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      Simple Expression Domains Are Regulated by Discrete CRMs During Drosophila Oogenesis

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          Abstract

          Eggshell patterning has been extensively studied in Drosophila melanogaster. However, the cis-regulatory modules (CRMs), which control spatiotemporal expression of these patterns, are vastly unexplored. The FlyLight collection contains >7000 intergenic and intronic DNA fragments that, if containing CRMs, can drive the transcription factor GAL4. We cross-listed the 84 genes known to be expressed during D. melanogaster oogenesis with the ∼1200 listed genes of the FlyLight collection, and found 22 common genes that are represented by 281 FlyLight fly lines. Of these lines, 54 show expression patterns during oogenesis when crossed to an UAS-GFP reporter. Of the 54 lines, 16 recapitulate the full or partial pattern of the associated gene pattern. Interestingly, while the average DNA fragment size is ∼3 kb in length, the vast majority of fragments show one type of spatiotemporal pattern in oogenesis. Mapping the distribution of all 54 lines, we found a significant enrichment of CRMs in the first intron of the associated genes’ model. In addition, we demonstrate the use of different anteriorly active FlyLight lines as tools to disrupt eggshell patterning in a targeted manner. Our screen provides further evidence that complex gene patterns are assembled combinatorially by different CRMs controlling the expression of genes in simple domains.

          Most cited references59

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          Transcription regulation and animal diversity.

          Whole-genome sequence assemblies are now available for seven different animals, including nematode worms, mice and humans. Comparative genome analyses reveal a surprising constancy in genetic content: vertebrate genomes have only about twice the number of genes that invertebrate genomes have, and the increase is primarily due to the duplication of existing genes rather than the invention of new ones. How, then, has evolutionary diversity arisen? Emerging evidence suggests that organismal complexity arises from progressively more elaborate regulation of gene expression.
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            Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31.

            The phiC31 integrase functions efficiently in vitro and in Escherichia coli, yeast, and mammalian cells, mediating unidirectional site-specific recombination between its attB and attP recognition sites. Here we show that this site-specific integration system also functions efficiently in Drosophila melanogaster in cultured cells and in embryos. Intramolecular recombination in S2 cells on transfected plasmid DNA carrying the attB and attP recognition sites occurred at a frequency of 47%. In addition, several endogenous pseudo attP sites were identified in the fly genome that were recognized by the integrase and used as substrates for integration in S2 cells. Two lines of Drosophila were created by integrating an attP site into the genome with a P element. phiC31 integrase injected into embryos as mRNA functioned to promote integration of an attB-containing plasmid into the attP site, resulting in up to 55% of fertile adults producing transgenic offspring. A total of 100% of these progeny carried a precise integration event at the genomic attP site. These experiments demonstrate the potential for precise genetic engineering of the Drosophila genome with the phiC31 integrase system and will likely benefit research in Drosophila and other insects.
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              Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.

              Although subcellular mRNA trafficking has been demonstrated as a mechanism to control protein distribution, it is generally believed that most protein localization occurs subsequent to translation. To address this point, we developed and employed a high-resolution fluorescent in situ hybridization procedure to comprehensively evaluate mRNA localization dynamics during early Drosophila embryogenesis. Surprisingly, of the 3370 genes analyzed, 71% of those expressed encode subcellularly localized mRNAs. Dozens of new and striking localization patterns were observed, implying an equivalent variety of localization mechanisms. Tight correlations between mRNA distribution and subsequent protein localization and function, indicate major roles for mRNA localization in nucleating localized cellular machineries. A searchable web resource documenting mRNA expression and localization dynamics has been established and will serve as an invaluable tool for dissecting localization mechanisms and for predicting gene functions and interactions.
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                Author and article information

                Journal
                G3 (Bethesda)
                Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes|Genomes|Genetics
                Genetics Society of America
                2160-1836
                20 June 2017
                August 2017
                : 7
                : 8
                : 2705-2718
                Affiliations
                [* ]Center for Computational and Integrative Biology, Rutgers, The State University of New Jersey, Camden, New Jersey 08103
                []Department of Biology, Rutgers, The State University of New Jersey, Camden, New Jersey 08103
                []Lewis-Sigler Institute for Integrative Genomics, Princeton University, New Jersey 08544
                Author notes
                [1 ]Corresponding author: Department of Biology and Center for Computational and Integrative Biology, Waterfront Technology Center, 200 Federal St., Rutgers, The State University of New Jersey, Camden, NJ 08103. E-mail: yakoby@ 123456camden.rutgers.edu
                Author information
                http://orcid.org/0000-0002-7959-7588
                Article
                GGG_043810
                10.1534/g3.117.043810
                5555475
                28634244
                8a406729-352e-4e78-ae64-0f54f7fd968b
                Copyright © 2017 Revaitis et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 04 December 2016
                : 09 June 2017
                Page count
                Figures: 5, Tables: 0, Equations: 0, References: 78, Pages: 14
                Categories
                Investigations

                Genetics
                gene regulation,eggshell patterning,gal4,genetic tools
                Genetics
                gene regulation, eggshell patterning, gal4, genetic tools

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