Rab GTPase is a paralog-rich gene family that controls the maintenance of the eukaryotic cell compartmentalization system. Diverse eukaryotes have varying numbers of Rab paralogs. Currently, little is known about the evolutionary pattern of Rab GTPase in most major eukaryotic 'supergroups'. Here, we present a comprehensive phylogenetic reconstruction of the Rab GTPase gene family in the eukaryotic 'supergroup' Amoebozoa, a diverse lineage represented by unicellular and multicellular organisms. We demonstrate that Amoebozoa conserved 20 of the 23 ancestral Rab GTPases predicted to be present in the last eukaryotic common ancestor and massively expanded several 'novel' in-paralogs. Due to these 'novel' in-paralogs, the Rab family composition dramatically varies between the members of Amoebozoa; as a consequence, 'supergroup'-based studies may significantly change our current understanding of the evolution and diversity of this gene family. The high diversity of the Rab GTPase gene family in Amoebozoa makes this 'supergroup' a key lineage to study and advance our knowledge of the evolution of Rab in Eukaryotes.

• Klíčová slova
• Phylogenomics, Rab therapeutic intervention, cellular biology, eukaryotic evolution,
• MeSH
• Amoebozoa * genetika   metabolismus   MeSH
• Eukaryota metabolismus   MeSH
• fylogeneze MeSH
• molekulární evoluce MeSH
• Rab proteiny vázající GTP * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Rab proteiny vázající GTP * MeSH

BACKGROUND: Comparing a parasitic lineage to its free-living relatives is a powerful way to understand how that evolutionary transition to parasitism occurred. Giardia intestinalis (Fornicata) is a leading cause of gastrointestinal disease world-wide and is famous for its unusual complement of cellular compartments, such as having peripheral vacuoles instead of typical endosomal compartments. Endocytosis plays an important role in Giardia's pathogenesis. Endosomal sorting complexes required for transport (ESCRT) are membrane-deforming proteins associated with the late endosome/multivesicular body (MVB). MVBs are ill-defined in G. intestinalis, and roles for identified ESCRT-related proteins are not fully understood in the context of its unique endocytic system. Furthermore, components thought to be required for full ESCRT functionality have not yet been documented in this species. RESULTS: We used genomic and transcriptomic data from several Fornicata species to clarify the evolutionary genome streamlining observed in Giardia, as well as to detect any divergent orthologs of the Fornicata ESCRT subunits. We observed differences in the ESCRT machinery complement between Giardia strains. Microscopy-based investigations of key components of ESCRT machinery such as GiVPS36 and GiVPS25 link them to peripheral vacuoles, highlighting these organelles as simplified MVB equivalents. Unexpectedly, we show ESCRT components associated with the endoplasmic reticulum and, for the first time, mitosomes. Finally, we identified the rare ESCRT component CHMP7 in several fornicate representatives, including Giardia and show that contrary to current understanding, CHMP7 evolved from a gene fusion of VPS25 and SNF7 domains, prior to the last eukaryotic common ancestor, over 1.5 billion years ago. CONCLUSIONS: Our findings show that ESCRT machinery in G. intestinalis is far more varied and complete than previously thought, associates to multiple cellular locations, and presents changes in ESCRT complement which pre-date adoption of a parasitic lifestyle.

• Klíčová slova
• ESCRT, Endomembrane, Evolutionary Cell Biology, Excavata, Giardia, PV, Parasitism,
• MeSH
• biologická evoluce MeSH
• endozomální třídící komplexy pro transport * genetika   metabolismus   MeSH
• endozomy metabolismus   MeSH
• Giardia lamblia * genetika   metabolismus   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• endozomální třídící komplexy pro transport * MeSH

The discovery that the protist Monocercomonoides exilis completely lacks mitochondria demonstrates that these organelles are not absolutely essential to eukaryotic cells. However, the degree to which the metabolism and cellular systems of this organism have adapted to the loss of mitochondria is unknown. Here, we report an extensive analysis of the M. exilis genome to address this question. Unexpectedly, we find that M. exilis genome structure and content is similar in complexity to other eukaryotes and less "reduced" than genomes of some other protists from the Metamonada group to which it belongs. Furthermore, the predicted cytoskeletal systems, the organization of endomembrane systems, and biosynthetic pathways also display canonical eukaryotic complexity. The only apparent preadaptation that permitted the loss of mitochondria was the acquisition of the SUF system for Fe-S cluster assembly and the loss of glycine cleavage system. Changes in other systems, including in amino acid metabolism and oxidative stress response, were coincident with the loss of mitochondria but are likely adaptations to the microaerophilic and endobiotic niche rather than the mitochondrial loss per se. Apart from the lack of mitochondria and peroxisomes, we show that M. exilis is a fully elaborated eukaryotic cell that is a promising model system in which eukaryotic cell biology can be investigated in the absence of mitochondria.

• Klíčová slova
• Monocercomonoides, oxymonads, protist genomics, amitochondrial eukaryote, cell biology,
• MeSH
• genom protozoální * MeSH
• intracelulární membrány * MeSH
• introny MeSH
• mikrofilamenta MeSH
• mitochondriální dynamika MeSH
• Oxymonadida enzymologie   genetika   ultrastruktura   MeSH
• proteom MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteom MeSH

G-quadruplexes (G4) are non-canonical DNA and/or RNA secondary structures formed in guanine-rich regions. Given their over-representation in specific regions in the genome such as promoters and telomeres, they are likely to play important roles in key processes such as transcription, replication or RNA maturation. Putative G4-forming sequences (G4FS) have been reported in humans, yeast, bacteria, viruses and many organisms. Here we present the first mapping of G-quadruplex sequences in Dictyostelium discoideum, the social amoeba. 'Dicty' is an ameboid protozoan with a small (34 Mb) and extremely AT rich genome (78%). As a consequence, very few G4-prone motifs are expected. An in silico analysis of the Dictyostelium genome with the G4Hunter software detected 249-1055 G4-prone motifs, depending on G4Hunter chosen threshold. Interestingly, despite an even lower GC content (as compared to the whole Dicty genome), the density of G4 motifs in Dictyostelium promoters and introns is significantly higher than in the rest of the genome. Fourteen selected sequences located in important genes were characterized by a combination of biophysical and biochemical techniques. Our data show that these sequences form highly stable G4 structures under physiological conditions. Five Dictyostelium genes containing G4-prone motifs in their promoters were studied for the effect of a new G4-binding porphyrin derivative on their expression. Our results demonstrated that the new ligand significantly decreased their expression. Overall, our results constitute the first step to adopt Dictyostelium discoideum as a 'G4-poor' model for studies on G-quadruplexes.

• MeSH
• Dictyostelium genetika   MeSH
• G-kvadruplexy * MeSH
• genom genetika   MeSH
• konformace nukleové kyseliny MeSH
• počítačová simulace MeSH
• porfyriny genetika   MeSH
• promotorové oblasti (genetika) * MeSH
• telomery genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• porfyriny MeSH

Guanine-rich DNA has the potential to fold into non-canonical G-quadruplex (G4) structures. Analysis of the genome of the social amoeba Dictyostelium discoideum indicates a low number of sequences with G4-forming potential (249-1055). Therefore, D. discoideum is a perfect model organism to investigate the relationship between the presence of G4s and their biological functions. As a first step in this investigation, we crystallized the dGGGGGAGGGGTACAGGGGTACAGGGG sequence from the putative promoter region of two divergent genes in D. discoideum. According to the crystal structure, this sequence folds into a four-quartet intramolecular antiparallel G4 with two lateral and one diagonal loops. The G-quadruplex core is further stabilized by a G-C Watson-Crick base pair and a A-T-A triad and displays high thermal stability (Tm > 90°C at 100 mM KCl). Biophysical characterization of the native sequence and loop mutants suggests that the DNA adopts the same structure in solution and in crystalline form, and that loop interactions are important for the G4 stability but not for its folding. Four-tetrad G4 structures are sparse. Thus, our work advances understanding of the structural diversity of G-quadruplexes and yields coordinates for in silico drug screening programs and G4 predictive tools.

• MeSH
• cirkulární dichroismus MeSH
• Dictyostelium genetika   MeSH
• G-kvadruplexy * MeSH
• genom MeSH
• konformace nukleové kyseliny * MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• mutace MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• promotorové oblasti (genetika) MeSH
• spektrofotometrie ultrafialová MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Bacteria influence eukaryotic biology as parasitic, commensal or beneficial symbionts. Aside from these organismal interactions, bacteria have also been important sources of new genetic sequences through horizontal gene transfer (HGT) for eukaryotes. In this Review, we focus on gene transfers from bacteria to eukaryotes, discuss how horizontally transferred genes become functional and explore what functions are endowed upon a broad diversity of eukaryotes by genes derived from bacteria. We classify HGT events into two broad types: those that maintain pre-existing functions and those that provide the recipient with new functionality, including altered host nutrition, protection and adaptation to extreme environments.

• MeSH
• Bacteria genetika   MeSH
• biologická evoluce MeSH
• Eukaryota genetika   MeSH
• fyziologická adaptace genetika   MeSH
• přenos genů horizontální fyziologie   MeSH
• regulace genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Amoebozoa is the eukaryotic supergroup sister to Obazoa, the lineage that contains the animals and Fungi, as well as their protistan relatives, and the breviate and apusomonad flagellates. Amoebozoa is extraordinarily diverse, encompassing important model organisms and significant pathogens. Although amoebozoans are integral to global nutrient cycles and present in nearly all environments, they remain vastly understudied. We present a robust phylogeny of Amoebozoa based on broad representative set of taxa in a phylogenomic framework (325 genes). By sampling 61 taxa using culture-based and single-cell transcriptomics, our analyses show two major clades of Amoebozoa, Discosea, and Tevosa. This phylogeny refutes previous studies in major respects. Our results support the hypothesis that the last common ancestor of Amoebozoa was sexual and flagellated, it also may have had the ability to disperse propagules from a sporocarp-type fruiting body. Overall, the main macroevolutionary patterns in Amoebozoa appear to result from the parallel losses of homologous characters of a multiphase life cycle that included flagella, sex, and sporocarps rather than independent acquisition of convergent features.

• Klíčová slova
• Amoebozoa, phylogenomics, phylotranscriptomics, reductive evolution, transcriptomes,
• MeSH
• Amoeba genetika   metabolismus   MeSH
• Amoebozoa genetika   MeSH
• bezobratlí genetika   MeSH
• biologická evoluce MeSH
• Eukaryota genetika   MeSH
• fylogeneze MeSH
• genová knihovna MeSH
• houby genetika   MeSH
• molekulární evoluce MeSH
• sekvenční analýza DNA metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

The moss Physcomitrella patens is part of an early divergent clade of land plants utilizing the plant hormone cytokinin for growth control. The rate-limiting step of cytokinin biosynthesis is mediated by isopentenyltransferases (IPTs), found in land plants either as adenylate-IPTs or as tRNA-IPTs. Although a dominant part of cytokinins in flowering plants are synthesized by adenylate-IPTs, the Physcomitrella genome only encodes homologues of tRNA-IPTs. This study therefore looked into the question of whether cytokinins in moss derive from tRNA exclusively. Targeted gene knockout of ipt1 (d|ipt1) along with localization studies revealed that the chloroplast-bound IPT1 was almost exclusively responsible for the A37 prenylation of tRNA in Physcomitrella. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)-based cytokinin profiling demonstrated that the total amount of all free cytokinins in tissue was almost unaffected. However, the knockout plants showed increased levels of the N (6) -isopentenyladenine (iP)- and trans-zeatin (tZ)-type cytokinins, considered to provide active forms, while cis-zeatin (cZ)-type cytokinins were reduced. The data provide evidence for an additional and unexpected tRNA-independent cytokinin biosynthetic pathway in moss. Comprehensive phylogenetic analysis indicates a diversification of tRNA-IPT-like genes in bryophytes probably related to additional functions.

• Klíčová slova
• Bryophyte, cytokinin, isopentenyladenosine, isopentenyltransferases, moss, tRNA.,
• MeSH
• alkyltransferasy a aryltransferasy genetika   metabolismus   MeSH
• biologická evoluce * MeSH
• chloroplasty enzymologie   genetika   MeSH
• cytokininy biosyntéza   MeSH
• fylogeneze * MeSH
• genový knockout MeSH
• mechy klasifikace   enzymologie   genetika   MeSH
• molekulární sekvence - údaje MeSH
• RNA transferová genetika   metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• rostliny klasifikace   enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylate isopentenyltransferase MeSH Prohlížeč
• alkyltransferasy a aryltransferasy MeSH
• cytokininy MeSH
• RNA transferová MeSH
• rostlinné proteiny MeSH