The Function and Evolution of Motile DNA Replication Systems in Ciliates

•The replication envelope is a new form of motile DNA system•Calcium signaling and cytoskeletal regulation may coordinate the band•Phylogenomics resolves deep phylogeny ciliate phylum ubiquitous conserved cellular process. However, only understood in small fraction organisms that poorly represent diversity genetic systems nature. Here we used computational experimental approaches to examine function evolution one such system, band (RB) spirotrich ciliates, which localized, hub traverses macronucleus while replicating DNA. We show RB can take unique forms different species, from polar bands “replication envelope,” where initiates at nuclear periphery before advancing inward. Furthermore, identify genes involved transport, including calcium transporters regulators, are associated with be its translocation. These findings highlight provide insights into organization processes. process by genomic duplicated anticipation cell division. 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Kolísko Čepička Genomics provides insight obligate anaerobiosis.Curr. 2020; 2037-2050.e6Abstract (13) Within SAL, branched formed deepest branches: next remaining topology, would probable assess could result sampling bias, sub-sampled set 5% (12 gene) concatenations jackknifing (i.e., random without replacement; n 58) alignments phylogenetics analysis. majority showed (93%), best (34%), not selection 2B 2C). sequential removal fast-evolving did influence paraphyly relationship CONThreeP, biased long branch attraction 2D). exact aside dependent site removal, making tentative 2B–2D) any way indicated lost studies failed observe it. discern possibilities, investigated cytological techniques species. agreement work, staining methyl green-rhodamine B 3A), Feulgen 3B), Protargol 3C) reveal visualize directly EdU (5-ethynyl-2′-deoxyuridine) incorporation followed Cy3-azide labeling. As positive Cy3-EdU labeling Hoechst applied revealed fluorescent characteristic passed exhibited Cy3 fluorescence 3D 3E). distinguished alternative appeared throughout inward 3F–3I, Figures 4A–4C; Figure S2). Re-evaluation preparations comparable structure, observations procedures 3C). Unlike lacked observed). initiate globally contrasting focality envelope.Figure 4Confocal Fluorescence Imaging Replication Envelope metchnikoffiShow caption(A–C) Confocal imaging montages separate cells.(A) phase.(B) Slightly later (A).(C) Completed phase.Scale bars, 50 μm; step 0.5 μm.View Large Image ViewerDownload Hi-res image Download (PPT) (A–C) cells. (A) phase. (B) (A). (C) Scale μm. divergence leverage function. particular, hypothesized presence/absence expansion/duplication rates families relatedness. end, clustered genomics (orthologous groups) compared frequency family Of 37,318 examined, 406 23 enriched depleted RB-containing clades, respectively (p < 0.05, permutation test, 10,000) 5A; Table those, 186 had, average, 1.8 2.7 times homologs per Consistent this, principal-component differentially present discernible RB-lacking species clusters 5B), RB-association distinct association S3). Functional annotation Gene Ontology (GO) functions ion cytoskeleton 5C; example, biological homeostasis (GO: 0006873; p 10−6), 0006811; 10−3.19), 0051049; 10−3.10) significantly prevalence (e.g., polycystin-2, sodium/calcium exchangers, channels) 5C 5D; S4A). Moreover, GO: 0007010; 10−2.10), homologs, regulate microtubules,38Vergnolle M.A.S. Taylor S.S. Cenp-F links kinetochores Ndel1/Nde1/Lis1/dynein microtubule motor complexes.Curr. 2007; 17: 1173-1179Abstract (121) gelsolin repeat proteins, calcium-dependent actin regulators,39Sun H.Q. Yamamoto Mejillano Yin H.L. Gelsolin, multifunctional regulatory protein.J. Chem. 274: 33179-33182Abstract (457) S4B; Other functions, responses chemicals 0042221; 10−2.39) 0016570, 10−1.30) presence containing, cyclic AMP/guanosine monophosphate (GMP) phosphodiesterase lysine-binding bromodomains, 5D). terms, transmembrane transporter activity 0022857; 10−3.60), enzyme binding 0019899; 10−2.62), 0008092; 10−1.47), phosphatase 0016791; 10−1.49), connected RB-related correlative preceding analysis, list RB-associated naturally contains investigate function, interference (RNAi)-induced knockdown assessed trifallax. selected most highly 10−5) S2), GO RNAi constructs capable knocking down expression paralogs (for total 64 knocked down). Compared non-specific RNAi, essential phase-initiating CDC6, decreased proportion 6A), observed, CDC6 resulted impaired (measured distance divided length lob