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MutS as a tool for mutation detection

100%

Stanisławska-Sachadyn A., Sachadyn P.

Acta Biochimica Polonica

2005

vol. 52

issue 3

575-583

MutS, a DNA mismatch-binding protein, seems to be a promising tool for mutation detection. We present three MutS based approaches to the detection of point mutations: DNA retardation, protection of mismatched DNA against exonuclease digestion, and chimeric MutS proteins. DNA retardation in polyacrylamide gels stained with SYBR-Gold allows mutation detection using 1-3 µg of Thermus thermophilus his6-MutS protein and 50-200 ng of a PCR product. The method enables the search for a broad range of mutations: from single up to several nucleotide, as mutations over three nucleotides could be detected in electrophoresis without MutS, due to the mobility shift caused by large insertion/deletion loops in heteroduplex DNA. The binding of DNA mismatches by MutS protects the complexed DNA against exonuclease digestion. The direct addition of the fluorescent dye, SYBR-Gold, allows mutation detection in a single-tube assay. The limited efficiency of T4 DNA polymerase as an exonuclease hampers the application of the method in practice. The assay required 300-400 ng of PCR products in the range of 200-700 bp and 1-3 µg of MutS. MutS binding to mismatched DNA immobilised on a solid phase can be observed thanks to the activity of a reporter domain linked to MutS. We obtained chimeric bifunctional proteins consisting of T. thermophilus MutS and reporter domains, like β-galactosidase or GFP. Very low detection limits for β-galactosidase could theoretically enable mutation detection not only by the examination of PCR products, but even of genomic DNA.

Application of 1H and 31P NMR to topological description of a model of biological membrane fusion Topological description of a model of biological membrane fusion

100%

Janiak-Osajca A., Timoszyk A.

Acta Biochimica Polonica

2012

vol. 59

issue 2

219-224

The process of biological membrane fusion can be analysed by topological methods. Mathematical analysis of the fusion process of vesicles indicated two significant facts: the formation of an inner, transient structure (hexagonal phase - HII) and a translocation of some lipids within the membrane. This shift had a vector character and only occurred from the outer to the inner layer. Model membrane composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) was studied. 31P- and 1H-NMR methods were used to describe the process of fusion. 31P-NMR spectra of multilamellar vesicles (MLV) were taken at various temperatures and concentrations of Ca2+ ions (natural fusiogenic agent). A 31P-NMR spectrum with the characteristic shape of the HII phase was obtained for the molar Ca2+/PS ratio of 2.0. During the study, 1H-NMR and 31P-NMR spectra for small unilamellar vesicle (SUV), which were dependent on time (concentration of Pr3+ ions was constant), were also recorded. The presence of the paramagnetic Pr3+ ions permits observation of separate signals from the hydrophilic part of the inner and outer lipid bilayers. The obtained results suggest that in the process of fusion translocation of phospholipid molecules takes place from the outer to the inner layer of the vesicle and size of the vesicles increase. The NMR study has showed that the intermediate state of the fusion process caused by Ca2+ ions is the HII phase. The experimental results obtained are in agreement with the topological model as well.

Heat load and deuterium plasma effects on SPS and WSP tungsten

86%

Vilémová M., Matějíček J., Nevrlá B., Chernyshova M., Gasior P., Kowalska-Strzeciwilk E., Jäger A.

Nukleonika

2015

vol. 60

issue 2

275-283

Tungsten is a prime choice for armor material in future nuclear fusion devices. For the realization of fusion, it is necessary to address issues related to the plasma–armor interactions. In this work, several types of tungsten material were studied, i.e. tungsten prepared by spark plasma sintering (SPS) and by water stabilized plasma spraying (WSP) technique. An intended surface porosity was created in the samples to model hydrogen/helium bubbles. The samples were subjected to a laser heat loading and a radiation loading of deuterium plasma to simulate edge plasma conditions of a nuclear fusion device (power density of 108 W/cm2 and 107 W/cm2, respectively, in the pulse intervals up to 200 ns). Thermally induced changes in the morphology and the damage to the studied surfaces are described. Possible consequences for the fusion device operation are pointed out.

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2 Acta Biochimica Polonica

1 Nukleonika

1 2015

1 2012

1 2005

1 Chernyshova M.

1 Gasior P.

1 Janiak-Osajca A.

1 Jäger A.

1 Kowalska-Strzeciwilk E.

1 Matějíček J.

1 Nevrlá B.

1 Sachadyn P.

1 Stanisławska-Sachadyn A.

1 Timoszyk A.

1 Vilémová M.

Search results

MutS as a tool for mutation detection

Application of 1H and 31P NMR to topological description of a model of biological membrane fusion Topological description of a model of biological membrane fusion

Heat load and deuterium plasma effects on SPS and WSP tungsten