Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques to explore SecY pore dynamics on multiple timescales in order to dissect the complex reaction pathway. The results show that SecA, both the signal sequence and mature components of the pre-protein, and ATP hydrolysis each have important and specific roles in channel unlocking, opening and priming for transport. After channel opening, translocation proceeds in two phases: a slow phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~40 amino acids per second for the proOmpA substrate. Broad translocation rate distributions reflect the stochastic nature of polypeptide transport.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• adenosintrifosfatasy chemie   genetika   metabolismus   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• fluorescenční mikroskopie metody   MeSH
• hydrolýza MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• mutace MeSH
• proteiny - lokalizační signály genetika   MeSH
• proteiny z Escherichia coli chemie   genetika   metabolismus   MeSH
• protonmotorická síla * MeSH
• translokační kanály SEC chemie   genetika   metabolismus   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The captive bred animal populations showing centric fusion polymorphism can serve as a model for analysis of the impact of the rearrangement on meiosis and reproduction. The synapsis of homologous chromosomes and the frequency and distribution of meiotic recombination events were studied in pachytene spermatocytes of captive bred male impalas (Aepyceros melampus) polymorphic for der(14;20) by immunofluorescent analysis and fluorescence in situ hybridization. The chromosomes 14 and 20 involved in the centric fusion were significantly shorter due to the loss of sat I repeats indicating ancient origin of the rearrangement. The fused chromosome and the normal acrocentric chromosomes 14 and 20 formed trivalent in pachynema which showed either protruding proximal ends of the acrocentric chromosomes or single axis with synaptic adjustment in the pericentromeric region. There was no significant difference in the number of recombination events per cell between the group of translocation heterozygotes and the animals with normal karyotype. A significant reduction in the number of recombination events was observed in the trivalent chromosomes compared to the normal chromosomes 14 and 20. The level of the recombination reduction was related to the trivalent configuration. The centric fusion der(14;20) was not apparently demonstrated by any spermatogenic defects or reproductive impairment in heterozygous impalas. However, the high incidence of the chromosomal polymorphism within the captive bred population shows the importance of cytogenetic examinations in captive breeding and wildlife conservation programs, especially in the case of reintroduction of the endangered species.

• MeSH
• hybridizace in situ fluorescenční MeSH
• jaderné proteiny genetika   MeSH
• lymfocyty metabolismus   MeSH
• meióza genetika   MeSH
• metafáze genetika   MeSH
• modely u zvířat MeSH
• pachytenní stadium MeSH
• přežvýkavci genetika   MeSH
• rekombinace genetická genetika   MeSH
• rozmnožování genetika   MeSH
• savčí chromozomy genetika   MeSH
• spermatocyty cytologie   metabolismus   MeSH
• synaptonemální komplex genetika   MeSH
• translokace genetická * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The crayfish plague agent, Aphanomyces astaci, has spread throughout Europe, causing a significant decline in native European crayfish. The introduction and dissemination of this pathogen is attributed to the spread of invasive North American crayfish, which can act as carriers for A. astaci. As native European crayfish often succumb to infection with A. astaci, determining the prevalence of this pathogen in non-native crayfish is vital to prioritize native crayfish populations for managed translocation. In the current study, 23 populations of invasive signal crayfish (Pacifastacus leniusculus) from the UK were tested for A. astaci presence using quantitative PCR. Altogether, 13 out of 23 (56·5%) populations were found to be infected, and pathogen prevalence within infected sites varied from 3 to 80%. Microsatellite pathogen genotyping revealed that at least one UK signal crayfish population was infected with the A. astaci genotype group B, known to include virulent strains. Based on recent crayfish distribution records and the average rate of signal crayfish population dispersal, we identified one native white-clawed crayfish (Austropotamobius pallipes) population predicted to come into contact with infected signal crayfish within 5 years. This population should be considered as a priority for translocation.

• MeSH
• Aphanomyces izolace a purifikace   MeSH
• interakce hostitele a parazita MeSH
• prevalence MeSH
• severní raci parazitologie   MeSH
• zachování přírodních zdrojů * MeSH
• zavlečené druhy MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Spojené království MeSH

The origin of protein import was a key step in the endosymbiotic acquisition of mitochondria. Though the main translocon of the mitochondrial outer membrane, TOM40, is ubiquitous among organelles of mitochondrial ancestry, the transit peptides, or N-terminal targeting sequences (NTSs), recognised by the TOM complex, are not. To better understand the nature of evolutionary conservation in mitochondrial protein import, we investigated the targeting behavior of Trichomonas vaginalis hydrogenosomal proteins in Saccharomyces cerevisiae and vice versa. Hydrogenosomes import yeast mitochondrial proteins even in the absence of their native NTSs, but do not import yeast cytosolic proteins. Conversely, yeast mitochondria import hydrogenosomal proteins with and without their short NTSs. Conservation of an NTS-independent mitochondrial import route from excavates to opisthokonts indicates its presence in the eukaryote common ancestor. Mitochondrial protein import is known to entail electrophoresis of positively charged NTSs across the electrochemical gradient of the inner mitochondrial membrane. Our present findings indicate that mitochondrial transit peptides, which readily arise from random sequences, were initially selected as a signal for charge-dependent protein targeting specifically to the mitochondrial matrix. Evolutionary loss of the electron transport chain in hydrogenosomes and mitosomes lifted the selective constraints that maintain positive charge in NTSs, allowing first the NTS charge, and subsequently the NTS itself, to be lost. This resulted in NTS-independent matrix targeting, which is conserved across the evolutionary divide separating trichomonads and yeast, and which we propose is the ancestral state of mitochondrial protein import.

• MeSH
• mitochondriální proteiny chemie   metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• molekulární evoluce * MeSH
• proteiny - lokalizační signály * MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• transport proteinů MeSH
• Trichomonas vaginalis metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

During translation, a conserved GTPase elongation factor-EF-G in bacteria or eEF2 in eukaryotes-translocates tRNA and mRNA through the ribosome. EF-G has been proposed to act as a flexible motor that propels tRNA and mRNA movement, as a rigid pawl that biases unidirectional translocation resulting from ribosome rearrangements, or by various combinations of motor- and pawl-like mechanisms. Using time-resolved cryo-EM, we visualized GTP-catalyzed translocation without inhibitors, capturing elusive structures of ribosome•EF-G intermediates at near-atomic resolution. Prior to translocation, EF-G binds near peptidyl-tRNA, while the rotated 30S subunit stabilizes the EF-G GTPase center. Reverse 30S rotation releases Pi and translocates peptidyl-tRNA and EF-G by ~20 Å. An additional 4-Å translocation initiates EF-G dissociation from a transient ribosome state with highly swiveled 30S head. The structures visualize how nearly rigid EF-G rectifies inherent and spontaneous ribosomal dynamics into tRNA-mRNA translocation, whereas GTP hydrolysis and Pi release drive EF-G dissociation.

• MeSH
• aminoacyl-tRNA metabolismus   MeSH
• elektronová kryomikroskopie * MeSH
• elongační faktor G chemie   metabolismus   MeSH
• Escherichia coli chemie   metabolismus   MeSH
• fosfáty metabolismus   MeSH
• guanosintrifosfát chemie   metabolismus   MeSH
• malé podjednotky ribozomu bakteriální chemie   metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• proteosyntéza MeSH
• ribozomy chemie   metabolismus   MeSH
• RNA transferová metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

BACKGROUND: Chromosomal rearrangements are a major driving force in shaping genome during evolution. Previous studies show that translocated genes could undergo elevated rates of evolution and recombination frequencies around these genes can be altered. Based on the recently released genome sequences of Triticum urartu, Aegilops tauschii, Brachypodium distachyon and bread wheat, an analysis of interchromosomal translocations in the hexaploid wheat genotype 'Chinese Spring' ('CS') was conducted based on chromosome shotgun sequences from individual chromosome arms of this genotype. RESULTS: A total of 720 genes representing putative interchromosomal rearrangements was identified. They were distributed across the 42 chromosome arms. About 59% of these translocated genes were those involved in the well-characterized translocations involving chromosomes 4A, 5A and 7B. The other 41% of the genes represent a large numbers of putative interchromosomal rearrangements which have not yet been described. The number of the putative translocation events in the D subgenome was about half of those presented in either the A or B subgenomes, which agreed well with that the times of interaction between the A and B subgenomes almost doubled that between either of them and the D subgenome. CONCLUSIONS: The possible existence of a large number of interchromosomal rearrangements detected in this study provide further evidence that caution should be taken when using synteny in ordering sequence contigs or in cloning genes in hexaploid wheat. The identification of these putative translocations in 'CS' also provide a base for a systematic evaluation of their presence or absence in the full spectrum of bread wheat and its close relatives, which could have significant implications in a wide array of fields ranging from studies of systematics and evolution to practical breeding.

• MeSH
• chromozomy rostlin * MeSH
• genom rostlinný MeSH
• lipnicovité genetika   MeSH
• mapování chromozomů MeSH
• pšenice klasifikace   genetika   MeSH
• rostlinné geny MeSH
• syntenie * MeSH
• translokace genetická MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The origin of Cardamine flexuosa (Wavy Bittercress) has been a conundrum for more than six decades. Here we identify its parental species, analyse its genome structure in comparison to parental genomes and describe intergenomic structural variations in C. flexuosa. Genomic in situ hybridization (GISH) and comparative chromosome painting (CCP) uncovered the parental genomes and the chromosome composition of C. flexuosa and its presumed diploid progenitors. Cardamine flexuosa is an allotetraploid (2n = 4x = 32), originating from two diploid species, Cardamine amara and Cardamine hirsuta (2n = 2x = 16). The two parental species display almost perfectly conserved chromosomal collinearity for seven out of the eight chromosomes. A 13 Mb pericentric inversion distinguishes chromosome CA1 from CH1. A comparative cytomolecular map was established for C. flexuosa by CCP/GISH. Whereas conserved chromosome collinearity between the C. amara and C. hirsuta subgenomes might have promoted intergenomic rearrangements through homeologous recombination, only one reciprocal translocation between two homeologues has occurred since the origin of C. flexuosa. The genome of C. flexuosa demonstrates that allopolyploids can maintain remarkably stable subgenomes over 10(4) -10(5) yr throughout a wide distribution range. By contrast, the rRNA genes underwent genome-specific elimination towards a diploid-like number of loci.

• MeSH
• Cardamine genetika   MeSH
• chromozomy rostlin genetika   MeSH
• délka genomu genetika   MeSH
• diploidie MeSH
• druhová specificita MeSH
• genetické lokusy genetika   MeSH
• genom rostlinný genetika   MeSH
• hybridizace in situ MeSH
• karyotypizace MeSH
• konzervovaná sekvence * MeSH
• malování chromozomů MeSH
• polyploidie * MeSH
• ribozomální DNA genetika   MeSH
• translokace genetická MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The Sec translocon is a highly conserved membrane assembly for polypeptide transport across, or into, lipid bilayers. In bacteria, secretion through the core channel complex-SecYEG in the inner membrane-is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the conformational state of SecYEG throughout the ATP hydrolysis cycle of SecA. We show that the SecYEG channel fluctuations between open and closed states are much faster (~20-fold during translocation) than ATP turnover, and that the nucleotide status of SecA modulates the rates of opening and closure. The SecY variant PrlA4, which exhibits faster transport but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore and thereby enhancing translocation efficiency. Thus, rapid SecYEG channel dynamics are allosterically coupled to SecA via modulation of the energy landscape, and play an integral part in protein transport. Loose coupling of ATP-turnover by SecA to the dynamic properties of SecYEG is compatible with a Brownian-rachet mechanism of translocation, rather than strict nucleotide-dependent interconversion between different static states of a power stroke.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• adenosintrifosfatasy genetika   metabolismus   MeSH
• bakteriální proteiny * metabolismus   MeSH
• nukleotidy metabolismus   MeSH
• proteiny SecA metabolismus   MeSH
• proteiny z Escherichia coli * metabolismus   MeSH
• translokační kanály SEC chemie   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH

Establishing translocated populations is a common process to preserve and maintain genetic diversity of threatened species. In 2001, three translocated populations of noble crayfish (Astacus astacus) were established in the Czech Republic, founded by either adult or juvenile individuals from three particular source populations. We assessed genetic diversity at seven microsatellite loci after one decade (assumed three generations) from establishment. Although the translocated populations exhibited a slight but non-significant reduction in genetic diversity (A R = 2.2-5.0; H O = 0.11-0.31), the most striking result was generally very low genetic diversity in source populations (A R = 3.0-5.3; H O = 0.15-0.38). Similarly, a high degree of inbreeding (F IS = 0.36-0.60) demonstrates the nature of source populations, already affected by isolation and small size. In spite of that, based on the results of this study, the establishment of new translocated noble crayfish populations was successful, since there is no significant decline in genetic variability and all populations are still viable. Although source populations did not exhibit high genetic diversity, their distinctiveness makes them possible to use for conservation purposes. Continued monitoring is necessary to track the long-term progress of the translocation program, including other parameters describing the state of the population, such as the occurrence and frequency of diseases or morphological changes.

• MeSH
• alely MeSH
• efekt zakladatele * MeSH
• genetická variace * MeSH
• hustota populace MeSH
• inbreeding MeSH
• mikrosatelitní repetice MeSH
• populační genetika * MeSH
• sekvenční analýza DNA MeSH
• severní raci genetika   MeSH
• zachování přírodních zdrojů MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

Potassium ion (K+) uptake in yeast is mediated mainly by the Trk1/2 proteins that enable cells to survive on external K+ concentration as low as a few μM. Fungal Trks are related to prokaryotic TRK and Ktr and plant HKT K+ transport systems. Overall sequence similarity is very low, thus requiring experimental verification of homology models. Here a refined structural model of the Saccharomyces cerevisiae Trk1 is presented that was obtained by combining homology modeling, molecular dynamics simulation and experimental verification through functional analysis of mutants. Structural models and experimental results showed that glycines within the selectivity filter, conserved among the K-channel/transporter family, are not only important for protein function, but are also required for correct folding/membrane targeting. A conserved aspartic acid in the PA helix (D79) and a lysine in the M2D helix (K1147) were proposed earlier to interact. Our results suggested individual roles of these residues in folding, structural integrity and function. While mutations of D79 completely abolished protein folding, mutations at position 1147 were tolerated to some extent. Intriguingly, a secondary interaction of D79 with R76 could enhance folding/stability of Trk1 and enable a fraction of Trk1[K1147A] to fold. The part of the ion permeation path containing the selectivity filter is shaped similar to that of ion channels. However below the selectivity filter it is obstructed or regulated by a proline containing loop. The presented model could provide the structural basis for addressing the long standing question if Trk1 is a passive or active ion-translocation system.

• MeSH
• buněčná membrána chemie   metabolismus   MeSH
• draslík metabolismus   MeSH
• gating iontového kanálu * MeSH
• glycin MeSH
• kinetika MeSH
• konformace proteinů MeSH
• konzervovaná sekvence MeSH
• kyselina asparagová MeSH
• lysin MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• mutageneze cílená MeSH
• permeabilita buněčné membrány MeSH
• proteiny přenášející kationty chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• stabilita proteinů MeSH
• výpočetní biologie MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH