Haploid yeast cells undergo a reversible phenotypic switch associated with chromosome II copy number

Background

SUP35 and SUP45 are essential genes encoding polypeptide chain release factors. However, mutants for these genes may be viable but display pleiotropic phenotypes which include, but are not limited to, nonsense suppressor phenotype due to translation termination defect. [PSI ⁺] prion formation is another Sup35p-associated mechanism leading to nonsense suppression through decreased availability of functional Sup35p. [PSI ⁺] differs from genuine sup35 mutations by the possibility of its elimination and subsequent re-induction. Some suppressor sup35 mutants had also been shown to undergo a reversible phenotypic switch in the opposite direction. This reversible switching had been attributed to a prion termed [ISP ⁺]. However, even though many phenotypic and molecular level features of [ISP ⁺] were revealed, the mechanism behind this phenomenon has not been clearly explained and might be more complex than suggested initially.

Results

Here we took a genomic approach to look into the molecular basis of the difference between the suppressor (Isp^?) and non-suppressor (Isp⁺) phenotypes. We report that the reason for the difference between the Isp⁺ and the Isp^? phenotypes is chromosome II copy number changes and support our finding with showing that these changes are indeed reversible by reproducing the phenotypic switch and tracking karyotypic changes. Finally, we suggest mechanisms that mediate elevation in nonsense suppression efficiency upon amplification of chromosome II and facilitate switching between these states.

Conclusions

(i) In our experimental system, amplification of chromosome II confers nonsense suppressor phenotype and guanidine hydrochloride resistance at the cost of overall decreased viability in rich medium. (ii) SFP1 might represent a novel regulator of chromosome stability, as SFP1 overexpression elevates frequency of the additional chromosome loss in our system. (iii) Prolonged treatment with guanidine hydrochloride leads to selection of resistant isolates, some of which are disomic for chromosome II.

     

Potential activity of the external pathway of NADH oxidation in mitochondria

High rate of exogenic NADH oxidation (up to 200 mg-at. oxygen for 1 min per 1 mg of protein and higher) along the rothenone, antimycin-nonsensitive pathway is observed under interaction of mitoplasts with the external membrane and cytochrome c. In the medium with low ionic strength the interaction of external and internal membranes is not a sufficient condition for activating the external pathway of the NADH oxidation: the presence of exogenic cytochrome c is also necessary. With saturated cytochrome c concentrations the addition of outer membranes leads to further stimulation of the NADH oxidation. In the medium with high ionic strength external membranes stimulate oxidation of NADH when exogenic cytochrome c is absent; the subsequent addition of cytochrome c stimulates the NADH oxidation in this medium to a greater extent than in the medium with the low ionic strength. Under the nonlimited interaction of external and internal membranes and cytochrome c the potential activity of the outer pathway of NADH oxidation in the liver mytoplasts of hybernating gophers is lower than in the liver mytoplasts of rats.     

The Epac protein inhibitor ESI-09 eliminates the tonic phase of aorta contraction induced by endogenic vasoconstrictors in rats

It has been shown that the Epac1 and Epac2 protein inhibitor ESI-09 has no effect on the amplitude of contraction of aortic rings caused by the influence of serotonin, noradrenaline, or KCl depolarizing solution, but changes the kinetics of the contractile response. It was noted that in the presence of ESI-09 the curve of the relaxation phase in intact and deendothelized vessels moved to the left under the impact of serotonin or KCl and the phase of prolonged tonic contraction, which developed after the exposure to noradrenalin, was canceled. It was found that ESI-09 exerted different effects on the induced growth in the concentration of cytoplasmic Ca²⁺ in the aortic smooth muscle cells of rats depending on the agonist, whereas the selective inhibitor Epac2 ESI-05 has no effect on vascular contractility and calcium metabolism in the aortic smooth muscle of rats. The cAMP-independent participation of Epac1 in the formation of the contractile response to the influence of vasoconstrictor compounds was revealed.     

Effects of manganese on potassium outflow from erythrocytes and on respiration of rat liver mitochondria

In this work, effects of manganese on respiration of rat liver mitochondria and the rate of K⁺ outflow from rat erythrocytes are studied in a broad range of concentrations. It is shown that manganese ions at low concentrations (1 × 10^–7–3 × 10^–5 М) inhibit K⁺ outflow from rat erythrocytes; this can be used to prevent their lysis. At high concentrations (1 × 10^–4–1 × 10^–3 M), manganese activates K⁺ outflow from the erythrocytes but inhibits the valinomycin-induced outflow of the ion from the erythrocytes. This fact is an indication of manganese influence on physicochemical properties of membranes. At low concentrations manganese does not affect parameters of respiration and oxidative phosphorylation of rat liver mitochondria, while at high concentrations it exerts acceleration of the mitochondrial respiration, i.e., uncouples respiration from phosphorilation and, hence, inhibits ATP synthesis.     

Statistical properties of the variation at linked microsatellite loci: implications for the history of human Y chromosomes [published erratum appears in Mol Biol Evol 1997 Mar;14(3):354]

It has recently been suggested that observed levels of variation at microsatellite loci can be used to infer patterns of selection in genomes and to assess demographic history. In order to evaluate the feasibility of these suggestions it is necessary to know something about how levels of variation at microsatellite loci are expected to fluctuate due simply to stochasticity in the processes of mutation and inheritance (genetic sampling). Here we use recently derived properties of the stepwise mutation model to place confidence intervals around the variance in repeat score that is expected at mutation-drift equilibrium and outline a statistical test for whether an observed value differs significantly from expectation. We also develop confidence intervals for the time course of the buildup of variation following a complete elimination of variation, such as might be caused by a selective sweep or an extreme population bottleneck. We apply these methods to the variation observed at human Y-specific microsatellites. Although a number of authors have suggested the possibility of a very recent sweep, our analyses suggest that a sweep or extreme bottleneck is unlikely to have occurred anytime during the last approximately 74,000 years. To generate this result we use a recently estimated mutation rate for microsatellite loci of 5.6 x 10(-4) along with the variation observed at autosomal microsatellite loci to estimate the human effective population size. This estimate is 18,000, implying an effective number of 4,500 Y chromosomes. One important general conclusion to emerge from this study is that in order to reject mutation-drift equilibrium at a set of linked microsatellite loci it is necessary to have an unreasonably large number of loci unless the observed variance is far below that expected at mutation-drift equilibrium.