To test this, we crossed CTPSH355A and CTPSWT transgenes into flies expressing an allele of CTPS tagged with GFP (Noree et al., 2010), to allow visualization of endogenous CTPS filaments. Together, these key findings demonstrate that CTPS assembly supports but is not absolutely essential for egg production under standard laboratory growth conditions and under the stress of inhibited glutamine metabolism. Here and below, error bars denote s.e.m. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. 3H, blue bars), indicating that germline disruption of CTPS assembly between stages 1 and 6 did not affect production of mature eggs under these conditions. Beyond this stage, expression of the transgene is reduced, allowing a return of CTPS assembly in later stage egg chambers (Fig. pPW constructs were used to generate transgenic flies (BestGene) and transgene expression was driven in the germline cells of the ovary with pCOG-Gal4 (Strochlic et al., 2014) or nosGal4:VP16 (Bloomington stock #4937).
,This subsection of the Function section describes the interaction between a single amino acid and another chemical entity.
However UniProtKB may contain entries with identical sequences in case
Press W.H., Flannery B.P., Teukolsky S.A. and Vetterling W.T.
(C) Quantification of the percent of stage 4 and 6 egg chambers with CTPS filaments in two independent Flag-CTPSWT transgenic lines (M1 and M4) and two Flag-CTPSH355A lines (M1 and M2). Help pages, FAQs, UniProtKB manual, documents, news archive and Biocuration projects. To assess whether expression of CTPSH355A in all tissues in the fly would have phenotypic consequences, we used act5c-Gal4 to drive ubiquitous expression of three different UAS-CTPSH355A alleles. CTP synthetase (CTPs) catalyzes the last step in CTP biosynthesis, in which ammonia generated at the glutaminase domain reacts with the ATP-phosphorylated UTP at the synthetase domain to give CTP. There are several major changes in the manuscript itself, although the conclusions remain the same. One possibility is that assembly may enhance Drosophila CTPS catalytic activity as it does for human CTPS1 (Lynch et al., 2017).
We are aware that the COVID-19 pandemic is having an unprecedented impact on researchers worldwide. It lists the nodes as they appear top-down in the taxonomic tree, with the more general grouping listed first.
This subsection of the Names and taxonomy section is present for entries that are part of a proteome, i.e. Stage 4, 6 and 8 egg chambers are bracketed. ), [R01HD065800] (A.M.O.R. These findings introduce a general approach to blocking the assembly of polymerizing enzymes without eliminating their catalytic activity and demonstrate a role for CTPS assembly in supporting egg production, particularly under conditions of limited glutamine metabolism. TaqMan gene expression assays (Life Technologies) were used to amplify dCTPS1 variant C (NM_168606.1 F: CTGTGGTCTGGATGTAACCTCG, R: CAAAACGTAAACCTCGCCATG), Flag/dCTPS1 (F: TACAAGGATGACGATGACAAGAAA, R: CTTGATCGAGGTTACATCCAGAC), GFP (F: CCCAGTCCGCCCTGAG, R: ACGAACTCCAGCAGGACCA) cDNA. In parallel, eggs laid during the 7-day incubation were maintained at 25°C for another 7 days and then pupae were counted. Eggs laid over 7 days were allowed to develop in the presence or absence of DON and pupae were counted after 7 days. P-values (*) left to right were 0.0358 and 0.0124. *, P-values in comparison to control pCOG-Gal4 flies are 0.029, 0.013, 0.007 (left to right). The Biological General Repository for Interaction Datasets (BioGRID), Protein interaction database and analysis system, STRING: functional protein association networks, SWISS-MODEL Repository - a database of annotated 3D protein structure models, Database of comparative protein structure models, Protein Data Bank in Europe - Knowledge Base, Relative evolutionary importance of amino acids within a protein sequence, evolutionary genealogy of genes: Non-supervised Orthologous Groups, The HOGENOM Database of Homologous Genes from Fully Sequenced Organisms, Database for complete collections of gene phylogenies, Gene3D Structural and Functional Annotation of Protein Families, Integrated resource of protein families, domains and functional sites, Superfamily database of structural and functional annotation, PROSITE; a protein domain and family database. CTP synthase (CTPS) can aggregate into an intracellular macrostructure, the cytoophidium, in various organisms including human cells. Here we demonstrate that a recently reported point mutant in CTPS, Histidine 355A (H355A), prevents CTPS filament assembly in vivo and dominantly inhibits the assembly of endogenous wild-type CTPS in the Drosophila ovary. The Editors of all The Company of Biologists’ journals have been considering ways in which we can alleviate concerns that members of our community may have around publishing activities during this time.
Whether assembly into these ultrastructures is important for CTPS function is unknown, in part because of a lack of tools to disrupt the assembly of CTP filaments in their native context completely but without eliminating CTPS catalytic activity. Yellow color indicates overlap between red and green channels. However, it remains unknown whether cytoophidia exist in archaea, the third domain of life. FLAG immunostaining revealed that, as expected, CTPSWT was expressed in early egg chambers, where it assembled into filaments morphologically similar to endogenous CTPS filaments (Strochlic et al., 2014) (Fig. NOTE: Your email address is requested solely to identify you as the sender of this article. Protein sets from fully sequenced genomes. To ensure that this phenotypic difference was not due to differences in CTPSWT and CTPSH355A expression levels, we screened additional ovary-specific drivers and found that the nosGal4:VP16 driver gave similar expression of the CTPSWT-M1 and CTPSH355A-M1 transgenes, both at the RNA (Fig. Several independent studies have demonstrated that CTP synthase can form an evolutionarily conserved subcellular structure termed cytoophidium. In stage 8 egg chambers, when pCOG-GAL4-driven transcription sharply decreases (Rørth, 1998), we observed a partial (10–17%) reassembly of GFP-CTPS into filaments (Fig. Strikingly, even in the absence of DON, flies expressing CTPSH355A-M1 produced significantly fewer eggs than flies expressing CTPSWT-M1 (Fig. CTP synthase (CTPS) is an important metabolic enzyme that catalyzes the rate-limiting reaction of de novo synthesis of the nucleotide CTP. CTP synthase (CTPS) is an essential enzyme mediating de novo pyrimidine synthesis by catalyzing the ATP-dependent amination of UTP into CTP. As a result, cells could express a mixture of diffuse mutant CTPS and polymerized wild-type CTPS. In flies fed standard food this comparison did not reach statistical significance, likely because the effects of disrupting CTPS assembly on egg production were less significant in the absence of DON and were further diluted over the subsequent week. However, it remains unknown whether cytoophidia exist in archaea, the third domain of life. Tissue-specific expression of the CTPSH355A allele using available GAL4 drivers can be used to rapidly probe the role of CTPS assembly in these other contexts. DNA Data Bank of Japan; a nucleotide sequence database, Protein sequence database of the Protein Information Resource, Ensembl bacterial and archaeal genome annotation project, Database of genes from NCBI RefSeq genomes, KEGG: Kyoto Encyclopedia of Genes and Genomes, Pathosystems Resource Integration Center (PATRIC), EchoBASE - an integrated post-genomic database for E. coli, ProtoNet; Automatic hierarchical classification of proteins, MobiDB: a database of protein disorder and mobility annotations. Likewise, feeding flies the glutamine antimetabolites DON or azaserine promotes CTPS filament assembly in many tissues (Chen et al., 2011).
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