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Yeast model analysis of novel polymerase gamma variants found in patients with autosomal recessive mitochondrial disease.
Get PDFReplication of the mitochondrial genome depends on the single DNA polymerase (pol gamma). Mutations in the POLG gene, encoding the catalytic subunit of the human polymerase gamma, have been linked to a wide variety of mitochondrial disorders that show remarkable heterogeneity, with more than 200 sequence variants, often very rare, found in patients. The pathogenicity and dominance status of many such mutations remain, however, unclear. Remarkable structural and functional conservation of human POLG and its S. cerevisiae ortholog (Mip1p) led to the development of many successful yeast models, enabling to study the phenotype of putative pathogenic mutations. In a group of patients with suspicion of mitochondrial pathology, we identified five novel POLG sequence variants, four of which (p.Arg869Ter, p.Gln968Glu, p.Thr1053Argfs*6, and p.Val1106Ala), together with one previously known but uncharacterised variant (p.Arg309Cys), were amenable to modelling in yeast. Familial analysis indicated causal relationship of these variants with disease, consistent with autosomal recessive inheritance. To investigate the effect of these sequence changes on mtDNA replication, we obtained the corresponding yeast mip1 alleles (Arg265Cys, Arg672Ter, Arg770Glu, Thr809Ter, and Val863Ala, respectively) and tested their effect on mitochondrial genome stability and replication fidelity. For three of them (Arg265Cys, Arg672Ter, and Thr809Ter), we observed a strong, partially dominant phenotype of a complete loss of functional mtDNA, whereas the remaining two led to partial mtDNA depletion and significant increase in point mutation frequencies. These results show good correlation with the severity of symptoms observed in patients and allow to establish these variants as pathogenic mutations
Co-regulation of translation in protein complexes
Get PDFBackground
Co-regulation of gene expression has been known for many years, and studied widely both globally and for individual genes. Nevertheless, most analyses concerned transcriptional control, which in case of physically interacting proteins and protein complex subunits may be of secondary importance. This research is the first quantitative analysis that provides global-scale evidence for translation co-regulation among associated proteins.
Results
By analyzing the results of our previous quantitative model of translation, we have demonstrated that protein production rates plus several other translational parameters, such as mRNA and protein abundance, or number of produced proteins from a gene, are well concerted between stable complex subunits and party hubs. This may be energetically favorable during synthesis of complex building blocks and ensure their accurate production in time. In contrast, for connections with regulatory particles and date hubs translational co-regulation is less visible, indicating that in these cases maintenance of accurate levels of interacting particles is not necessarily beneficial.
Conclusions
Similar results obtained for distantly related model organisms, Saccharomyces cerevisiae and Homo sapiens, suggest that the phenomenon of translational co-regulation applies to the variety of living organisms and concerns many complex constituents. This phenomenon was also observed among the set of functionally linked proteins from Escherichia coli operons. This leads to the conclusion that translational regulation of a protein should always be studied with respect to the expression of its primary interacting partners.
Reviewers
This article was reviewed by Sandor Pongor and Claus Wilke
Indicator species population monitoring in Antarctica with UAV.
Get PDFA program to monitor bird and pinniped species in the vicinity of Arctowski Station, King George Island, South Shetlands, Antarctica, has been conducted over the past 38 years. Annual monitoring of these indicator species includes estimations of breeding population sizes of three Pygoscelis penguin species: Adélie, gentoo and chinstrap. Six penguin colonies situated on the western shores of two bays: Admiralty and King George are investigated.
To study changes in penguin populations Unmanned Aerial Vehicles were used for the first time in the 2014/15 austral summer season. During photogrammetric flights the high-resolution images of eight penguin breeding colonies were taken. Obtained high resolution images were used for estimation of breeding population size and compared with the results of measurements taken at the same time from the ground. During this Antarctic expedition eight successful photogrammetry missions (total distance 1500 km) were performed. Images were taken with digital SLR Canon 700D, Nikon D5300, Nikon D5100 with a 35mm objective lens. Flights altitude at 350 – 400 AGL, allowed images to be taken with a resolution GSD (ground sample distance) less than 5 cm. The Image J software analysis method was tested to provide automatic population estimates from obtained images. The use of UAV for monitoring of indicator species, enabled data acquisition from areas inaccessible by ground methods
SWP73 Subunits of Arabidopsis SWI/SNF Chromatin Remodeling Complexes Play Distinct Roles in Leaf and Flower Development.
Get PDFArabidopsis thaliana SWP73A and SWP73B are homologs of mammalian BRAHMA-associated factors (BAF60s) that tether SWITCH/SUCROSE NONFERMENTING chromatin remodeling complexes to transcription factors of genes regulating various cell differentiation pathways. Here, we show that Arabidopsis thaliana SWP73s modulate several important developmental pathways. While undergoing normal vegetative development, swp73a mutants display reduced expression of FLOWERING LOCUS C and early flowering in short days. By contrast, swp73b mutants are characterized by retarded growth, severe defects in leaf and flower development, delayed flowering, and male sterility. MNase-Seq, transcript profiling, and ChIP-Seq studies demonstrate that SWP73B binds the promoters of ASYMMETRIC LEAVES1 and 2, KANADI1 and 3, and YABBY2, 3, and 5 genes, which regulate leaf development and show coordinately altered transcription in swp73b plants. Lack of SWP73B alters the expression patterns of APETALA1, APETALA3, and the MADS box gene AGL24, whereas other floral organ identity genes show reduced expression correlating with defects in flower development. Consistently, SWP73B binds to the promoter regions of APETALA1 and 3, SEPALLATA3, LEAFY, UNUSUAL FLORAL ORGANS, TERMINAL FLOWER1, AGAMOUS-LIKE24, and SUPPRESSOR OF CONSTANS OVEREXPRESSION1 genes, and the swp73b mutation alters nucleosome occupancy on most of these loci. In conclusion, SWP73B acts as important modulator of major developmental pathways, while SWP73A functions in flowering time control
Identification and characterization of tetracycline resistance in Lactococcus lactis isolated from Polish raw milk and fermented artisanal products
Get PDFTo assess the occurrence of antibiotic-resistant Lactic Acid Bacteria (LAB) in Polish raw milk and fermented artisanal products, a collection comprising 500 isolates from these products was screened. Among these isolates, six strains (IBB28, IBB160, IBB161, IBB224, IBB477 and IBB487) resistant to tetracycline were identified. The strains showing atypical tetracycline resistance were classified as Lactococcus lactis: three of them were identified as L. lactis subsp. cremoris (IBB224, IBB477 and IBB487) and the other three (IBB28, IBB160, IBB161) were identified as L. lactis subsp. lactis. The mechanism involving Ribosomal Protection Proteins (RPP) was identified as responsible for tetracycline resistance. Three of the tested strains (IBB28, IBB160 and IBB224) had genes encoding the TetS protein, whereas the remaining three (IBB161, IBB477 and IBB487) expressed TetM. The results also demonstrated that the genes encoding these proteins were located on genetic mobile elements. The tet(S) gene was found to be located on plasmids, whereas tet(M) was found within the Tn916 transposon
Use of biomolecules delivery vector, adenovirus dodecahedron, for targeted cancer therapy.
Get PDFAdenoviral dodecahedron (Dd) is a non-enveloped symmetrical virus-like particle (VLP). VLPs are multimeric stable proteinaceous nanostructures devoid of any genetic material, formed from functional viral proteins responsible for cell penetration, which ensures VLPs efficient cell entry. Dd, composed of twelve copies of the pentameric penton base (Pb) protein, is spontaneously generated in the baculovirus system upon the expression of the Pb gene of adenovirus serotype 3 (Ad3). The particle shows remarkable cell penetration ability with 200,000–300,000 Dd internalized into one cell in culture, conceivably delivering several millions of foreign cargo molecules to the target cell. For this reason, they are of great interest as a delivery vector. Stability studies show that Dds can be conveniently stored and transported, and can potentially be used for therapeutic purposes under various climates. We have used Dd for delivery of small drugs for targeted cancer therapy. A cell-impermeant oncogene inhibitor or anti-cancer antibiotics doxorubicin and bleomycin were delivered as Dd conjugates, demonstrating significantly improved drug bioavailability. Recently we undertook some improvements in the protocols of Dd expression and purification, leading to considerable savings in time, improved yield and allowing the scaling-up of the protein production
Purification of eukaryotic exoribonucleases following heterologous expression in bacteria and analysis of their biochemical properties by in vitro enzymatic assays
Get PDFExoribonucleases-among the other RNases-play a crucial role in the regulation of different aspects of RNA metabolism in the eukaryotic cell. To fully understand the exact mechanism of activity exhibited by such enzymes, it is crucial to determine their detailed biochemical properties, notably their substrate specificity and optimal conditions for enzymatic action. One of the most significant features of exoribonucleases is the direction of degradation of RNA substrates, which can proceed either from 5'-end to 3'-end or in the opposite way. Here, we present methods allowing the efficient production and purification of eukaryotic exoribonucleases, the preparation and labeling of various RNA substrates, and the biochemical characterization of exonucleolytic activity. We also explain how the exonucleolytic activity may be distinguished from that of endonucleases
Microbial community development on the surface of Hans and Werenskiold Glaciers (Svalbard, Arctic) – a comparison.
Get PDFSurface ice and cryoconite holes of two types of polythermal Svalbard Glaciers (Hans Glacier - grounded tidewater glacier and Werenskiold Glacier - land-based valley glacier) were investigated in terms of chemical composition, microbial abundance and diversity. Gathered data served to describe supraglacial habitats and to compare microbe-environment interactions on those different type glaciers. Hans Glacier samples displayed elevated nutrient levels (DOC, nitrogen and seston) compared to Werenskiold Glacier. Adjacent tundra formations, bird nesting sites and marine aerosol were candidates for allochtonic enrichment sources. Microbial numbers were comparable on both glaciers, with surface ice containing cells in the range of 104 mL-1 and cryoconite sediment 108 g-1 dry weight. Denaturating gradient gel electrophoresis band-based clustering revealed differences between glaciers in terms of dominant bacterial taxa structure. Microbial community on Werenskiold Glacier benefited from the snow-released substances. On Hans Glacier this effect was not as pronounced, affecting mainly the photoautotrophs. Over-fertilization of Hans Glacier surface was proposed as the major factor, desensitizing the microbial community to the snow melt event. Nitrogen emerged as a limiting factor in surface ice habitats, especially to Eukaryotic algae
Noteworthy Facts about a Methane-Producing Microbial Community Processing Acidic Effluent from Sugar Beet Molasses Fermentation
Get PDFAnaerobic digestion is a complex process involving hydrolysis, acidogenesis, acetogenesis and methanogenesis. The separation of the hydrogen-yielding (dark fermentation) and methane-yielding steps under controlled conditions permits the production of hydrogen and methane from biomass. The characterization of microbial communities developed in bioreactors is crucial for the understanding and optimization of fermentation processes. Previously we developed an effective system for hydrogen production based on long-term continuous microbial cultures grown on sugar beet molasses. Here, the acidic effluent from molasses fermentation was used as the substrate for methanogenesis in an upflow anaerobic sludge blanket bioreactor. This study focused on the molecular analysis of the methane-yielding community processing the non-gaseous products of molasses fermentation. The substrate for methanogenesis produces conditions that favor the hydrogenotrophic pathway of methane synthesis. Methane production results from syntrophic metabolism whose key process is hydrogen transfer between bacteria and methanogenic Archaea. High-throughput 454 pyrosequencing of total DNA isolated from the methanogenic microbial community and bioinformatic sequence analysis revealed that the domain Bacteria was dominated by Firmicutes (mainly Clostridia), Bacteroidetes, δ- and γ-Proteobacteria, Cloacimonetes and Spirochaetes. In the domain Archaea, the order Methanomicrobiales was predominant, with Methanoculleus as the most abundant genus. The second and third most abundant members of the Archaeal community were representatives of the Methanomassiliicoccales and the Methanosarcinales. Analysis of the methanogenic sludge by scanning electron microscopy with Energy Dispersive X-ray Spectroscopy and X-ray diffraction showed that it was composed of small highly heterogeneous mineral-rich granules. Mineral components of methanogenic granules probably modulate syntrophic metabolism and methanogenic pathways. A rough functional analysis from shotgun data of the metagenome demonstrated that our knowledge of methanogenesis is poor and/or the enzymes responsible for methane production are highly effective, since despite reasonably good sequencing coverage, the details of the functional potential of the microbial community appeared to be incomplet
Potato Annexin STANN1 Promotes Drought Tolerance and Mitigates Light Stress in Transgenic Solanum tuberosum L. Plants.
Get PDFAnnexins are a family of calcium- and membrane-binding proteins that are important for plant tolerance to adverse environmental conditions. Annexins function to counteract oxidative stress, maintain cell redox homeostasis, and enhance drought tolerance. In the present study, an endogenous annexin, STANN1, was overexpressed to determine whether crop yields could be improved in potato (Solanum tuberosum L.) during drought. Nine potential potato annexins were identified and their expression characterized in response to drought treatment. STANN1 mRNA was constitutively expressed at a high level and drought treatment strongly increased transcription levels. Therefore, STANN1 was selected for overexpression analysis. Under drought conditions, transgenic potato plants ectopically expressing STANN1 were more tolerant to water deficit in the root zone, preserved more water in green tissues, maintained chloroplast functions, and had higher accumulation of chlorophyll b and xanthophylls (especially zeaxanthin) than wild type (WT). Drought-induced reductions in the maximum efficiency and the electron transport rate of photosystem II (PSII), as well as the quantum yield of photosynthesis, were less pronounced in transgenic plants overexpressing STANN1 than in the WT. This conferred more efficient non-photochemical energy dissipation in the outer antennae of PSII and probably more efficient protection of reaction centers against photooxidative damage in transgenic plants under drought conditions. Consequently, these plants were able to maintain effective photosynthesis during drought, which resulted in greater productivity than WT plants despite water scarcity. Although the mechanisms underlying this stress protection are not yet clear, annexin-mediated photoprotection is probably linked to protection against light-induced oxidative stress