The SEVA platform (https://seva-plasmids.com) was launched one decade ago, both as a database (DB) and as a physical repository of plasmid vectors for genetic analysis and engineering of Gram-negative bacteria with a structure and nomenclature that follows a strict, fixed architecture of functional DNA segments. While the current update keeps the basic features of earlier versions, the platform has been upgraded not only with many more ready-to-use plasmids but also with features that expand the range of target species, harmonize DNA assembly methods and enable new applications. In particular, SEVA 4.0 includes (i) a sub-collection of plasmids for easing the composition of multiple DNA segments with MoClo/Golden Gate technology, (ii) vectors for Gram-positive bacteria and yeast and [iii] off-the-shelf constructs with built-in functionalities. A growing collection of plasmids that capture part of the standard-but not its entirety-has been compiled also into the DB and repository as a separate corpus (SEVAsib) because of its value as a resource for constructing and deploying phenotypes of interest. Maintenance and curation of the DB were accompanied by dedicated diffusion and communication channels that make the SEVA platform a popular resource for genetic analyses, genome editing and bioengineering of a large number of microorganisms.
The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity. The nuclear magnetic resonance-derived solution structure of the CC1 domain represents a three-helix bundle stabilized by interhelical contacts, which are absent in the Stormorken disease-related STIM1 R304W mutant. Two interhelical sites between the CC1α1 and CC1α2 helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. Nuclear magnetic resonance-directed mutations within these interhelical interactions restore the physiological, store-dependent activation behavior of the gain-of-function STIM1 R304W mutant. This study reveals the functional impact of interhelical interactions within the CC1 domain for modifying the CC1-CC3 clamp strength to control the activation of STIM1.
- MeSH
- abnormální erytrocyty MeSH
- dyslexie genetika MeSH
- HEK293 buňky MeSH
- ichtyóza genetika MeSH
- kanály aktivované uvolněním vápníku metabolismus MeSH
- klonování DNA MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie MeSH
- metoda terčíkového zámku MeSH
- migréna genetika MeSH
- mióza genetika MeSH
- molekulární modely MeSH
- mutace genetika MeSH
- nádorové proteiny genetika MeSH
- protein ORAI1 genetika MeSH
- protein STIM1 genetika MeSH
- slezina abnormality MeSH
- svalová únava genetika MeSH
- trombocytopatie genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In Europe, Ixodes ricinus is the most important vector of human infectious diseases, most notably Lyme borreliosis and tick-borne encephalitis virus. Multiple non-natural hosts of I. ricinus have shown to develop immunity after repeated tick bites. Tick immunity has also been shown to impair B. burgdorferi transmission. Most interestingly, multiple tick bites reduced the likelihood of contracting Lyme borreliosis in humans. A vaccine that mimics tick immunity could therefore potentially prevent Lyme borreliosis in humans. A yeast surface display library (YSD) of nymphal I. ricinus salivary gland genes expressed at 24, 48 and 72 h into tick feeding was constructed and probed with antibodies from humans repeatedly bitten by ticks, identifying twelve immunoreactive tick salivary gland proteins (TSGPs). From these, three proteins were selected for vaccination studies. An exploratory vaccination study in cattle showed an anti-tick effect when all three antigens were combined. However, immunization of rabbits did not provide equivalent levels of protection. Our results show that YSD is a powerful tool to identify immunodominant antigens in humans exposed to tick bites, yet vaccination with the three selected TSGPs did not provide protection in the present form. Future efforts will focus on exploring the biological functions of these proteins, consider alternative systems for recombinant protein generation and vaccination platforms and assess the potential of the other identified immunogenic TSGPs.
- MeSH
- antigeny krev imunologie izolace a purifikace MeSH
- Borrelia burgdorferi izolace a purifikace MeSH
- imunizace MeSH
- infestace klíšťaty imunologie parazitologie MeSH
- klíště imunologie MeSH
- kousnutí klíštětem imunologie MeSH
- králíci MeSH
- lidé MeSH
- lymeská nemoc krev parazitologie přenos MeSH
- metody zobrazení buněčného povrchu metody MeSH
- peptidová knihovna MeSH
- peptidové fragmenty imunologie MeSH
- Saccharomyces cerevisiae MeSH
- skot MeSH
- slinné proteiny a peptidy imunologie MeSH
- slinné žlázy imunologie MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- lidé MeSH
- mužské pohlaví MeSH
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Animal or human protothecosis belongs to rather rare, endemic, pro-inflammatory infections. It is caused by achlorophyllous algae of the genus Prototheca. Especially, P. bovis (formerly P. zopfii genotype 2) is often inflected as a non-bacterial causative agent of dairy cattle mastitis. In this study, we present a multiplex real-time PCR (qPCR) system for rapid and exact Prototheca spp. detection and quantification. Limit of detection, diagnostic sensitivity, and specificity were determined. For the first time, specific sequences of AccD (encoding acetyl CoA reductase) for P. bovis, cox1 (encoding cytochrome C oxidase subunit 1) for P. wickerhamii, cytB (encoding cytochrome B) for P. blashkeae and atp6 (encoding transporting ATPase F0 subunit 6) for P. ciferrii (formerly P. zopfii genotype 1) were used for species identification and quantification together with 28S rRNA sequence detecting genus Prototheca. The developed qPCR assay was applied to 55 individual cow milk samples from a herd suspected of protothecosis, 41 bulk milk samples from different Czech farms, 16 boxed milk samples purchased in supermarkets and 21 environmental samples originating from a farm suspected of protothecosis. Our work thus offers the possibility to diagnose protothecosis in the samples, where bacterial mastitis is the most commonly presumed and thereby assisting adequate corrective measures to be taken.
- MeSH
- DNA chemie izolace a purifikace MeSH
- farmy MeSH
- klonování DNA MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- limita detekce MeSH
- mikrobiologie životního prostředí MeSH
- mlékárenství MeSH
- mléko mikrobiologie MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- plazmidy genetika MeSH
- Prototheca genetika růst a vývoj izolace a purifikace MeSH
- senzitivita a specificita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Due to the inappropriate use of florfenicol in agricultural practice, florfenicol resistance has become increasingly serious. In this work, we studied the novel florfenicol resistance mechanism of an animal-derived Leclercia adecarboxylata strain R25 with high-level florfenicol resistance. A random genomic DNA library was constructed to screen the novel florfenicol resistance gene. Gene cloning, gene knockout, and complementation combined with the minimum inhibitory concentration (MIC) detection were conducted to determine the function of the resistance-related gene. Sequencing and bioinformatics methods were applied to analyze the structure of the resistance gene-related sequences. Finally, we obtained a regulatory gene of an RND (resistance-nodulation-cell division) system, ramA, that confers resistance to florfenicol and other antibiotics. The ramA-deleted variant (LA-R25ΔramA) decreased the level of resistance against florfenicol and several other antibiotics, while a ramA-complemented strain (pUCP24-prom-ramA/LA-R25ΔramA) restored the drug resistance. The whole-genome sequencing revealed that there were five RND efflux pump genes (mdtABC, acrAB, acrD, acrEF, and acrAB-like) encoded over the chromosome, and ramA located upstream of the acrAB-like genes. The results of this work suggest that ramA confers resistance to florfenicol and other structurally unrelated antibiotics, presumably by regulating the RND efflux pump genes in L. adecarboxylata R25.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Enterobacteriaceae účinky léků genetika metabolismus MeSH
- Escherichia coli genetika MeSH
- genový knockout MeSH
- klonování DNA MeSH
- mikrobiální testy citlivosti MeSH
- mnohočetná bakteriální léková rezistence účinky léků genetika MeSH
- RNA ribozomální 16S MeSH
- sekvenování celého genomu MeSH
- thiamfenikol analogy a deriváty MeSH
- trans-aktivátory genetika MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
Flap endonuclease is a structure-specific nuclease which cleaves 5'-flap of bifurcated DNA substrates. Genome sequence of Thermococcus kodakarensis harbors an open reading frame, Tk1281, exhibiting high homology with archaeal flap endonucleases 1. The corresponding gene was cloned and expressed in Escherichia coli, and the gene product was purified to apparent homogeneity. Tk1281 was a monomer of 38 kDa and catalyzed the cleavage of 5'-flap from double-stranded DNA substrate containing single-stranded DNA flap. The highest cleavage activity was observed at 80 °C and pH 7.5. Under optimal conditions, Tk1281 exhibited apparent Vmax and Km values of 278 nmol/min/mg and 37 μM, respectively, against a 54-nucleotide double-stranded substrate containing a single-stranded 5'-flap of 27 nucleotides. A unique feature of Tk1281 is its highest activation in the presence of Co2+ and no activation with Mn2+. To the best of our knowledge, this is the first cloning and characterization of a flap endonuclease from the genus Thermococcus.
- MeSH
- "flap" endonukleasy chemie genetika metabolismus MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- kinetika MeSH
- klonování DNA * MeSH
- molekulová hmotnost MeSH
- stabilita enzymů MeSH
- substrátová specifita MeSH
- Thermococcus chemie enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
The SMC (Structural Maintenance of Chromosomes) complexes are composed of SMC dimers, kleisin and kleisin-interacting (HAWK or KITE) subunits. Mutual interactions of these subunits constitute the basal architecture of the SMC complexes. In addition, binding of ATP molecules to the SMC subunits and their hydrolysis drive dynamics of these complexes. Here, we developed new systems to follow the interactions between SMC5/6 subunits and the relative stability of the complex. First, we show that the N-terminal domain of the Nse4 kleisin molecule binds to the SMC6 neck and bridges it to the SMC5 head. Second, binding of the Nse1 and Nse3 KITE proteins to the Nse4 linker increased stability of the ATP-free SMC5/6 complex. In contrast, binding of ATP to SMC5/6 containing KITE subunits significantly decreased its stability. Elongation of the Nse4 linker partially suppressed instability of the ATP-bound complex, suggesting that the binding of the KITE proteins to the Nse4 linker constrains its limited size. Our data suggest that the KITE proteins may shape the Nse4 linker to fit the ATP-free complex optimally and to facilitate opening of the complex upon ATP binding. This mechanism suggests an important role of the KITE subunits in the dynamics of the SMC5/6 complexes.
- MeSH
- adenosintrifosfatasy metabolismus MeSH
- jaderné proteiny genetika metabolismus MeSH
- makromolekulární látky metabolismus MeSH
- mutageneze cílená MeSH
- proteiny buněčného cyklu genetika metabolismus MeSH
- Schizosaccharomyces pombe - proteiny genetika metabolismus MeSH
- Schizosaccharomyces genetika metabolismus MeSH
- sekvenční seřazení MeSH
- techniky dvojhybridového systému MeSH
- transportní proteiny genetika metabolismus MeSH
- vazba proteinů genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Characterization of protein-protein and protein-DNA interactions is critical to understand mechanisms governing the biology of cells. Here we describe optimized methods and their mutual combinations for this purpose: bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP), yeast two-hybrid systems (Y2H), and chromatin immunoprecipitation (ChIP). These improved protocols detect trimeric complexes in which two proteins of interest interact indirectly via a protein sandwiched between them. They also allow isolation of low-abundance chromatin proteins and confirmation that proteins of interest are associated with specific DNA sequences, for example telomeric tracts. Here we describe these methods and their application to map interactions of several telomere- and telomerase-associated proteins and to purify a sufficient amount of chromatin from Arabidopsis thaliana for further investigations (e.g., next-generation sequencing, hybridization).
- MeSH
- Arabidopsis genetika metabolismus MeSH
- buněčné jádro metabolismus MeSH
- chromatin metabolismus MeSH
- chromatinová imunoprecipitace metody MeSH
- DNA rostlinná metabolismus MeSH
- DNA vazebné proteiny izolace a purifikace metabolismus MeSH
- imunoprecipitace metody MeSH
- mapování interakce mezi proteiny metody MeSH
- optické zobrazování metody MeSH
- proteiny huseníčku genetika metabolismus MeSH
- techniky dvojhybridového systému MeSH
- telomerasa metabolismus MeSH
- telomery metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mats of the green alga Trentepohlia, a genus widely distributed in the tropics as well as temperate regions, have always been perceived as homogeneous (i.e., formed by only one species). As such, their general nature and specific feature play a supportive role in the species delimitation. However, the presence of morphologically dissimilar thalli was observed under the light microscope and when cultivating a piece of a single mat. To address this, we performed DNA cloning of the rbcL gene on mat fragments of T. abietina, T. annulata, T. jolithus and T. umbrina sampled in Europe to reveal if they are composed of one or more species. We revealed that more Trentepohlia haplotypes may coexist in a single mat. In consideration of this, we conclude that the use of material isolated in unialgal culture will be almost mandatory for a taxonomic reassessment of this complicated genus. Another direct implication of this problem is that herbarium specimens consisting of field-collected material should not be used for direct sequencing. We further hypothesize the reasons why multiple haplotypes of Trentepohlia occur more frequently in the tuft-like mats. Possibly, fragments and/or cells of other microalgae, including other species of Trentepohlia, might be retained in such mats more easily than in the crustose trentepohlialean mats.
- MeSH
- Chlorophyta * MeSH
- DNA MeSH
- fylogeneze MeSH
- genetická heterogenita * MeSH
- klonování DNA MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
A segment of Triticum militinae chromosome 7G harbors a gene(s) conferring powdery mildew resistance which is effective at both the seedling and the adult plant stages when transferred into bread wheat (T. aestivum). The introgressed segment replaces a piece of wheat chromosome arm 4AL. An analysis of segregating materials generated to positionally clone the gene highlighted that in a plant heterozygous for the introgression segment, only limited recombination occurs between the introgressed region and bread wheat 4A. Nevertheless, 75 genetic markers were successfully placed within the region, thereby confining the gene to a 0.012 cM window along the 4AL arm. In a background lacking the Ph1 locus, the localized rate of recombination was raised 33-fold, enabling the reduction in the length of the region containing the resistance gene to a 480 kbp stretch harboring 12 predicted genes. The substituted segment in the reference sequence of bread wheat cv. Chinese Spring is longer (640 kbp) and harbors 16 genes. A comparison of the segments' sequences revealed a high degree of divergence with respect to both their gene content and nucleotide sequence. Of the 12 T. militinae genes, only four have a homolog in cv. Chinese Spring. Possible candidate genes for the resistance have been identified based on function predicted from their sequence.
- MeSH
- anotace sekvence MeSH
- Ascomycota fyziologie MeSH
- chléb MeSH
- chromozomy rostlin genetika MeSH
- genetická variace * MeSH
- genetické lokusy * MeSH
- klonování DNA MeSH
- mapování chromozomů MeSH
- nemoci rostlin genetika imunologie mikrobiologie MeSH
- odolnost vůči nemocem genetika MeSH
- pšenice genetika imunologie mikrobiologie MeSH
- rostlinné geny * MeSH
- Publikační typ
- časopisecké články MeSH
