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Lysis

About: Lysis is a research topic. Over the lifetime, 6072 publications have been published within this topic receiving 216978 citations.


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Book ChapterDOI
TL;DR: This chapter discusses a procedure for isolation of DNA from the yeast Saccharomyces cerevisiae that yields a preparation of total-yeast DNA including all three buoyant density species observed in neutral isopycnic cesium chloride gradients.
Abstract: Publisher Summary This chapter discusses a procedure for isolation of DNA from the yeast Saccharomyces cerevisiae This procedure is a modification of an earlier method for isolating DNA from bacteria and has general operations: (1) preparation of osmotically fragile spheroplasts by enzymatic digestion of the cell wall, (2) lysis of the spheroplasts and partial proteolysis of the lysate, (3) deproteinization and extraction of lipids from the lysate, and (4) enzymatic digestion of RNA and elimination of polysaccharides by centrifugation or digestion, followed by separation of the DNA from the digestion products by selective isopropanol precipitation This procedure yields a preparation of total-yeast DNA including all three buoyant density species observed in neutral isopycnic cesium chloride gradients: nuclear, nuclear heavy satellite, and mitochondrial If spheroplasting of the cells is adequate and lysis is complete, there are no preferential losses of any of the observed DNA species Preparations free of DNA of mitochondrial density can be obtained from petite yeast strains that lack mitochondrial DNA The various DNA species obtained from normal strains by this method may be separated and purified using cesium chloride gradients, cesium chloride–ethidium bromide gradients, cesium sulfate–mercury, or cesium sulfate–silver gradients, and hydroxyapatite chromatography

446 citations

Journal ArticleDOI
TL;DR: This simple, one-step DNA extraction procedure can be utilized in conjunction with Hoechst reagent to obtain quantitative estimates of DNA levels in cell or tissue extracts to eliminate the need for enzyme treatment or exposure to high salt solutions.

445 citations

Journal ArticleDOI
TL;DR: Using a GFP-based approach to visualize chlamydial inclusions within cells by live fluorescence videomicroscopy, it is identified that Chlamydia release occurred by two mutually exclusive pathways: lysis and extrusion.
Abstract: The mechanisms that mediate the release of intracellular bacteria from cells are poorly understood, particularly for those that live within a cellular vacuole. The release pathway of the obligate intracellular bacterium Chlamydia from cells is unknown. Using a GFP-based approach to visualize chlamydial inclusions within cells by live fluorescence videomicroscopy, we identified that Chlamydia release occurred by two mutually exclusive pathways. The first, lysis, consisted of an ordered sequence of membrane permeabilizations: inclusion, nucleus and plasma membrane rupture. Treatment with protease inhibitors abolished inclusion lysis. Intracellular calcium signaling was shown to be important for plasma membrane breakdown. The second release pathway was a packaged release mechanism, called extrusion. This slow process resulted in a pinching of the inclusion, protrusion out of the cell within a cell membrane compartment, and ultimately detachment from the cell. Treatment of Chlamydia-infected cells with specific pharmacological inhibitors of cellular factors demonstrated that extrusion required actin polymerization, neuronal Wiskott-Aldrich syndrome protein, myosin II and Rho GTPase. The participation of Rho was unique in that it functioned late in extrusion. The dual nature of release characterized for Chlamydia has not been observed as a strategy for intracellular bacteria.

441 citations

Journal ArticleDOI
TL;DR: The characterisation of extracellular DNA can integrate information on the composition of the microbial community of soil and sediments obtained by analysing intracellular DNA.
Abstract: The review discusses origin, state and function of extracellular DNA in soils and sediments Extracellular DNA can be released from prokaryotic and eukaryotic cells and can be protected against nuclease degradation by its adsorption on soil colloids and sand particles Laboratory experiments have shown that DNA adsorbed by colloids and sand particles can be taken up by prokaryotic competent cells and be involved in natural transformation Most of these experiments have been carried out under artificial conditions with pure DNA molecules and pure adsorbing matrices, but in soils and sediments, pure surface-reactive colloids are not present and DNA is present with other cellular components (wall debris, proteins, lipids, RNA, etc) especially if released after cell lysis The presence of inorganic compounds and organic molecules on both soil particles and DNA molecules can influence the DNA adsorption, degradation and transformation of competent cells Extracellular DNA can be used as C, N and P sources by heterotrophic microorganisms and plays a significant role in bacterial biofilm formation The nucleotides and nucleosides originated from the degradation of extracellular DNA can be re-assimilated by soil microorganisms Extracellular DNA in soil can be leached and moved by water through the soil profile by capillarity In this way, the extracellular DNA secreted by a cell can reach a competent bacterial cell far from the donor cell Finally, the characterisation of extracellular DNA can integrate information on the composition of the microbial community of soil and sediments obtained by analysing intracellular DNA

429 citations

Journal ArticleDOI
TL;DR: This unit provides two protocols for extraction of RNA from yeast that differ primarily in the method for lysing the yeast cells, and describes the scaling up of the first two procedures to isolate enough total RNA for poly (A)+ RNA preparation.
Abstract: This unit provides two protocols for extraction of RNA from yeast that differ primarily in the method for lysing the yeast cells. The first protocol isolates RNA directly from intact yeast cells by extraction with hot acidic phenol. This yields RNA that is relatively free of contaminating DNA, is convenient to perform with multiple samples, and gives little or no sample-to-sample variation. In contrast, an alternate protocol relies upon disruption of cells by vigorous mixing with glass beads and denaturing agents. Although this procedure results in efficient breaking of the cells, the product is associated with residual DNA, and the procedure itself is troublesome when one is working with multiple samples. A second alternate protocol describes the scaling up of the first two procedures to isolate enough total RNA for poly (A)+ RNA preparation.

428 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
2023389
2022607
2021123
2020142
2019139
2018161