Protein evolution and protein engineering techniques are of great interest in basic science and industrial applications such as pharmacology, medicine, or biotechnology. Ancestral sequence reconstruction (ASR) is a powerful technique for probing evolutionary relationships and engineering robust proteins with good thermostability and broad substrate specificity. The following protocol describes the setting up and execution of an automated FireProtASR workflow using a dedicated web site. The service allows for inference of ancestral proteins automatically, from a single protein sequence. Once a protein sequence is submitted, the server will build a dataset of homology sequences, perform a multiple sequence alignment (MSA), build a phylogenetic tree, and reconstruct ancestral nodes. The protocol is also highly flexible and allows for multiple forms of input, advanced settings, and the ability to start jobs from: (i) a single sequence, (ii) a set of homologous sequences, (iii) an MSA, and (iv) a phylogenetic tree. This approach automates all necessary steps and offers a way for novices with limited exposure to ASR techniques to improve the properties of a protein of interest. The technique can even be used to introduce catalytic promiscuity into an enzyme. A web server for accessing the fully automated workflow is freely accessible at https://loschmidt.chemi.muni.cz/fireprotasr/. © 2021 Wiley Periodicals LLC. Basic Protocol: ASR using the Web Server FireProtASR.

• Klíčová slova
• ancestral sequence reconstruction, automation, protein engineering, protein evolution, thermostability,
• MeSH
• fylogeneze MeSH
• molekulární evoluce * MeSH
• proteiny * genetika   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny * MeSH

Ancestral sequence reconstruction (ASR) represents a powerful approach for empirical testing structure-function relationships of diverse proteins. We employed ASR to predict sequences of five ancestral haloalkane dehalogenases (HLDs) from the HLD-II subfamily. Genes encoding the inferred ancestral sequences were synthesized and expressed in Escherichia coli, and the resurrected ancestral enzymes (AncHLD1-5) were experimentally characterized. Strikingly, the ancestral HLDs exhibited significantly enhanced thermodynamic stability compared to extant enzymes (ΔTm up to 24 °C), as well as higher specific activities with preference for short multi-substituted halogenated substrates. Moreover, multivariate statistical analysis revealed a shift in the substrate specificity profiles of AncHLD1 and AncHLD2. This is extremely difficult to achieve by rational protein engineering. The study highlights that ASR is an efficient approach for the development of novel biocatalysts and robust templates for directed evolution.

• Klíčová slova
• ancestral sequence reconstruction, haloalkane dehalogenase, protein engineering, robustness, substrate specificity,
• MeSH
• genetický kód MeSH
• hydrolasy chemie   genetika   metabolismus   MeSH
• multivariační analýza MeSH
• proteinové inženýrství MeSH
• řízená evoluce molekul MeSH
• substrátová specifita MeSH
• termodynamika MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH

Ancestral sequence reconstruction is a powerful method for inferring ancestors of modern enzymes and for studying structure-function relationships of enzymes. We have previously applied this approach to haloalkane dehalogenases (HLDs) from the subfamily HLD-II and obtained thermodynamically highly stabilized enzymes (ΔT m up to 24 °C), showing improved catalytic properties. Here we combined crystallographic structural analysis and computational molecular dynamics simulations to gain insight into the mechanisms by which ancestral HLDs became more robust enzymes with novel catalytic properties. Reconstructed ancestors exhibited similar structure topology as their descendants with the exception of a few loop deviations. Strikingly, molecular dynamics simulations revealed restricted conformational dynamics of ancestral enzymes, which prefer a single state, in contrast to modern enzymes adopting two different conformational states. The restricted dynamics can potentially be linked to their exceptional stabilization. The study provides molecular insights into protein stabilization due to ancestral sequence reconstruction, which is becoming a widely used approach for obtaining robust protein catalysts.

• Klíčová slova
• Ancestral sequence reconstruction, Conformational flexibility, Enzyme, Haloalkane dehalogenase, Protein design, Protein simulations, Thermostability, X-ray crystallography,
• Publikační typ
• časopisecké články MeSH

Tropical forests of Central and South America represent hotspots of biological diversity. Tree squirrels of the tribe Sciurini are an excellent model system for the study of tropical biodiversity as these squirrels disperse exceptional distances, and after colonizing the tropics of the Central and South America, they have diversified rapidly. Here, we compare signals from DNA sequences with morphological signals using pictures of skulls and computational simulations. Phylogenetic analyses reveal step-wise geographic divergence across the Northern Hemisphere. In Central and South America, tree squirrels form two separate clades, which split from a common ancestor. Simulations of ancestral distributions show western Amazonia as the epicenter of speciation in South America. This finding suggests that wet tropical forests on the foothills of Andes possibly served as refugia of squirrel diversification during Pleistocene climatic oscillations. Comparison of phylogeny and morphology reveals one major discrepancy: Microsciurus species are a single clade morphologically but are polyphyletic genetically. Modeling of morphology-diet relationships shows that the only group of species with a direct link between skull shape and diet are the bark-gleaning insectivorous species of Microsciurus. This finding suggests that the current designation of Microsciurus as a genus is based on convergent ecologically driven changes in morphology.

• Klíčová slova
• Ancestral range reconstruction, Sciurini, diet modeling, geometric morphometry, multilocus phylogeny, speciation,
• MeSH
• dieta * MeSH
• fylogeneze MeSH
• fyziologická adaptace MeSH
• lebka anatomie a histologie   MeSH
• počítačová simulace * MeSH
• rozšíření zvířat * MeSH
• Sciuridae * anatomie a histologie   klasifikace   genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Regulation of gene expression is arguably the main mechanism underlying the phenotypic diversity of tissues within and between species. Here we assembled an extensive transcriptomic dataset covering 8 tissues across 20 bilaterian species and performed analyses using a symmetric phylogeny that allowed the combined and parallel investigation of gene expression evolution between vertebrates and insects. We specifically focused on widely conserved ancestral genes, identifying strong cores of pan-bilaterian tissue-specific genes and even larger groups that diverged to define vertebrate and insect tissues. Systematic inferences of tissue-specificity gains and losses show that nearly half of all ancestral genes have been recruited into tissue-specific transcriptomes. This occurred during both ancient and, especially, recent bilaterian evolution, with several gains being associated with the emergence of unique phenotypes (for example, novel cell types). Such pervasive evolution of tissue specificity was linked to gene duplication coupled with expression specialization of one of the copies, revealing an unappreciated prolonged effect of whole-genome duplications on recent vertebrate evolution.

• MeSH
• fylogeneze MeSH
• hmyz * genetika   MeSH
• molekulární evoluce * MeSH
• obratlovci * genetika   MeSH
• orgánová specificita MeSH
• transkriptom MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Body growth is typically thought to be indeterminate in ectothermic vertebrates. Indeed, until recently, this growth pattern was considered to be ubiquitous in ectotherms. Our recent observations of a complete growth plate cartilage (GPC) resorption, a reliable indicator of arrested skeletal growth, in many species of lizards clearly reject the ubiquity of indeterminate growth in reptiles and raise the question about the ancestral state of the growth pattern. Using X-ray micro-computed tomography (µCT), here we examined GPCs of long bones in three basally branching clades of squamate reptiles, namely in Gekkota, Scincoidea and Lacertoidea. A complete loss of GPC, indicating skeletal growth arrest, was the predominant finding. Using a dataset of 164 species representing all major clades of lizards and the tuataras, we traced the evolution of determinate growth on the phylogenetic tree of Lepidosauria. The reconstruction of character states suggests that determinate growth is ancestral for the squamate reptiles (Squamata) and remains common in the majority of lizard lineages, while extended (potentially indeterminate) adult growth evolved several times within squamates. Although traditionally associated with endotherms, determinate growth is coupled with ectothermy in this lineage. These findings combined with existing literature suggest that determinate growth predominates in both extant and extinct amniotes.

• Klíčová slova
• determinate growth, endothermy, growth plate cartilage, indeterminate growth, micro-CT, squamata,
• MeSH
• biologická evoluce MeSH
• fylogeneze MeSH
• hadi MeSH
• ještěři MeSH
• plazi růst a vývoj   fyziologie   MeSH
• rentgenová mikrotomografie MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Bacterial division initiates at the site of a contractile Z-ring composed of polymerized FtsZ. The location of the Z-ring in the cell is controlled by a system of three mutually antagonistic proteins, MinC, MinD, and MinE. Plastid division is also known to be dependent on homologs of these proteins, derived from the ancestral cyanobacterial endosymbiont that gave rise to plastids. In contrast, the mitochondria of model systems such as Saccharomyces cerevisiae, mammals, and Arabidopsis thaliana seem to have replaced the ancestral α-proteobacterial Min-based division machinery with host-derived dynamin-related proteins that form outer contractile rings. Here, we show that the mitochondrial division system of these model organisms is the exception, rather than the rule, for eukaryotes. We describe endosymbiont-derived, bacterial-like division systems comprising FtsZ and Min proteins in diverse less-studied eukaryote protistan lineages, including jakobid and heterolobosean excavates, a malawimonad, stramenopiles, amoebozoans, a breviate, and an apusomonad. For two of these taxa, the amoebozoan Dictyostelium purpureum and the jakobid Andalucia incarcerata, we confirm a mitochondrial localization of these proteins by their heterologous expression in Saccharomyces cerevisiae. The discovery of a proteobacterial-like division system in mitochondria of diverse eukaryotic lineages suggests that it was the ancestral feature of all eukaryotic mitochondria and has been supplanted by a host-derived system multiple times in distinct eukaryote lineages.

• Klíčová slova
• Min proteins, MinCDE, mitochondria, mitochondrial division, mitochondrial fission,
• MeSH
• adenosintrifosfatasy metabolismus   MeSH
• Arabidopsis genetika   MeSH
• Bacteria cytologie   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• buněčné dělení MeSH
• cytoskeletální proteiny genetika   MeSH
• databáze genetické MeSH
• Dictyostelium metabolismus   MeSH
• DNA bakterií genetika   MeSH
• fylogeneze MeSH
• mitochondriální dynamika * MeSH
• mitochondrie metabolismus   MeSH
• molekulární evoluce MeSH
• molekulární sekvence - údaje MeSH
• plastidy metabolismus   MeSH
• pravděpodobnostní funkce MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• proteiny z Escherichia coli metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• bakteriální proteiny MeSH
• cytoskeletální proteiny MeSH
• DNA bakterií MeSH
• FtsZ protein, Bacteria MeSH Prohlížeč
• MinC protein, Bacteria MeSH Prohlížeč
• MinD protein, E coli MeSH Prohlížeč
• MinE protein, E coli MeSH Prohlížeč
• proteiny buněčného cyklu MeSH
• proteiny z Escherichia coli MeSH

The dentitions of extant fishes and land vertebrates vary in both pattern and type of tooth replacement. It has been argued that the common ancestral condition likely resembles the nonmarginal, radially arranged tooth files of arthrodires, an early group of armoured fishes. We used synchrotron microtomography to describe the fossil dentitions of so-called acanthothoracids, the most phylogenetically basal jawed vertebrates with teeth, belonging to the genera Radotina, Kosoraspis, and Tlamaspis (from the Early Devonian of the Czech Republic). Their dentitions differ fundamentally from those of arthrodires; they are marginal, carried by a cheekbone or a series of short dermal bones along the jaw edges, and teeth are added lingually as is the case in many chondrichthyans (cartilaginous fishes) and osteichthyans (bony fishes and tetrapods). We propose these characteristics as ancestral for all jawed vertebrates.

• MeSH
• biologická evoluce * MeSH
• čelisti anatomie a histologie   MeSH
• dentice * MeSH
• fylogeneze MeSH
• obratlovci anatomie a histologie   klasifikace   MeSH
• synchrotrony MeSH
• tomografie elektronová MeSH
• zkameněliny MeSH
• zuby anatomie a histologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

We searched for the previously defined uncoupling protein (UCP) signatures [Jezek, P. and Urbánková, E. (2000) IUBMB Life 49, 63-70] in genomes of Drosophila melanogaster, Caenorhabditis elegans, Dictyostelium discoideum, and Arabidopsis thaliana. We identified four UCPs in Drosophila and one in Caenorhabditis or Dictyostelium as close relatives of human UCP4 (BMCP), but distant from UCP1, UCP2, UCP3, and two plant UCPs of Arabidopsis. But the third Arabidopsis UCP is the closest UCP4 relative. This suggests that UCP4 represents the ancestral UCP from which other mammalian and plant UCPs diverged. Speculations on UCP4 participation in apoptosis are thus supported by its early phylogenetic occurrence.

• MeSH
• aminokyselinové motivy MeSH
• Arabidopsis genetika   MeSH
• Caenorhabditis elegans genetika   MeSH
• Dictyostelium genetika   MeSH
• Drosophila melanogaster genetika   MeSH
• fylogeneze * MeSH
• geny helmintů MeSH
• hmyzí geny MeSH
• membránové transportní proteiny * MeSH
• mitochondriální odpřahující proteiny MeSH
• molekulární evoluce * MeSH
• molekulární sekvence - údaje MeSH
• protozoální geny MeSH
• rostlinné geny MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• transportní proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové transportní proteiny * MeSH
• mitochondriální odpřahující proteiny MeSH
• SLC25A27 protein, human MeSH Prohlížeč
• transportní proteiny MeSH

Nonequilibrium dynamics and non-neutral processes, such as trait-dependent dispersal, are often missing from quantitative island biogeography models despite their potential explanatory value. One of the most influential nonequilibrium models is the taxon cycle, but it has been difficult to test its validity as a general biogeographical framework. Here, we test predictions of the taxon cycle model using six expected phylogenetic patterns and a time-calibrated phylogeny of Indo-Pacific Odontomachus (Hymenoptera: Formicidae: Ponerinae), one of the ant genera that E.O. Wilson used when first proposing the hypothesis. We used model-based inference and a newly developed trait-dependent dispersal model to jointly estimate ancestral biogeography, ecology (habitat preferences for forest interiors, vs. "marginal" habitats, such as savannahs, shorelines, disturbed areas) and the linkage between ecology and dispersal rates. We found strong evidence that habitat shifts from forest interior to open and disturbed habitats increased macroevolutionary dispersal rate. In addition, lineages occupying open and disturbed habitats can give rise to both island endemics re-occupying only forest interiors and taxa that re-expand geographical ranges. The phylogenetic predictions outlined in this study can be used in future work to evaluate the relative weights of neutral (e.g., geographical distance and area) and non-neutral (e.g., trait-dependent dispersal) processes in historical biogeography and community ecology.

• Klíčová slova
• Formicidae, Melanesia, biogeography, diversification, insect, taxon cycle,
• MeSH
• ekosystém MeSH
• Formicidae klasifikace   genetika   MeSH
• fylogeneze * MeSH
• fylogeografie 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