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      A novel mistranslating tRNA model in Drosophila melanogaster has diverse, sexually dimorphic effects

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          Abstract

          Transfer RNAs (tRNAs) are the adaptor molecules required for reading the genetic code and producing proteins. Transfer RNA variants can lead to genome-wide mistranslation, the misincorporation of amino acids not specified by the standard genetic code into nascent proteins. While genome sequencing has identified putative mistranslating transfer RNA variants in human populations, little is known regarding how mistranslation affects multicellular organisms. Here, we create a multicellular model of mistranslation by integrating a serine transfer RNA variant that mistranslates serine for proline ( tRNA UGG , G 26 A Ser ) into the Drosophila melanogaster genome. We confirm mistranslation via mass spectrometry and find that tRNA UGG , G 26 A Ser misincorporates serine for proline at a frequency of ∼0.6% per codon. tRNA UGG , G 26 A Ser extends development time and decreases the number of flies that reach adulthood. While both sexes of adult flies containing tRNA UGG , G 26 A Ser present with morphological deformities and poor climbing performance, these effects are more pronounced in female flies and the impact on climbing performance is exacerbated by age. This model will enable studies into the synergistic effects of mistranslating transfer RNA variants and disease-causing alleles.

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          The PRIDE database and related tools and resources in 2019: improving support for quantification data

          Abstract The PRoteomics IDEntifications (PRIDE) database (https://www.ebi.ac.uk/pride/) is the world’s largest data repository of mass spectrometry-based proteomics data, and is one of the founding members of the global ProteomeXchange (PX) consortium. In this manuscript, we summarize the developments in PRIDE resources and related tools since the previous update manuscript was published in Nucleic Acids Research in 2016. In the last 3 years, public data sharing through PRIDE (as part of PX) has definitely become the norm in the field. In parallel, data re-use of public proteomics data has increased enormously, with multiple applications. We first describe the new architecture of PRIDE Archive, the archival component of PRIDE. PRIDE Archive and the related data submission framework have been further developed to support the increase in submitted data volumes and additional data types. A new scalable and fault tolerant storage backend, Application Programming Interface and web interface have been implemented, as a part of an ongoing process. Additionally, we emphasize the improved support for quantitative proteomics data through the mzTab format. At last, we outline key statistics on the current data contents and volume of downloads, and how PRIDE data are starting to be disseminated to added-value resources including Ensembl, UniProt and Expression Atlas.
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            Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.

            The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line-restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25-100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.
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              A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.

              A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae. Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trp1, leu2 and ura3 mutations. A set of YCp and YIp vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT. These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HIS3, TRP1, LEU2 and URA3). They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well. Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                G3 (Bethesda)
                Genetics
                g3journal
                G3: Genes|Genomes|Genetics
                Oxford University Press
                2160-1836
                May 2022
                10 February 2022
                10 February 2022
                : 12
                : 5
                : jkac035
                Affiliations
                [1 ] Department of Biology, The University of Western Ontario , London, ON N6A 5B7, Canada
                [2 ] Department of Biochemistry, The University of Western Ontario , London, ON N6A 5B7, Canada
                [3 ] Department of Genome Sciences, University of Washington , Seattle, WA 98195, USA
                Author notes
                Corresponding author: 1151 Richmond Street, Biological and Geological Sciences Building Room 2082, London, Ontario N6A 5B7, Canada. Email: jisaacso@ 123456uwo.ca
                Author information
                https://orcid.org/0000-0002-1323-0257
                https://orcid.org/0000-0002-7924-9241
                https://orcid.org/0000-0002-1005-1739
                https://orcid.org/0000-0001-8015-9668
                Article
                jkac035
                10.1093/g3journal/jkac035
                9073681
                35143655
                7fdebab2-9f47-48f3-8245-24cf6e8330ce
                © The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 16 December 2021
                : 03 February 2022
                : 18 March 2022
                Page count
                Pages: 9
                Funding
                Funded by: Natural Sciences and Engineering Research Council of Canada (NSERC);
                Award ID: RGPIN-2015-04394
                Award ID: RGPIN-2020-06464
                Funded by: University of Western Ontario Medical & Health Science Research Board seed grant to AJM;
                Funded by: Keck Foundation, National Institutes of Health;
                Award ID: R35 GM119536
                Funded by: NSERC Postgraduate Scholarship (Doctoral) and NSERC Canada Graduate Scholarship (Doctoral);
                Categories
                Investigation
                AcademicSubjects/SCI01180
                AcademicSubjects/SCI01140
                AcademicSubjects/SCI00010
                AcademicSubjects/SCI00960

                Genetics
                trna,mistranslation,drosophila melanogaster,development,locomotion,proteostasis,deformity,sex-specific

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