacetylase SIR2 and its homolog HST1 in the CTG clade of yeasts. HST1 has been consistently retained throughout the clade, whereas SIR2 is only present in a subset of species. These NAD+-dependent deacetylases generate condensed chromatin that represses transcription and stabilizes tandemly repeated sequences. By analyzing phylogenetic trees and gene order, we found that a single duplication of the SIR2/HST1 gene occurred, likely prior to the emergence of the CTG clade. This ancient duplication was followed by at least two independent losses of SIR2. Functional characterization of Sir2 and Hst1 in three species revealed that these proteins have not maintained consistent functions since the duplication. In particular, the rDNA locus is deacetylated by Sir2 in Candida albicans, by Hst1 in C. lusitaniae, and by neither paralog in C. parapsilosis. In addition, the subtelomeres in C. albicans are deacetylated by Sir2 rather than by Hst1, which is orthologous to the sirtuin associated with Saccharomyces cerevisiae subtelomeres. These differences in function support the model that sirtuin deacetylases can regain ancestral functions to compensate for gene loss. Thursday, October 13 2016 09:06:55 AM Mutations in the Motile Cilia Gene DNAAF1 Are Associated with Neural Tube Defects in Humans Miao, C., Jiang, Q., Li, H., Zhang, Q., Bai, B., Bao, Y., Zhang, T. Neural tube defects (NTDs) are severe malformations of the central nervous system caused by complex genetic and environmental factors. Among genes involved in NTD, cilia-related genes have been well defined and found to be essential for the completion of neural tube closure (NTC). We have carried out next-generation sequencing on target genes in 373 NTDs and 222 healthy controls, and discovered eight disease-specific rare mutations in cilia-related gene DNAAF1. DNAAF1 plays a central role in cytoplasmic preassembly of distinct dynein-arm complexes, and is expressed in some key tissues involved in neural system development, such as neural tube, floor plate, embryonic node, and brain ependyma epithelial cells in zebrafish and mouse. Therefore, we evaluated the expression and functions of mutations in DNAAF1 in transfected cells to analyze the potential correlation of these mutants to NTDs in humans. One rare frameshift mutation (p.Gln341Argfs*10) resulted in significantly diminished DNAAF1 protein expression, compared to the wild type. Another mutation, p.Lys231Gln, disrupted cytoplasmic preassembly of the dynein-arm complexes in cellular assay. Furthermore, results from NanoString assay on mRNA from NTD samples indicated that DNAAF1 mutants altered the expression level of NTC-related genes. Altogether, these findings suggest that the rare mutations in DNAAF1 may contribute to the susceptibility for NTDs in humans. Thursday, October 13 2016 09:06:55 AM Global Fitness Profiling Identifies Arsenic and Cadmium Tolerance Mechanisms in Fission Yeast Guo, L., Ganguly, A., Sun, L., Suo, F., Du, L.-L., Russell, P. Heavy metals and metalloids such as cadmium [Cd(II)] and arsenic [As(III)] are widespread environmental toxicants responsible for multiple adverse health effects in humans. However, the molecular mechanisms underlying metal-induced cytotoxicity and carcinogenesis, as well as the detoxification and tolerance pathways, are incompletely understood. Here, we use global fitness profiling by barcode sequencing to quantitatively survey the Schizosaccharomyces pombe haploid deletome for genes that confer tolerance of cadmium or arsenic. We identified 106 genes required for cadmium resistance and 110 genes required for arsenic resistance, with a highly significant overlap of 36 genes. A subset of these 36 genes account for almost all proteins required for incorporating sulfur into the cysteine-rich glutathione and phytochelatin peptides that chelate cadmium and arsenic. A requirement for Mms19 is explained by its role in directing iron–sulfur cluster assembly into sulfite reductase as opposed to promoting DNA repair, as DNA damage response genes were not enriched among those required for cadmium or arsenic tolerance. Ubiquinone, siroheme, and pyridoxal 5'-phosphate biosynthesis were also identified as critical for Cd/As tolerance. Arsenic-specific pathways included prefoldin-mediated assembly of unfolded proteins and protein targeting to the peroxisome, whereas cadmium-specific pathways included plasma membrane and vacuolar transporters, as well as Spt–Ada–Gcn5-acetyltransferase (SAGA) transcriptional coactivator that controls expression of key genes required for cadmium tolerance. Notable differences are apparent with corresponding screens in the budding yeast Saccharomyces cerevisiae, underscoring the utility of analyzing toxic metal defense mechanisms in both organisms. Thursday, October 13 2016 09:06:55 AM Obp56h Modulates Mating Behavior in Drosophila melanogaster Shorter, J. R., Dembeck, L. M., Everett, L. J., Morozova, T. V., Arya, G. H., Turlapati, L., St. Armour, G. E., Schal, C., Mackay, T. F. C., Anholt, R. R. H. Social interactions in insects are driven by conspecific chemical signals that are detected via olfactory and gustatory neurons. Odorant binding proteins (Obps) transport volatile odorants to chemosensory receptors, but their effects on behaviors remain poorly characterized. Here, we report that RNAi knockdown of Obp56h gene expression in Drosophila melanogaster enhances mating behavior by reducing courtship latency. The change in mating behavior that results from inhibition of Obp56h expression is accompanied by significant alterations in cuticular hydrocarbon (CHC) composition, including reduction in 5-tricosene (5-T), an inhibitory sex pheromone produced by males that increases copulation latency during courtship. Whole genome RNA sequencing confirms that expression of Obp56h is virtually abolished in Drosophila heads. Inhibition of Obp56h expression also affects expression of other chemoreception genes, including upregulation of lush in both sexes and Obp83ef in females, and reduction in expression of Obp19b and Or19b in males. In addition, several genes associated with lipid metabolism, which underlies the production of cuticular hydrocarbons, show altered transcript abundances. Our data show that modulation of mating behavior through reduction of Obp56h is accompanied by altered cuticular hydrocarbon profiles and implicate 5-T as a possible ligand for Obp56h. Thursday, October 13 2016 09:06:55 AM Genomes of Candidatus Wolbachia bourtzisii wDacA and Candidatus Wolbachia pipientis wDacB from the Cochineal Insect Dactylopius coccus (Hemiptera: Dactylopiidae) Ramirez-Puebla, S. T., Ormeno-Orrillo, E., Vera-Ponce de Leon, A., Lozano, L., Sanchez-Flores, A., Rosenblueth, M., Martinez-Romero, E. Dactylopius species, known as cochineal insects, are the source of the carminic acid dye used worldwide. The presence of two Wolbachia strains in Dactylopius coccus from Mexico was revealed by PCR amplification of wsp and sequencing of 16S rRNA genes. A metagenome analysis recovered the genome sequences of Candidatus Wolbachia bourtzisii wDacA (supergroup A) and Candidatus Wolbachia pipientis wDacB (supergroup B). Genome read coverage, as well as 16S rRNA clone sequencing, revealed that wDacB was more abundant than wDacA. The strains shared similar predicted metabolic capabilities that are common to Wolbachia, including riboflavin, ubiquinone, and heme biosynthesis, but lacked other vitamin and cofactor biosynthesis as well as glycolysis, the oxidative pentose phosphate pathway, and sugar uptake systems. A complete tricarboxylic acid cycle and gluconeogenesis were predicted as well as limited amino acid biosynthesis. Uptake and catabolism of proline were evidenced in Dactylopius Wolbachia strains. Both strains possessed WO-like phage regions and type I and type IV secretion systems. Several efflux systems found suggested the existence of metal toxicity within their host. Besides already described putative virulence factors like ankyrin domain proteins, VlrC homologs, and patatin-like proteins, putative novel virulence factors related to those found in intracellular pathogens like Legionella and Mycobacterium are highlighted for the first time in Wolbachia. Candidate genes identified in other Wolbachia that are likely involved in cytoplasmic incompatibility were found in wDacB but not in wDacA. Thursday, October 13 2016 09:06:55 AM Evaluation of Ligand-Inducible Expression Systems for Conditional Neuronal Manipulations of Sleep in Drosophila Li, Q., Stavropoulos, N. Drosophila melanogaster is a powerful model organism for dissecting the molecular mechanisms that regulate sleep, and numerous studies in the fly have identified genes that impact sleep–wake cycles. Conditional genetic analysis is essential to distinguish the mechanisms by which these genes impact sleep: some genes might exert their effects developmentally, for instance by directing the assembly of neuronal circuits that regulate sleep; other genes may regulate sleep in adulthood; and yet other genes might influence sleep by both developmental and adult mechanisms. Here we have assessed two ligand-inducible expression systems, Geneswitch and the Q-system, for conditional and neuronally restricted manipulations of sleep in Drosophila. While adult-specific induction of a neuronally expressed Geneswitch transgene (elav-GS) is compatible with studies of sleep as shown previously, developmental induction of elav-GS strongly and nonspecifically perturbs sleep in adults. The alterations of sleep in elav-GS animals occur at low doses of Geneswitch agonist and in the presence of transgenes unrelated to sleep, such as UAS-CD8-GFP. Furthermore, developmental elav-GS induction is toxic and reduces brood size, indicating multiple adverse effects of neuronal Geneswitch activation. In contrast, the transgenes and ligand of the Q-system do not significantly impact sleep–wake cycles when used for constitutive, developmental, or adult-specific neuronal induction. The nonspecific effects of developmental elav-GS activation on sleep indicate that such manipulations require cautious interpretation, and suggest that the Q-system or other strategies may be more suitable for conditional genetic analysis of sleep and other behaviors in Drosophila. Thursday, October 13 2016 09:06:55 AM Preservation Analysis of Macrophage Gene Coexpression Between Human and Mouse Identifies PARK2 as a Genetically Controlled Master Regulator of Oxidative Phosphorylation in Humans Codoni, V., Blum, Y., Civelek, M., Proust, C., Franzen, O., Cardiogenics Consortium, IDEM Leducq Consortium CADGenomics, Bjorkegren, J. L. M., Le Goff, W., Cambien, F., Lusis, A. J., Tregouet, D.-A. Macrophages are key players involved in numerous pathophysiological pathways and an in-depth characterization of their gene regulatory networks can help in better understanding how their dysfunction may impact on human diseases. We here conducted a cross-species network analysis of macrophage gene expression data between human and mouse to identify conserved networks across both species, and assessed whether such networks could reveal new disease-associated regulatory mechanisms. From a sample of 684 individuals processed for genome-wide macrophage gene expression profiling, we identified 27 groups of coexpressed genes (modules). Six modules were found preserved (P < 10–4) in macrophages from 86 mice of the Hybrid Mouse Diversity Panel. One of these modules was significantly [false discovery rate (FDR) = 8.9 x 10–11] enriched for genes belonging to the oxidative phosphorylation (OXPHOS) pathway. This pathway was also found significantly (FDR < 10–4) enriched in susceptibility genes for Alzheimer, Parkinson, and Huntington diseases. We further conducted an expression quantitative trait loci analysis to identify SNP that could regulate macrophage OXPHOS gene expression in humans. This analysis identified the PARK2 rs192804963 as a trans-acting variant influencing (minimal P-value = 4.3 x 10–8) the expression of most OXPHOS genes in humans. Further experimental work demonstrated that PARK2 knockdown expression was associated with increased OXPHOS gene expression in THP1 human macrophages. This work provided strong new evidence that PARK2 participates to the regulatory networks associated with oxidative phosphorylation and suggested that PARK2 genetic variations could act as a trans regulator of OXPHOS gene macrophage expression in humans. Thursday, October 13 2016 09:06:55 AM Main Effect QTL with Dominance Determines Heterosis for Dynamic Plant Height in Upland Cotton Shang, L., Ma, L., Wang, Y., Su, Y., Wang, X., Li, Y., Abduweli, A., Cai, S., Liu, F., Wang, K., Hua, J. Plant height, which shows dynamic development and heterosis, is a major trait affecting plant architecture and has an indirect influence on economic yield related to biological yield in cotton. In the present study, we carried out dynamic analysis for plant height and its heterosis by quantitative trait loci (QTL) mapping at multiple developmental stages using two recombinant inbred lines (RILs) and their backcross progeny. At the single-locus level, 47 QTL were identified at five developmental stages in two hybrids. In backcross populations, QTL identified at an early stage mainly showed partial effects and QTL detected at a later stage mostly displayed overdominance effects. At the two-locus level, we found that main effect QTL played a more important role than epistatic QTL in the expression of heterosis in backcross populations. Therefore, this study implies that the genetic basis of plant height heterosis shows dynamic character and main effect QTL with dominance determines heterosis for plant height in Upland cotton. Thursday, October 13 2016 09:06:55 AM Seizure Suppression by High Temperature via cAMP Modulation in Drosophila Saras, A., Tanouye, M. A. Bang-sensitive (BS) Drosophila mutants display characteristic seizure-like activity (SLA) and paralysis after mechanical shock . After high-frequency electrical stimulation (HFS) of the brain, they generate robust seizures at very low threshold voltage. Here we report an important phenomenon, which effectively suppresses SLA in BS mutants. High temperature causes seizure suppression in all BS mutants (parabss1, eas, sda) examined in this study. This effect is fully reversible and flies show complete recovery from BS paralysis once the temperature effect is nullified. High temperature induces an increase in seizure threshold after a brief pulse of heat shock (HS). By genetic screening, we identified the involvement of cAMP in the suppression of seizures by high temperature. We propose that HS induces adenylyl cyclase which in turn increases cAMP concentration which eventually suppresses seizures in mutant flies. In summary, we describe an unusual phenomenon, where high temperature can suppress SLA in flies by modulating cAMP concentration. Thursday, October 13 2016 09:06:55 AM A Genome-Wide Association Study Identifies Multiple Regions Associated with Head Size in Catfish Geng, X., Liu, S., Yao, J., Bao, L., Zhang, J., Li, C., Wang, R., Sha, J., Zeng, P., Zhi, D., Liu, Z. Skull morphology is fundamental to evolution and the biological adaptation of species to their environments. With aquaculture fish species, head size is also important for economic reasons because it has a direct impact on fillet yield. However, little is known about the underlying genetic basis of head size. Catfish is the primary aquaculture species in the United States. In this study, we performed a genome-wide association study using the catfish 250K SNP array with backcross hybrid catfish to map the QTL for head size (head length, head width, and head depth). One significantly associated region on linkage group (LG) 7 was identified for head length. In addition, LGs 7, 9, and 16 contain suggestively associated regions for head length. For head width, significantly associated regions were found on LG9, and additional suggestively associated regions were identified on LGs 5 and 7. No region was found associated with head depth. Head size genetic loci were mapped in catfish to genomic regions with candidate genes involved in bone development. Comparative analysis indicated that homologs of several candidate genes are also involved in skull morphology in various other species ranging from amphibian to mammalian species, suggesting possible evolutionary conservation of those genes in the control of skull morphologies. Thursday, October 13 2016 09:06:55 AM A Genetic Screen for Fission Yeast Gene Deletion Mutants Exhibiting Hypersensitivity to Latrunculin A Asadi, F., Michalski, D., Karagiannis, J. Fission yeast cells treated with low doses of the actin depolymerizing drug, latrunculin A (LatA), delay entry into mitosis via a mechanism that is dependent on both the Clp1p and Rad24p proteins. During this delay, cells remain in a cytokinesis-competent state that is characterized by continuous repair and/or reestablishment of the actomyosin ring. In this manner, cells ensure the faithful completion of the preceding cytokinesis in response to perturbation of the cell division machinery. To uncover other genes with a role in this response, or simply genes with roles in adapting to LatA-induced stress, we carried out a genome-wide screen and identified a group of 38 gene deletion mutants that are hyper-sensitive to the drug. As expected, we found genes affecting cytokinesis and/or the actin cytoskeleton within this set (ain1, acp2, imp2). We also identified genes with roles in histone modification (tra1, ngg1), intracellular transport (apl5, aps3), and glucose-mediated signaling (git3, git5, git11, pka1, cgs2). Importantly, while the identified gene deletion mutants are prone to cytokinesis failure in the presence of LatA, they are nevertheless fully capable of cell division in the absence of the drug. These results indicate that fission yeast cells make use of a diverse set of regulatory modules to counter abnormal cytoskeletal perturbations, and furthermore, that these modules act redundantly to ensure cell survival and proliferation. Thursday, October 13 2016 09:06:55 AM A Forward Genetic Screen and Whole Genome Sequencing Identify Deflagellation Defective Mutants in Chlamydomonas, Including Assignment of ADF1 as a TRP Channel Hilton, L. K., Meili, F., Buckoll, P. D., Rodriguez-Pike, J. C., Choutka, C. P., Kirschner, J. A., Warner, F., Lethan, M., Garces, F. A., Qi, J., Quarmby, L. M. With rare exception, ciliated cells entering mitosis lose their cilia, thereby freeing basal bodies to serve as centrosomes in the formation of high-fidelity mitotic spindles. Cilia can be lost by shedding or disassembly, but either way, it appears that the final release may be via a coordinated severing of the nine axonemal outer doublet microtubules linking the basal body to the ciliary transition zone. Little is known about the mechanism or regulation of this important process. The stress-induced deflagellation response of Chlamydomonas provides a basis to identifying key players in axonemal severing. In an earlier screen we uncovered multiple alleles for each of three deflagellation genes, ADF1, FA1, and FA2. Products of the two FA genes localize to the site of axonemal severing and encode a scaffolding protein and a member of the NIMA-related family of ciliary-cell cycle kinases. The identity of the ADF1 gene remained elusive. Here, we report a new screen using a mutagenesis that yields point mutations in Chlamydomonas, an enhanced screening methodology, and whole genome sequencing. We isolated numerous new alleles of the three known genes, and one or two alleles each of at least four new genes. We identify ADF1 as a TRP ion channel, which we suggest may reside at the flagellar transition zone. Thursday, October 13 2016 09:06:55 AM Potential Direct Regulators of the Drosophila yellow Gene Identified by Yeast One-Hybrid and RNAi Screens Kalay, G., Lusk, R., Dome, M., Hens, K., Deplancke, B., Wittkopp, P. J. The regulation of gene expression controls development, and changes in this regulation often contribute to phenotypic evolution. Drosophila pigmentation is a model system for studying evolutionary changes in gene regulation, with differences in expression of pigmentation genes such as yellow that correlate with divergent pigment patterns among species shown to be caused by changes in cis- and trans-regulation. Currently, much more is known about the cis-regulatory component of divergent yellow expression than the trans-regulatory component, in part because very few trans-acting regulators of yellow expression have been identified. This study aims to improve our understanding of the trans-acting control of yellow expression by combining yeast-one-hybrid and RNAi screens for transcription factors binding to yellow cis-regulatory sequences and affecting abdominal pigmentation in adults, respectively. Of the 670 transcription factors included in the yeast-one-hybrid screen, 45 showed evidence of binding to one or more sequence fragments tested from the 5' intergenic and intronic yellow sequences from D. melanogaster, D. pseudoobscura, and D. willistoni, suggesting that they might be direct regulators of yellow expression. Of the 670 transcription factors included in the yeast-one-hybrid screen, plus another TF previously shown to be genetically upstream of yellow, 125 were also tested using RNAi, and 32 showed altered abdominal pigmentation. Nine transcription factors were identified in both screens, including four nuclear receptors related to ecdysone signaling (Hr78, Hr38, Hr46, and Eip78C). This finding suggests that yellow expression might be directly controlled by nuclear receptors influenced by ecdysone during early pupal development when adult pigmentation is forming. Thursday, October 13 2016 09:06:55 AM RNAi-Based Suppressor Screens Reveal Genetic Interactions Between the CRL2LRR-1 E3-Ligase and the DNA Replication Machinery in Caenorhabditis elegans Ossareh-Nazari, B., Katsiarimpa, A., Merlet, J., Pintard, L. Cullin-RING E3-Ligases (CRLs), the largest family of E3 ubiquitin-Ligases, regulate diverse cellular processes by promoting ubiquitination of target proteins. The evolutionarily conserved Leucine Rich Repeat protein 1 (LRR-1) is a substrate-recognition subunit of a CRL2LRR-1 E3-ligase. Here we provide genetic evidence supporting a role of this E3-enzyme in the maintenance of DNA replication integrity in Caenorhabditis elegans. Through RNAi-based suppressor screens of lrr-1(0) and cul-2(or209ts) mutants, we identified two genes encoding components of the GINS complex, which is part of the Cdc45-MCM-GINS (CMG) replicative helicase, as well as CDC-7 and MUS-101, which drives the assembly of the CMG helicase during DNA replication. In addition, we identified the core components of the ATR/ATL-1 DNA replication checkpoint pathway (MUS-101, ATL-1, CLSP-1, CHK-1). These results suggest that the CRL2LRR-1 E3-ligase acts to modify or degrade factor(s) that would otherwise misregulate the replisome, eventually leading to the activation of the DNA replication checkpoint.