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. Thermal cycling of Na-pyrophosphate (Na-PPi)- and Mg(2+)-containing mixtures (without DNA polymerase and dNTPs) under the standard PCR regime yielded crystalline oval or lenticular microdisks and 3D MPs composed from magnesium pyrophosphate (Mg-PPi). As shown by scanning electron microscopy (SEM), the produced Mg-PPi microparticles consisted of intersecting disks or their segments. They were morphologically similar but simpler than DNA-containing MPs generated in PCR. The incorporation of dNTPs, primers, or dsDNA into Mg-pyrophosphate particles resulted in the structural diversification of 3D microparticles. Thus, the unusual and complex structure of DNA MPs generated in PCR is governed by the unique feature of Mg-pyrophosphate to form supramolecular particles during thermal cycling. We hypothesize the Mg-pyrophosphate particles that are produced during thermal cycling serve as scaffolds for amplicon DNA condensation.

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. The essentiality of polyphosphate kinases in the virulence of pathogenic bacteria is a basis for the discovery of new antibiotics. The properties of the major enzymes of PolyP metabolism, first of all polyphosphate kinases and exopolyphosphatases, are described in the review. The main differences between the enzymes of PolyP biosynthesis and utilization of prokaryotic and eukaryotic cells, as well as the multiple functions of some enzymes of PolyP metabolism, are considered.

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. fusiformata secreted mainly 16-[6-O-acetyl-2'-O-(3-hydroxyhexanoyl)-beta-cellobiosyloxy)-2,15-dihydroxyhexadecanoic acid. These compounds exhibit similar fungicidal activities against different yeasts including pathogenic Cryptococcus and Candida species. The cells of Filobasidiella neoformans causing systemic cryptococcosis completely died after 30-min incubation with 0.02 mg mL(-1) of cellobiose lipids. The same effect on ascomycetous yeast, including pathogenic Candida species, is achieved at 0.1-0.3 mg mL(-1) of cellobiose lipids depending on the test culture used. Cellobiose lipid of Ps. fusiformata inhibits the growth of phytopathogenic fungi Sclerotinia sclerotiorum and Phomopsis helianthi more efficiently than cellobiose lipids from Cr. humicola. Fully O-deacylated analogue, namely 16-(beta-cellobiosyloxy)-2-hydroxyhexadecanoic acid, and totally synthetic compound, 16-(beta-cellobiosyloxy)-hexadecanoic acid, do not inhibit the growth of F. neoformans and Saccharomyces cerevisiae, while 16-(beta-cellobiosyloxy)-2,15-dihydroxyhexadecanoic acid inhibits the growth of both test cultures but at higher concentrations than cellobiose lipids of Cr. humicola and Ps. fusiformata. The amide of 16-(beta-cellobiosyloxy)-2,15-dihydroxyhexadecanoic acid possessed no fungicide activity. Thus, the structures of both the carbohydrate part and fatty acid aglycon moiety are important for the fungicidal activity of cellobiose lipids.

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. Transition of the yeast cells from stationary phase to active growth at orthophosphate (P(i)) excess in the medium is followed by the synthesis of the shortest polyP (<15 phosphate residues) and hydrolysis of the high-molecular polyP (>45 phosphate residues) in the nuclei. Nuclear exopolyphosphatase (exopolyPase) activity does not depend on the growth phase. The PPX1 gene encoding the major cytosolic exopolyPase does not encode the nuclear one and its inactivation has no effect on polyP metabolism in this compartment. Under inactivation of the PPN1 gene encoding another yeast exopolyPase, elimination of the nuclear exopolyPase is observed. The effect of PPN1 inactivation on the polyP level in the nuclei is insignificant in the stationary phase, while in the exponential phase this level increases 2.3-fold as compared with the parent strain of S. cerevisiae. In the active growth phase, no hydrolysis of high-molecular polyP is detected while the synthesis of short-chain polyP is retained. The data obtained indicate substantial changes in polyP metabolism in nuclei under the renewal of active growth, which only partially depends on the genes of polyP metabolism known to date.

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. This inactivation slightly increased the levels of polyphosphates in the cytosol and vacuoles and had no effect on polyphosphate chain lengths in all compartments. Exopolyphosphatase activities in all yeast compartments under study critically depended on the PPN1 gene encoding an endopolyphosphatase. In the single PPN1 mutant, a considerable decrease of exopolyphosphatase activity was observed in all the compartments under study. Inactivation of PPN1 decreased the polyphosphate level in the cytosol 1.4-fold and increased it 2- and 2.5-fold in mitochondria and vacuoles, respectively. This inactivation was accompanied by polyphosphate chain elongation. In nuclei, this mutation had no effect on polyphosphate level and chain length as compared with the parent strain CRY. In the double mutant of PPX1 and PPN1, no exopolyphosphatase activity was detected in the cytosol, nuclei, and mitochondria and further elongation of polyphosphates was observed in all compartments.

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. The average chain length of mitochondrial polyphosphates at the stationary growth stage on glucose was about 15-20 and about 130-180 phosphate residues in the parent strain and PPN1 mutants, respectively. Inactivation of the PPX1 gene encoding exopolyphosphatase had no effect on respiration functions and on polyphosphate level and chain length 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. In the single endopolyphosphatase mutant CRN, the expression of exopolyphosphatase PPX1 decreases 6.5-fold and 2.5-fold at the stationary and exponential growth phases, respectively, as compared with the parent strain CRY. In this mutant, the activity of the high-molecular-mass exopolyphosphatase of approximately 1000 kDa decreases approximately 10-fold as compared with that in strains with the PPN1 gene. In a double mutant of PPX1 and PPN1, no exopolyphosphatase activity is detected in the cytosol at the stationary growth phase. Thus, the exopolyPase activity in cell cytosol depends on the endopolyPase gene PPN1.

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 0.8 mM P(i) as compared with 19.3 mM P(i). When spheroplasts isolated from P(i)-starved cells were incubated in the P(i)-complete medium, they accumulated polyP and exhibited a phosphate overplus effect. Under phosphate overplus the polyP level in mitochondria was two times higher than in the complete medium without preliminary P(i) starvation. The average chain length of polyP in mitochondria was of <15 phosphate residues at 19.3 mM P(i) in the culture medium and increased at phosphate overplus. Deoxyglucose inhibited polyP accumulation in spheroplasts, but had no effect on polyP accumulation in mitochondria. Uncouplers (FCCP, dinitrophenol) and ionophores (monensin, nigericin) inhibited polyP accumulation in mitochondria more efficiently than in spheroplasts. Fast hydrolysis of polyP was observed after sonication of isolated mitochondria. Probably, the accumulation of polyP in mitochondria depended on the proton-motive force of their membranes.

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.

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. Inactivation of PPX1 encoding a major exopolyPase (PPX1) in S. cerevisiae did not result in considerable changes in the content and properties of nuclear exopolyPase as compared to the parental strain of S. cerevisiae. Consequently, the nuclear exopolyPase was not encoded by PPX1. In the CRX strain, the exopolyPase was stimulated by bivalent metal cations. Co2+, the best activator, stimulated it by approximately 2.5-fold. The exopolyPase activity was nearly the same with polyphosphate (polyP) chain lengths ranging from 3 to 208 orthophosphate when measured with Mg2+. With Co 2+, the exopolyPase activity increased along with the increase in polymerization degree of the substrate.

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. Inactivation of PPX1 does not result in any changes in the content and properties of vacuolar exopolyphosphatase as compared with the wild strain of S. cerevisiae. In response to PPX1 mutation, exopolyphosphatase properties in the cell as a whole undergo modifications including the ability to hydrolyze polyphosphates with different lengths of the chain.