Some generalizations based on stratification and vertical mixing in meromictic Lake Shira,Russia, in the period 2002–2009

In a brackish, temperate, 24-m-deep Lake Shira, the profiles of salinity, temperature, oxygen and sulfide concentrations were measured on a seasonal basis from 2002 to 2009. The lake was shown to be meromictic with autumnal overturn restricted to mixolimnion. The depth of mixolimnion and position of oxic–anoxic interface varied annually. The spring mixing processes contribute to the formation of mixolimnion in autumn. The exceptionally windy spring of 2007 caused the deepening of mixolimnion in the winter of 2008. The winter position of oxic–anoxic interface was affected by the position of lower boundary of mixolimnion in all winters. The salinity in the winter mixolimnion increased compared with the autumn because of freezing out of salts from the upper water layers meters during ice formation and their dissolution in water below. The profiles of salinity and temperature were simulated by the mathematical 1-D model of temperature and salinity conditions taking into account ice formation. The simulated profiles generally coincided with the measured ones. The coincidence implies that simplified one-dimensional model can be applied to roughly describe salinity and density profiles and mixing behavior of Lake Shira.     

Mismatch repair-independent increase in spontaneous mutagenesis in yeast lacking non-essential subunits of DNA polymerase ε

Yeast DNA polymerase ε (Pol ε) is a highly accurate and processive enzyme that participates in nuclear DNA replication of the leading strand template. In addition to a large subunit (Pol2) harboring the polymerase and proofreading exonuclease active sites, Pol ε also has one essential subunit (Dpb2) and two smaller, non-essential subunits (Dpb3 and Dpb4) whose functions are not fully understood. To probe the functions of Dpb3 and Dpb4, here we investigate the consequences of their absence on the biochemical properties of Pol ε in vitro and on genome stability in vivo. The fidelity of DNA synthesis in vitro by purified Pol2/Dpb2, i.e. lacking Dpb3 and Dpb4, is comparable to the four-subunit Pol ε holoenzyme. Nonetheless, deletion of DPB3 and DPB4 elevates spontaneous frameshift and base substitution rates in vivo, to the same extent as the loss of Pol ε proofreading activity in a pol2-4 strain. In contrast to pol2-4, however, the dpb3Δdpb4Δ does not lead to a synergistic increase of mutation rates with defects in DNA mismatch repair. The increased mutation rate in dpb3Δdpb4Δ strains is partly dependent on REV3, as well as the proofreading capacity of Pol δ. Finally, biochemical studies demonstrate that the absence of Dpb3 and Dpb4 destabilizes the interaction between Pol ε and the template DNA during processive DNA synthesis and during processive 3' to 5'exonucleolytic degradation of DNA. Collectively, these data suggest a model wherein Dpb3 and Dpb4 do not directly influence replication fidelity per se, but rather contribute to normal replication fork progression. In their absence, a defective replisome may more frequently leave gaps on the leading strand that are eventually filled by Pol ζ or Pol δ, in a post-replication process that generates errors not corrected by the DNA mismatch repair system.     

Mutational hotspots in the TP53 gene and, possibly, other tumor suppressors evolve by positive selection

Background  

The mutation spectra of the TP53 gene and other tumor suppressors contain multiple hotspots, i.e., sites of non-random, frequent mutation in tumors and/or the germline. The origin of the hotspots remains unclear, the general view being that they represent highly mutable nucleotide contexts which likely reflect effects of different endogenous and exogenous factors shaping the mutation process in specific tissues. The origin of hotspots is of major importance because it has been suggested that mutable contexts could be used to infer mechanisms of mutagenesis contributing to tumorigenesis.     

The mutational spectra of gene p53 in different types of tumors]

The comparative analysis of the frequencies of nucleotide exchanges in mutational spectra of gene p53 (5-8 exons) between germline cancer-prone families (Li-Fraumeni syndrome), between somatic mutations in the tumors of different histogenesis and cell lines, obtained from them, was carried out. The nucleotide positions with high level of mutation events (mutational "hot spots"), typical for germline mutational spectra, tissue-specific patterns of their disappearing and appearing of new ones in solid tumors in vivo, nearly complete absence of "hot spots" in lymphomas and cell lines in vitro were revealed. The obtained results allowed to suggest, that one of the leading factor controlling hot-spots distribution in 5-8 exons of gene p53 is the specificity of cell division conditions in vivo and in vitro.     

Microbial metabolism of the carbon and sulfur cycles in Shira Lake (Khakasia)

Microbiological and biogeochemical studies of the meromictic saline Lake Shira (Khakasia) were conducted. In the upper part of the hydrogen-sulfide zone, at a depth of 13.5-14 m, there was a pale pink layer of water due to the development of purple bacteria (6 x 10(5) cells/ml), which were assigned by their morphological and spectral characteristics to Lamprocystis purpureus (formerly Amoebobacter purpurea). In August, the production of organic matter (OM) in Lake Shira was estimated to be 943 mg C/(m2 day). The contribution of anoxygenic photosynthesis was insignificant (about 7% of the total OM production). The share of bacterial chemosynthesis was still less (no more than 2%). In the anaerobic zone, the community of sulfate-reducing bacteria played a decisive role in the terminal decomposition of OM. The maximal rates of sulfate reduction were observed in the near-bottom water (114 micrograms S/(1 day)) and in the surface layer of bottom sediments (901 micrograms S/(dm3 day)). The daily expenditure of Corg for sulfate reduction was 73% of Corg formed daily in the processes of oxygenic and anoxygenic photosynthesis and bacterial chemosynthesis. The profile of methane distribution in the water column and bottom sediments was typical of meromictic reservoirs. The methane content in the water column increased beginning with the thermocline (7-8 m), and reached maximum values in the near-bottom water (17 microliters/l). In bottom sediments, the greatest methane concentrations (57 microliters/l) were observed in the surface layer (0-3 cm). The integral rate of methane formation in the water column and bottom sediments was almost an order of magnitude higher than the rate of its oxidation by aerobic and anaerobic methanotrophic microorganisms.     

Remarkable interkingdom conservation of intron positions and massive,lineage-specific intron loss and gain in eukaryotic evolution

Sequencing of eukaryotic genomes allows one to address major evolutionary problems, such as the evolution of gene structure. We compared the intron positions in 684 orthologous gene sets from 8 complete genomes of animals, plants, fungi, and protists and constructed parsimonious scenarios of evolution of the exon-intron structure for the respective genes. Approximately one-third of the introns in the malaria parasite Plasmodium falciparum are shared with at least one crown group eukaryote; this number indicates that these introns have been conserved through >1.5 billion years of evolution that separate Plasmodium from the crown group. Paradoxically, humans share many more introns with the plant Arabidopsis thaliana than with the fly or nematode. The inferred evolutionary scenario holds that the common ancestor of Plasmodium and the crown group and, especially, the common ancestor of animals, plants, and fungi had numerous introns. Most of these ancestral introns, which are retained in the genomes of vertebrates and plants, have been lost in fungi, nematodes, arthropods, and probably Plasmodium. In addition, numerous introns have been inserted into vertebrate and plant genes, whereas, in other lineages, intron gain was much less prominent.     

Unique error signature of the four-subunit yeast DNA polymerase epsilon

We have purified wild type and exonuclease-deficient four-subunit DNA polymerase epsilon (Pol epsilon) complex from Saccharomyces cerevisiae and analyzed the fidelity of DNA synthesis by the two enzymes. Wild type Pol epsilon synthesizes DNA accurately, generating single-base substitutions and deletions at average error rates of 5' exonuclease activity is less accurate to a degree suggesting that wild type Pol epsilon proofreads at least 92% of base substitution errors and at least 99% of frameshift errors made by the polymerase. Surprisingly the base substitution fidelity of exonuclease-deficient Pol epsilon is severalfold lower than that of proofreading-deficient forms of other replicative polymerases. Moreover the spectrum of errors shows a feature not seen with other A, B, C, or X family polymerases: a high proportion of transversions resulting from T.dTTP, T.dCTP, and C.dTTP mispairs. This unique error specificity and amino acid sequence alignments suggest that the structure of the polymerase active site of Pol epsilon differs from those of other B family members. We observed both similarities and differences between the spectrum of substitutions generated by proofreading-deficient Pol epsilon in vitro and substitutions occurring in vivo in a yeast strain defective in Pol epsilon proofreading and DNA mismatch repair. We discuss the implications of these findings for the role of Pol epsilon polymerase activity in DNA replication.     

Microbial sulfate reduction in a brackish meromictic steppe lake

Patterns of sulfate reduction were studied in water and sediments of Lake Shira, South Siberia, Russia. The lake was characterized by a high level of sulfate (91-116 mM). The concentration of hydrogen sulfide in the anoxic waters of the lake reached 0.6 mM. In summer the sulfate reduction rate in the water column, measured by radiometric technique, varied from 0.25 to 9.81 mol sulfate l^-1 d^-1. There were two peaks of sulfate reduction activity: just below the chemocline and near the sediment surface. Sulfate reduction rate in the profundal silts ranged from 4.1 to 90.6 mol l^-1 d^-1. The zone of the most active sulfate reduction was restricted to the surface sediment layers. The acceleration of sulfate reduction rate (up to 236 mol l^-1 d^-1) and the increase of density of viable sulfate reducers (up to 2 x 10⁵ cells ml^-1) were recorded in the littoral sediments adjacent to the mouth of the Son River and sewage discharge. It was apparently caused by the input of allochthonous organic substrates and also by a high environmental temperature. On an areal basis, sulfate reduction rate in the water was approximately 8 times higher than that in the profundal sediments. Sulfate reduction was the most important process of anaerobic oxidation of organic carbon in Lake Shira. In summer in the profundal zone of the lake, sulfate reducers were able to mineralize about 67% of the daily integrated primary production of phototrophic and chemotrophic organisms.