In endolysosomal networks, two hetero-hexameric tethers called HOPS and CORVET are found widely throughout eukaryotes. The unicellular ciliate Tetrahymena thermophila possesses elaborate endolysosomal structures, but curiously both it and related protozoa lack the HOPS tether and several other trafficking proteins, while retaining the related CORVET complex. Here, we show that Tetrahymena encodes multiple paralogs of most CORVET subunits, which assemble into six distinct complexes. Each complex has a unique subunit composition and, significantly, shows unique localization, indicating participation in distinct pathways. One pair of complexes differ by a single subunit (Vps8), but have late endosomal versus recycling endosome locations. While Vps8 subunits are thus prime determinants for targeting and functional specificity, determinants exist on all subunits except Vps11. This unprecedented expansion and diversification of CORVET provides a potent example of tether flexibility, and illustrates how 'backfilling' following secondary losses of trafficking genes can provide a mechanism for evolution of new pathways.This article has an associated First Person interview with the first author of the paper.

• Klíčová slova
• Backfilling, CORVET, Ciliate, Cryomilling, Endosomal trafficking, Evolutionary cell biology, Mass spectrometry, Paralogous expansion,
• MeSH
• endozomy MeSH
• lidé MeSH
• lyzozomy MeSH
• Tetrahymena thermophila * genetika   MeSH
• vezikulární transportní proteiny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• vezikulární transportní proteiny MeSH

Kit ligand (Kitlg) is pleiotropic cytokine with a prominent role in vertebrate erythropoiesis. Although the role of Kitlg in this process has not been reported in Danio rerio (zebrafish), in the present study we show that its function is evolutionarily conserved. Zebrafish possess 2 copies of Kitlg genes (Kitlga and Kitlgb) as a result of whole-genome duplication. To determine the role of each ligand in zebrafish, we performed a series of ex vivo and in vivo gain- and loss-of-function experiments. First, we tested the biological activity of recombinant Kitlg proteins in suspension culture from zebrafish whole-kidney marrow, and we demonstrate that Kitlga is necessary for expansion of erythroid progenitors ex vivo. To further address the role of kitlga and kitlgb in hematopoietic development in vivo, we performed gain-of-function experiments in zebrafish embryos, showing that both ligands cooperate with erythropoietin (Epo) to promote erythroid cell expansion. Finally, using the kita mutant (kitab5/b5 or sparse), we show that the Kita receptor is crucial for Kitlga/b cooperation with Epo in erythroid cells. In summary, using optimized suspension culture conditions with recombinant cytokines (Epo, Kitlga), we report, for the first time, ex vivo suspension cultures of zebrafish hematopoietic progenitor cells that can serve as an indispensable tool to study normal and aberrant hematopoiesis in zebrafish. Furthermore, we conclude that, although partial functional diversification of Kit ligands has been described in other processes, in erythroid development, both paralogs play a similar role, and their function is evolutionarily conserved.

• MeSH
• dánio pruhované MeSH
• erythropoetin * MeSH
• erytroidní buňky MeSH
• faktor růstu kmenových buněk genetika   MeSH
• ligandy MeSH
• proteiny dánia pruhovaného genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• erythropoetin * MeSH
• faktor růstu kmenových buněk MeSH
• kitlga protein, zebrafish MeSH Prohlížeč
• ligandy MeSH
• proteiny dánia pruhovaného MeSH

Imaginal disc growth factors (IDGFs) are a small protein family found in insects. They are related to chitinases and implicated in multiple functions, including cell growth stimulation, antimicrobial activity, insect hemolymph clotting, and maintenance of the extracellular matrix. A number of new IDGFs have been found in several insect species and their detailed phylogenetic analysis provides a good basis for further functional studies. To achieve this goal, we sequenced Idgf cDNAs from several lepidopteran and trichopteran species and supplemented our data with sequences retrieved from public databases. A comparison of Idgf genes in different species showed that Diptera typically contain several Idgf paralogs with a simple exon-intron structure (2-3 exons), whereas lepidopteran Idgfs appear as a single copy per genome and contain a higher number of exons (around 9). Our results show that, while lepidopteran Idgfs, having single orthologs, are characterized by low divergence and stronger purifying selection over most of the molecule, the duplicated Idgf genes in Diptera, Idgf1 and Idgf4, exhibit signs of positive selection. This characterization of IDGF evolution provides, to our knowledge, the first information on the changes that formed these important molecules.

• Klíčová slova
• Drosophila, IDGF, chitinase, chitinase-like protein, phylogeny, positive selection,
• Publikační typ
• časopisecké články MeSH

SMG7 proteins are evolutionary conserved across eukaryotes and primarily known for their function in nonsense mediated RNA decay (NMD). In contrast to other NMD factors, SMG7 proteins underwent independent expansions during evolution indicating their propensity to adopt novel functions. Here we characterized SMG7 and SMG7-like (SMG7L) paralogs in Arabidopsis thaliana. SMG7 retained its role in NMD and additionally appears to have acquired another function in meiosis. We inactivated SMG7 by CRISPR/Cas9 mutagenesis and showed that, in contrast to our previous report, SMG7 is not an essential gene in Arabidopsis. Furthermore, our data indicate that the N-terminal phosphoserine-binding domain is required for both NMD and meiosis. Phenotypic analysis of SMG7 and SMG7L double mutants did not indicate any functional redundancy between the two genes, suggesting neofunctionalization of SMG7L. Finally, protein sequence comparison together with a phenotyping of T-DNA insertion mutants identified several conserved regions specific for SMG7 that may underlie its role in NMD and meiosis. This information provides a framework for deciphering the non-canonical functions of SMG7-family proteins.

• Klíčová slova
• Arabidopsis, SMG7, gene duplication, meiosis, nonsense mediated RNA decay,
• Publikační typ
• časopisecké články MeSH

Background: The eukaryotic endomembrane system most likely arose via paralogous expansions of genes encoding proteins that specify organelle identity, coat complexes and govern fusion specificity. While the majority of these gene families were established by the time of the last eukaryotic common ancestor (LECA), subsequent evolutionary events has moulded these systems, likely reflecting adaptations retained for increased fitness. As well as sequence evolution, these adaptations include loss of otherwise canonical components, the emergence of lineage-specific proteins and paralog expansion. The exocyst complex is involved in late exocytosis and additional trafficking pathways and a member of the complexes associated with tethering containing helical rods (CATCHR) tethering complex family. CATCHR includes the conserved oligomeric Golgi (COG) complex, homotypic fusion and vacuole protein sorting (HOPS)/class C core vacuole/endosome tethering (CORVET) complexes and several others. The exocyst is integrated into a complex GTPase signalling network in animals, fungi and other lineages. Prompted by discovery of Exo99, a non-canonical subunit in the excavate protist Trypanosoma brucei, and availability of significantly increased genome sequence data, we re-examined evolution of the exocyst. Methods: We examined the evolution of exocyst components by comparative genomics, phylogenetics and structure prediction. Results: The exocyst composition is highly conserved, but with substantial losses of subunits in the Apicomplexa and expansions in Streptophyta plants, Metazoa and land plants, where for the latter, massive paralog expansion of Exo70 represents an extreme and unique example. Significantly, few taxa retain a partial complex, suggesting that, in general, all subunits are probably required for functionality. Further, the ninth exocyst subunit, Exo99, is specific to the Euglenozoa with a distinct architecture compared to the other subunits and which possibly represents a coat system. Conclusions: These data reveal a remarkable degree of evolutionary flexibility within the exocyst complex, suggesting significant diversity in exocytosis mechanisms.

• Klíčová slova
• Exocytosis, comparative genomics, eukaryotes, exocyst, membrane transport, molecular evolution,
• Publikační typ
• časopisecké články MeSH

Gene duplication is an important evolutionary mechanism and no eukaryote has more duplicated gene families than the parasitic protist Trichomonas vaginalis. Iron is an essential nutrient for Trichomonas and plays a pivotal role in the establishment of infection, proliferation, and virulence. To gain insight into the role of iron in T. vaginalis gene expression and genome evolution, we screened iron-regulated genes using an oligonucleotide microarray for T. vaginalis and by comparative EST (expressed sequence tag) sequencing of cDNA libraries derived from trichomonads cultivated under iron-rich (+Fe) and iron-restricted (-Fe) conditions. Among 19,000 ESTs from both libraries, we identified 336 iron-regulated genes, of which 165 were upregulated under +Fe conditions and 171 under -Fe conditions. The microarray analysis revealed that 195 of 4,950 unique genes were differentially expressed. Of these, 117 genes were upregulated under +Fe conditions and 78 were upregulated under -Fe conditions. The results of both methods were congruent concerning the regulatory trends and the representation of gene categories. Under +Fe conditions, the expression of proteins involved in carbohydrate metabolism, particularly in the energy metabolism of hydrogenosomes, and in methionine catabolism was increased. The iron-sulfur cluster assembly machinery and certain cysteine proteases are of particular importance among the proteins upregulated under -Fe conditions. A unique feature of the T. vaginalis genome is the retention during evolution of multiple paralogous copies for a majority of all genes. Although the origins and reasons for this gene expansion remain unclear, the retention of multiple gene copies could provide an opportunity to evolve differential expression during growth in variable environmental conditions. For genes whose expression was affected by iron, we found that iron influenced the expression of only some of the paralogous copies, whereas the expression of the other paralogs was iron independent. This finding indicates a very stringent regulation of the differentially expressed paralogous genes in response to changes in the availability of exogenous nutrients and provides insight into the evolutionary rationale underlying massive paralog retention in the Trichomonas genome.

• MeSH
• cysteinové proteasy genetika   metabolismus   MeSH
• duplikace genu MeSH
• exprimované sekvenční adresy MeSH
• genom protozoální MeSH
• genová dávka MeSH
• genová knihovna MeSH
• glykolýza genetika   MeSH
• molekulární evoluce MeSH
• proteiny obsahující železo a síru genetika   metabolismus   MeSH
• protozoální geny * MeSH
• regulace genové exprese * MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• transkriptom * MeSH
• Trichomonas vaginalis genetika   metabolismus   MeSH
• železo metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cysteinové proteasy MeSH
• proteiny obsahující železo a síru MeSH
• železo MeSH

The rapid expansion of whole genome sequencing in bacterial taxonomy has revealed deep evolutionary relationships and speciation signals, but assembly methods often miss true nucleotide diversity in the ribosomal operons. Though it lacks sufficient phylogenetic signal at the species level, the 16S ribosomal RNA gene is still much used in bacterial taxonomy. In cyanobacterial taxonomy, comparisons of 16S-23S Internal Transcribed Spacer (ITS) regions are used to bridge this information gap. Although ITS rRNA region analyses are routinely being used to identify species, researchers often do not identify orthologous operons, which leads to improper comparisons. No method for delineating orthologous operon copies from paralogous ones has been established. A new method for recognizing orthologous ribosomal operons by quantifying the conserved paired nucleotides in a helical domain of the ITS, has been developed. The D1' Index quantifies differences in the ratio of pyrimidines to purines in paired nucleotide sequences of this helix. Comparing 111 operon sequences from 89 strains of Brasilonema, four orthologous operon types were identified. Plotting D1' Index values against the length of helices produced clear separation of orthologs. Most orthologous operons in this study were observed both with and without tRNA genes present. We hypothesize that genomic rearrangement, not gene duplication, is responsible for the variation among orthologs. This new method will allow cyanobacterial taxonomists to utilize ITS rRNA region data more correctly, preventing erroneous taxonomic hypotheses. Moreover, this work could assist genomicists in identifying and preserving evident sequence variability in ribosomal operons, which is an important proxy for evolution in prokaryotes.

• Klíčová slova
• Brasilonema, 16S‐23S ITS, cyanobacteria, evolution, homology, taxonomy,
• MeSH
• fylogeneze * MeSH
• mezerníky ribozomální DNA analýza   genetika   MeSH
• operon * MeSH
• RNA ribozomální 16S genetika   MeSH
• sinice genetika   klasifikace   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mezerníky ribozomální DNA MeSH
• RNA ribozomální 16S MeSH

BACKGROUND: Photosynthetic euglenids are major contributors to fresh water ecosystems. Euglena gracilis in particular has noted metabolic flexibility, reflected by an ability to thrive in a range of harsh environments. E. gracilis has been a popular model organism and of considerable biotechnological interest, but the absence of a gene catalogue has hampered both basic research and translational efforts. RESULTS: We report a detailed transcriptome and partial genome for E. gracilis Z1. The nuclear genome is estimated to be around 500 Mb in size, and the transcriptome encodes over 36,000 proteins and the genome possesses less than 1% coding sequence. Annotation of coding sequences indicates a highly sophisticated endomembrane system, RNA processing mechanisms and nuclear genome contributions from several photosynthetic lineages. Multiple gene families, including likely signal transduction components, have been massively expanded. Alterations in protein abundance are controlled post-transcriptionally between light and dark conditions, surprisingly similar to trypanosomatids. CONCLUSIONS: Our data provide evidence that a range of photosynthetic eukaryotes contributed to the Euglena nuclear genome, evidence in support of the 'shopping bag' hypothesis for plastid acquisition. We also suggest that euglenids possess unique regulatory mechanisms for achieving extreme adaptability, through mechanisms of paralog expansion and gene acquisition.

• Klíčová slova
• Cellular evolution, Euglena gracilis, Excavata, Gene architecture, Horizontal gene transfer, Plastid, Secondary endosymbiosis, Splicing, Transcriptome,
• MeSH
• buněčné jádro MeSH
• Euglena gracilis genetika   metabolismus   MeSH
• genom * MeSH
• plastidy MeSH
• proteom * MeSH
• transkriptom * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteom * MeSH

Exocyst is an evolutionarily conserved vesicle tethering complex functioning especially in the last stage of exocytosis. Homologs of its eight canonical subunits - Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 - were found also in higher plants and confirmed to form complexes in vivo, and to participate in cell growth including polarized expansion of pollen tubes and root hairs. Here we present results of a phylogenetic study of land plant exocyst subunits encoded by a selection of completely sequenced genomes representing a variety of plant, mostly angiosperm, lineages. According to their evolution histories, plant exocyst subunits can be divided into several groups. The core subunits Sec6, Sec8, and Sec10, together with Sec3 and Sec5, underwent few, if any fixed duplications in the tracheophytes (though they did amplify in the moss Physcomitrella patens), while others form larger families, with the number of paralogs ranging typically from two to eight per genome (Sec15, Exo84) to several dozens per genome (Exo70). Most of the diversity, which can be in some cases traced down to the origins of land plants, can be attributed to the peripheral subunits Exo84 and, in particular, Exo70. As predicted previously, early land plants (including possibly also the Rhyniophytes) encoded three ancestral Exo70 paralogs which further diversified in the course of land plant evolution. Our results imply that plants do not have a single "Exocyst complex" - instead, they appear to possess a diversity of exocyst variants unparalleled among other organisms studied so far. This feature might perhaps be directly related to the demands of building and maintenance of the complicated and spatially diverse structures of the endomembranes and cell surfaces in multicellular land plants.

• Klíčová slova
• co-evolution, exocyst, gene duplication, land plants, phylogeny,
• Publikační typ
• časopisecké články MeSH

Spatially directed cell division and expansion is important for plant growth and morphogenesis and relies on cooperation between the cytoskeleton and the secretory pathway. The phylogenetically conserved octameric complex exocyst mediates exocytotic vesicle tethering at the plasma membrane. Unlike other exocyst subunits of land plants, the core exocyst subunit SEC6 exists as a single paralog in Physcomitrium patens and Arabidopsis thaliana genomes. Arabidopsis SEC6 (AtSEC6) loss-of-function (LOF) mutation causes male gametophytic lethality. Our attempts to inactivate the P. patens SEC6 gene, PpSEC6, using targeted gene replacement produced two independent partial LOF ('weak allele') mutants via perturbation of the PpSEC6 gene locus. These mutants exhibited the same pleiotropic developmental defects: protonema with dominant chloronema stage; diminished caulonemal filament elongation rate; and failure in post-initiation gametophore development. Mutant gametophore buds, mostly initiated from chloronema cells, exhibited disordered cell file organization and cross-wall perforations, resulting in arrested development at the eight- to 10-cell stage. Complementation of both sec6 moss mutant lines by both PpSEC6 and AtSEC6 cDNA rescued gametophore development, including sexual organ differentiation. However, regular sporophyte formation and viable spore production were recovered only by the expression of PpSEC6, whereas the AtSEC6 complementants were only rarely fertile, indicating moss-specific SEC6 functions.

• Klíčová slova
• Physcomitrium patens, SEC6, cytokinesis, exocyst, exocytosis, gametophore development, polarity,
• MeSH
• exocytóza * genetika   MeSH
• mechy genetika   růst a vývoj   MeSH
• mutace MeSH
• rostlinné geny genetika   MeSH
• rostlinné proteiny genetika   fyziologie   MeSH
• zárodečné buňky rostlin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• rostlinné proteiny MeSH