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      Inducible degradation of dosage compensation protein DPY-27 facilitates isolation of Caenorhabditis elegans males for molecular and biochemical analyses

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          Abstract

          Biological sex affects numerous aspects of biology, yet how sex influences different biological processes have not been extensively studied at the molecular level . Caenorhabditis elegans, with both hermaphrodites (functionally females as adults) and males, is an excellent system to uncover how sex influences physiology. Here, we describe a method to isolate large quantities of C. elegans males by conditionally degrading DPY-27, a component of the dosage compensation complex essential for hermaphrodite, but not male, development. We show that germ cells from males isolated following DPY-27 degradation undergo meiosis and spermiogenesis like wild type and these males are competent to mate and sire viable offspring. We further demonstrate the efficacy of this system by analyzing gene expression and performing affinity pull-downs from male worm extracts.

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          Most cited references54

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          MSFragger: ultrafast and comprehensive peptide identification in shotgun proteomics

          There is a need to better understand and handle the “dark matter” of proteomics – the vast diversity of post-translational and chemical modifications that are unaccounted in a typical analysis and thus remain unidentified. We present a novel fragment-ion indexing method, and its implementation in peptide identification tool MSFragger, that enables an over 100-fold improvement in speed over most existing tools. Using some of the largest proteomic datasets to date, we demonstrate how MSFragger empowers the open database search concept for comprehensive identification of peptides and all their modified forms, uncovering dramatic differences in the modification rates across experimental samples and conditions. We further illustrate its utility using protein-RNA crosslinked peptide data, and using affinity purification experiments where we observe on average a 300% increase in the number of identified spectra for enriched proteins. We also discuss the benefits of open searching for improved false discovery rate estimation in proteomics.
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            An auxin-based degron system for the rapid depletion of proteins in nonplant cells.

            Plants have evolved a unique system in which the plant hormone auxin directly induces rapid degradation of the AUX/IAA family of transcription repressors by a specific form of the SCF E3 ubiquitin ligase. Other eukaryotes lack the auxin response but share the SCF degradation pathway, allowing us to transplant the auxin-inducible degron (AID) system into nonplant cells and use a small molecule to conditionally control protein stability. The AID system allowed rapid and reversible degradation of target proteins in response to auxin and enabled us to generate efficient conditional mutants of essential proteins in yeast as well as cell lines derived from chicken, mouse, hamster, monkey and human cells, thus offering a powerful tool to control protein expression and study protein function.
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              Online Parallel Accumulation–Serial Fragmentation (PASEF) with a Novel Trapped Ion Mobility Mass Spectrometer *

              PASEF multiplies the sequencing speed without any loss in sensitivity and is implemented in the timsTOF Pro instrument introduced here. Sequencing speeds above 100 Hz enable single run proteome analysis at a depth of 6000 proteins, making the instrument particularly attractive for rapid and highly sensitive proteomics. Collisional cross sections can be determined with up to 0.1% precision and acquired on a scale of 100,000s, which opens exciting areas for proteomics exploration.
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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
                11 April 2022
                11 April 2022
                : 12
                : 5
                : jkac085
                Affiliations
                [1 ] Department of Molecular and Cellular Biology, University of California, Davis , Davis, CA 95616, USA
                [2 ] Biochemistry, Molecular, Cellular and Developmental Biology Graduate Group, University of California, Davis , Davis, CA 95616, USA
                Author notes
                Corresponding author: University of California, Davis, One Shields Avenue, Davis, CA 95616, USA. Email: jengebrecht@ 123456ucdavis.edu

                Present address for Benjamin Mallory: Genome Sciences Program, University of Washington, Seattle, WA 98195, USA.

                Author information
                https://orcid.org/0000-0003-4865-0101
                https://orcid.org/0000-0002-9846-0675
                https://orcid.org/0000-0003-1572-6181
                https://orcid.org/0000-0003-4693-2359
                https://orcid.org/0000-0002-2733-7506
                Article
                jkac085
                10.1093/g3journal/jkac085
                9073673
                35404452
                367a9b0c-05b3-42e4-be37-2ed3aaf55a5d
                © 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
                : 04 April 2022
                : 26 January 2022
                : 19 April 2022
                Page count
                Pages: 9
                Funding
                Funded by: The Caenorhabditis Genetic Center;
                Funded by: NIH Office of Research Infrastructure Programs;
                Award ID: P40 OD010440
                Funded by: National Institutes of Health, DOI 10.13039/100000002;
                Award ID: GM103860
                Award ID: GM103860S1
                Categories
                Investigation
                AcademicSubjects/SCI01180
                AcademicSubjects/SCI01140
                AcademicSubjects/SCI00010
                AcademicSubjects/SCI00960

                Genetics
                caenorhabditis elegans,dosage compensation,dpy-27,males,meiosis,spermiogenesis,genetics of sex
                Genetics
                caenorhabditis elegans, dosage compensation, dpy-27, males, meiosis, spermiogenesis, genetics of sex

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