New insight into formation of DNA-containing microparticles during PCR: the scaffolding role of magnesium pyrophosphate crystals.

This work aims to study molecular mechanisms involved in the formation of DNA-containing microparticles and nanoparticles during PCR. Both pyrophosphate and Mg(2+) ions proved to play an important role in the generation of DNA microparticles (MPs) with a unique and sophisticated structure in PCR with Taq polymerase. Thus, the addition of Tli thermostable pyrophosphatase to a PCR mixture inhibited this process and caused the destruction of synthesized DNA MPs.

Enzymes of inorganic polyphosphate metabolism.

Inorganic polyphosphate (PolyP) is a linear polymer containing a few to several hundred orthophosphate residues linked by energy-rich phosphoanhydride bonds. Investigation of PolyP-metabolizing enzymes is important for medicine, because PolyPs perform numerous functions in the cells. In human organism, PolyPs are involved in the regulation of Ca(2+) uptake in mitochondria, bone tissue development, and blood coagulation.

Extracellular cellobiose lipid from yeast and their analogues: structures and fungicidal activities.

Basidiomycetous yeasts Cryptococcus humicola and Pseudozyma fusiformata secrete cellobiose lipids into the culture broth. In the case of Cr. humicola, 16-(tetra-O-acetyl-beta-cellobiosyloxy)-2-hydroxyhexadecanoic acid was defined as major product and 16-(tetra-O-acetyl-beta-cellobiosyloxy)-2,15-dihydrohexadecanoic acid was defined as minor product, while Ps.

Inorganic polyphosphate and exopolyphosphatase in the nuclei of Saccharomyces cerevisiae: dependence on the growth phase and inactivation of the PPX1 and PPN1 genes.

Nuclei of the yeast Saccharomyces cerevisiae possess inorganic polyphosphates (polyP) with chain lengths of ca. 10-200 phosphate residues. Subfractionation of the nuclei reveals that the most part of polyP is not associated with DNA.

Inorganic polyphosphates and exopolyphosphatases in cell compartments of the yeast Saccharomyces cerevisiae under inactivation of PPX1 and PPN1 genes.

Purified fractions of cytosol, vacuoles, nuclei, and mitochondria of Saccharomyces cerevisiae possessed inorganic polyphosphates with chain lengths characteristic of each individual compartment. The most part (80-90%) of the total polyphosphate level was found in the cytosol fractions. Inactivation of a PPX1 gene encoding ~40-kDa exopolyphosphatase substantially decreased exopolyphosphatase activities only in the cytosol and soluble mitochondrial fraction, the compartments where PPX1 activity was localized.

Effects of inactivation of the PPN1 gene on exopolyphosphatases, inorganic polyphosphates and function of mitochondria in the yeast Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae with inactivated endopolyphosphatase gene PPN1 did not grow on lactate and ethanol, and stopped growth on glucose earlier than the parent strain. Their mitochondria were defective in respiration functions and in metabolism of inorganic polyphosphates. The PPN1 mutants lacked exopolyphosphatase activity and possessed a double level of inorganic polyphosphates in mitochondria.

Inactivation of endopolyphosphatase gene PPN1 results in inhibition of expression of exopolyphosphatase PPX1 and high-molecular-mass exopolyphosphatase not encoded by PPX1 in Saccharomyces cerevisiae.

Saccharomyces cerevisiae possesses multiple forms of exopolyphosphatases, the enzymes involved in the metabolism of inorganic polyphosphates, which are important regulatory compounds. In S. cerevisiae, inactivation of endopolyphosphatase gene PPN1 leads to the inhibition of expression of both exopolyphosphatase PPX1 and high-molecular-mass exopolyphosphatase of approximately 1000 kDa not encoded by PPX1.

Inorganic polyphosphate in mitochondria of Saccharomyces cerevisiae at phosphate limitation and phosphate excess.

Isolated mitochondria of Saccharomyces cerevisiae cells grown on glucose possess acid-soluble inorganic polyphosphate (polyP). Its level strongly depends on phosphate (P(i)) concentration in the culture medium. The polyP level in mitochondria showed 11-fold decrease under 0.

Nuclear exopolyphosphatase of Saccharomyces cerevisiae is not encoded by the PPX1 gene encoding the major yeast exopolyphosphatase.

Intact nuclei from a parental strain CRY and a PPX1-mutant CRX of Saccharomyces cerevisiae were isolated and found to be essentially free of cytoplasmic, mitochondrial and vacuolar marker enzymes. The protein-to-DNA ratios of the nuclei were 22 and 30 for CRY and CRX nuclei, respectively. An exopolyphosphatase (exopolyPase) with molecular mass of approximately 57 kDa and a pyrophosphatase (PPase) of approximately 41 kDa were detected in the parental strain CRY.

Exopolyphosphatases of the yeast Saccharomyces cerevisiae.

Separate compartments of the yeast cell possess their own exopolyphosphatases differing from each other in their properties and dependence on culture conditions. The low-molecular-mass exopolyphosphatases of the cytosol, cell envelope, and mitochondrial matrix are encoded by the PPX1 gene, while the high-molecular-mass exopolyphosphatase of the cytosol and those of the vacuoles, mitochondrial membranes, and nuclei are presumably encoded by their own genes. Based on recent works, a preliminary classification of the yeast exopolyphosphatases is proposed.

Effect of PPX1 inactivation on exopolyphosphatases of different cell compartments of the yeast Saccharomyces cerevisiae.

Inactivation of PPX1 encoding a major exopolyphosphatase (PPX1) in Saccharomyces cerevisiae results in a change of exopolyphosphatase spectra in the yeast cells. In the PPX1-deficient strain, an elimination of approximately 45 kDa enzyme is observed in cytosol and cell envelopes, and the activity of an exopolyphosphatase with a molecular mass of approximately 830 kDa increases 5-fold in the cytosol. These two enzyme activities differ greatly from each other not only in molecular masses but also in biochemical properties.