Preferential Binding of Hot Spot Mutant p53 Proteins to Supercoiled DNA In Vitro and in Cells

Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed.     

Protein phosphorylation in Streptomyces albus

The phosphorylated proteins of Streptomyces albus, radioactively labeled with [32P]orthophosphate have been analyzed by gel electrophoresis and autoradiography. More than 10 protein species were found to be phosphorylated. With [32P]ATP as substrate cell free extracts phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. From cell extract which exhibited active phosphorylated in vitro, a protein kinase has been partially purified. The kinase activity was identified in fractions corresponding to a 90 kDa protein.     

A 323-year long reconstruction of drought for SW Romania based on black pine (Pinus Nigra) tree-ring widths

Increase in temperature and decrease in precipitation pose a major future challenge for sustainable ecosystem management in Romania. To understand ecosystem response and the wider social consequences of environmental change, we constructed a 396-year long (1615–2010) drought sensitive tree-ring width chronology (TRW) of Pinus nigra var. banatica (Georg. et Ion.) growing on steep slopes and shallow organic soil. We established a statistical relationship between TRW and two meteorological parameters—monthly sum of precipitation (PP) and standardised precipitation index (SPI). PP and SPI correlate significantly with TRW (r?=?0.54 and 0.58) and are stable in time. Rigorous statistical tests, which measure the accuracy and prediction ability of the model, were all significant. SPI was eventually reconstructed back to 1688, with extreme dry and wet years identified using the percentile method. By means of reconstruction, we identified two so far unknown extremely dry years in Romania—1725 and 1782. Those 2 years are almost as dry as 1946, which was known as the “year of great famine.” Since no historical documents for these 2 years were available in local archives, we compared the results with those from neighbouring countries and discovered that both years were extremely dry in the wider region (Slovakia, Hungary, Anatolia, Syria, and Turkey). While the 1800–1900 period was relatively mild, with only two moderately extreme years as far as weather is concerned, the 1900–2009 period was highly salient owing to the very high number of wet and dry extremes—five extremely wet and three extremely dry events (one of them in 1946) were identified.     

Engineering TaqII bifunctional endonuclease DNA recognition fidelity: the effect of a single amino acid substitution within the methyltransferase catalytic site

The aim of this study was to improve a useful molecular tool—TaqII restriction endonuclease-methyltransferase—by rational protein engineering, as well as to show an application of our novel method of restriction endonuclease activity modulation through a single amino acid change in the NPPY motif of methyltransferase. An amino acid change was introduced using site-directed mutagenesis into the taqIIRM gene. The mutated gene was expressed in Escherichia coli. The protein variant was purified and characterized. Previously, we described a TspGWI variant with an amino acid change in the methyltransferase motif IV. Here, we investigate a complex, pleiotropic effect of an analogous amino acid change on its homologue—TaqII. The methyltransferase activity is reduced, but not abolished, while TaqII restriction endonuclease can be reactivated by sinefungin, with an increased DNA recognition fidelity. The general method for engineering of the IIS/IIC/IIG restriction endonuclease activity/fidelity is developed along with the generation of an improved TaqII enzyme for biotechnological applications. A successful application of our novel strategy for restriction endonuclease activity/fidelity alteration, based on bioinformatics analyses, mutagenesis and the use of cofactor-analogue activity modulation, is presented.     

Effects of plant density and nutrient levels on fruit polymorphism in Atriplex sagittata

Atriplex sagittata is a heterocarpic species producing three types of fruits which differ in morphology and ecological properties. This study focused on variation in biomass allocation into particular fruit types under different density and fertilization levels. The reduction in total weight under stressful conditions was accompanied by a reduction of reproductive structures in terms of both total fruit production and mean fruit weight. Allocation of biomass to particular fruit types under different density-fertilization treatments showed considerable variation. The non-dormant, bracteolate fruit type (termed C) contributed 80–90% to the total fecundity and its production was not affected by density-fertilization treatments. The production of this fruit type is higher in the upper part of the maternal plant stem. Production of the very dormant ebracteate fruit type (termed A) increased under favourable conditions and was greater on the lower part of the stem, whereas more of the so-called type B fruit (covered by bracteoles and exhibiting dispersal and dormancy characteristics intermediate between types A and C) was produced under suboptimal conditions and was concentrated in the middle part of the plant body. This pattern contradicts to some extent the theory that deeply dormant and less dispersible fruits will be produced under stressful conditions and may be considered a new model for the behaviour of plants with more than two heterocarpic fruits. The type B fruit, intermediate in both morphology and ecological behaviour, provides a continuum between extreme strategies. Received: 8 February 1998 / Accepted: 19 November 1998     

Lipid remodelling is a widespread strategy in marine heterotrophic bacteria upon phosphorus deficiency

Upon phosphorus (P) deficiency, marine phytoplankton reduce their requirements for P by replacing membrane phospholipids with alternative non-phosphorus lipids. It was very recently demonstrated that a SAR11 isolate also shares this capability when phosphate starved in culture. Yet, the extent to which this process occurs in other marine heterotrophic bacteria and in the natural environment is unknown. Here, we demonstrate that the substitution of membrane phospholipids for a variety of non-phosphorus lipids is a conserved response to P deficiency among phylogenetically diverse marine heterotrophic bacteria, including members of the Alphaproteobacteria and Flavobacteria. By deletion mutagenesis and complementation in the model marine bacterium Phaeobacter sp. MED193 and heterologous expression in recombinant Escherichia coli, we confirm the roles of a phospholipase C (PlcP) and a glycosyltransferase in lipid remodelling. Analyses of the Global Ocean Sampling and Tara Oceans metagenome data sets demonstrate that PlcP is particularly abundant in areas characterized by low phosphate concentrations. Furthermore, we show that lipid remodelling occurs seasonally and responds to changing nutrient conditions in natural microbial communities from the Mediterranean Sea. Together, our results point to the key role of lipid substitution as an adaptive strategy enabling heterotrophic bacteria to thrive in the vast P-depleted areas of the ocean.     

Effect of the preweaning nutritional state on the cardiac protein profile and functional performance of the rat heart

The aim of the study was to find out whether the changes in nutritional status induced by different litter size during early postnatal development can influence quantitative and qualitative protein remodeling and contractile performance of the myocardium. Male Wistar rats born at the same day were pooled together at 2 days postbirth and assigned by random selection to dams in groups of 4, 8 or 16 rats/litter. The animals were investigated at the age of 4 and 16 weeks. The results revealed that the early postnatal nutritional modification altered weight parameters: whereas lower heart weight persisted in slow-growing rats until 16 weeks, higher body weight of fast-growing rats returned to the control level at the age of 16 weeks. Altered nutritional status influenced also protein remodeling of the myocardium: the concentration of all noncollagenous proteins (fractions of metabolic and contractile proteins) significantly increased in slow-growing rats, on the other hand, the concentration of collagenous proteins (pepsin-soluble and -insoluble fractions) was higher in fast-growing animals. The changes were, however, only transitional: three months after the end of the weaning period most protein changes returned to the control level. However, higher concentration of total blood lipids and triglycerides in fast-growing rats persisted until adulthood. Nutritional changes had, however, only minor effect on ventricular performance. No differences among groups were observed in basal values of the left ventricular pressure, while the maximum pressure attained after an acute ventricular loading and the contractile reserve were significantly decreased in slowgrowing 4 week old rats. The functional consequence of altered nutritional status during weaning was only transitional, in agreement with the transient character of most structural and biochemical markers of myocardial remodeling.