Identification of tissue-specific DNA-protein binding sites by means of two-dimensional electrophoretic mobility shift assay display

We developed a technique of differential electrophoretic mobility shift assay (EMSA) display allowing identification of tissue-specific protein-binding sites within long genomic sequences. Using this approach, we identified 10 cell type-specific protein-binding sites (protein target sites [PTSs]) within a 137-kb human chromosome 19 region. In general, tissue-specific binding of proteins from different nuclear extracts by individual PTSs did not follow the all-or-nothing principle. Most often, PTS-protein complexes were formed in all cases, but they were different for different nuclear extracts used.     

A tag-based approach for high-throughput analysis of CCWGG methylation

Non-CpG methylation occurring in the context of CNG sequences is found in plants at a large number of genomic loci. However, there is still little information available about non-CpG methylation in mammals. Efficient methods that would allow detection of scarcely localized methylated sites in small quantities of DNA are required to elucidate the biological role of non-CpG methylation in both plants and animals. In this study, we tested a new whole genome approach to identify sites of CCWGG methylation (W is A or T), a particular case of CNG methylation, in genomic DNA. This technique is based on digestion of DNAs with methylation-sensitive restriction endonucleases EcoRII-C and AjnI. Short DNAs flanking methylated CCWGG sites (tags) are selectively purified and assembled in tandem arrays of up to nine tags. This allows high-throughput sequencing of tags, identification of flanking regions, and their exact positions in the genome. In this study, we tested specificity and efficiency of the approach.     

N.BspD6I DNA nickase strongly stimulates template-independent synthesis of non-palindromic repetitive DNA by Bst DNA polymerase

Highly efficient DNA synthesis without template and primer DNAs occurs when N.BspD6I DNA nickase is added to a reaction mixture containing deoxynucleoside triphosphates and the large fragment of Bst DNA polymerase. Over a period of 2 h, virtually all the deoxynucleoside triphosphates (dNTPs) become incorporated into DNA. Inactivation of N.BspD6I nickase by heating inhibits DNA synthesis. Optimal N.BspD6I activity is required to achieve high yields of synthesized DNA. Electron microscopy data revealed that the majority of DNA molecules have a branched structure. Cloning and sequencing of the fragments synthesized demonstrated that the DNA product mainly consists of multiple hexanucleotide non-palindromic tandem repeats containing nickase recognition sites. A possible mechanism is discussed that addresses template-independent DNA synthesis stimulated by N.BspD6I nickase.     

The latitude-zonal and spatial-successional trends in the distribution of yeasts

The distribution of yeasts in natural habitats is analyzed in the different nature zones of the former USSR (from tundra to desert) using the results of long-term research. Yeast community structure is changing in parallel to different stages of plant debris decay as well as to latitude-zonal gradient. These changes are not fluctuating but trend ones. As mineralization of plant debris proceeds the availability of sugars decreases and habitat become more extremal for yeasts which are typical saccharolytics. It causes decrease in species abundance and species diversity. At the same time in zonal gradient the most significant changes take place in the relative abundance of dominant species, genera or higher taxonomic groups. The thermotolerant species occurred more often in the southern regions while psychrophilic species dominate in the north. Soil yeast communities become more polytrophic in the north latitudes where mineralization of organic matter is rather low. Species inhabiting climatic pessimum areas usually form chlamidospores.     

SuUR protein binds to the boundary regions separating forum domains in Drosophila melanogaster

Forum domains are 50-150 kb DNA fragments that are released during spontaneous fragmentation of chromosomes. They are separated by islands of putative heterochromatin boundary regions. The SuUR protein, which is involved in the control of chromosome organization, is localized exclusively in heterochromatin and often colocalizes on chromosomes with Polycomb group proteins. To test whether the SuUR protein is associated with boundary regions, we used gel retardation assays and found that the SuUR protein binds specifically to boundary regions and that boundary regions are under-replicated. These results suggest that the regular distribution of boundary regions in chromosomes may represent the dispersion of sites designed for chromosomal silencing.     

The ratio of fungi and bacteria in the biomass of different types of soil determined by selective inhibition

Tundra, chernozem (virgin and arable), sod-podzolic (coniferous forest, meadow, and arable), and grey forest (larch forest) soils were used to separate the contributions of fungi and bacteria to substrate-induced respiration (SIR) with the help of antibiotics. For soils with a high content of organic matter (tundra and chernozem: 12 and 8%, respectively), the procedure of selective inhibition of SIR has been optimized. The optimized procedure includes the application of high concentrations of streptomycin (50-120 mg/g of soil) and cycloheximide (50-80 mg/g of soil) and decreasing the weight of the analyzed soil sample. Soils under study have shown the predominant contribution of fungi (63-82%) to the total SIR. The fungal-bacterial ratio in the soils of natural ecosystems (0-5 cm, without litter) was 4.32, 2.19, 1.5, and 1.5 for tundra soil, virgin chernozem, coniferous (sod-podzolic soil), and larch (grey forest soil) forests, respectively. The lower layers of sod-podzolic (5-10 cm) and grey forest (48-58 cm) soils showed a decrease in the fungal and increase in the bacterial component in the total SIR.     

Investigation of beta-endorphin nonopioid reception in preimplantation development of mouse embryo in vitro

The effect of beta-endorphin on 2-, 4- and 8-cell embryo development in vitro was studied. It is shown, that hormone has no effect on 2-cell embryos development, but it has enhanced viability of 4- and 8-cell mouse embryos. The number ofblastocyst formation increases in presence of 0.1 microM beta-endorphin in embryo cultured medium but the number of blastocyst with abnormal structure decreases. The effect of hormone on the change of intracellular concentration of Ca2+ ion in 2-, 4- and 8-cell mouse embryo has been studied with the help of fluorescent microscopy. The effect of adenylate cyclase, and phospholipase activity blockers and opioid blocker naloxone on the change of intracellular concentration of Ca2+ ion in early mouse embryo in the presence of beta-endorphin have been also studied. It is shown that 2-cell embryo has opioid and nonopioid beta-endorphin receptors, whereas 4- and 8-cell mouse embryos have only nonopoioid beta-endorphin receptors. It is also shown that the effect of beta-endorphin in the early mouse embryo through a nonopioid receptors occurs with the participation of intracellular Ca2+ and adenylate cyclase signaling system.     

Characterization of yeast groupings in the phyllosphere of Sphagnum mosses

Significant differences were revealed in the taxonomic structure of the epiphytic yeast communities formed on Sphagnum mosses and on the leaves of vascular plants. On mosses, low abundance of red yeasts was found (the most typical epiphytes on vascular plant leaves), along with a relatively high content and diversity of nonpigmented dimorphic basidiomycetes related to the order Leucosporidiales. The species composition of epiphytic yeasts from mosses is different from that of both forest and meadow grasses and of the parts of vascular plants submerged in the turf. The specific composition of the Sphagnum mosses yeast community is probably determined by the biochemical characteristics of this environment, rather than by the hydrothermal regime in the turf.     

Characterization of yeast groupings in the phyllosphere of <Emphasis Type="Italic">Sphagnum</Emphasis> mosses

Significant differences were revealed in the taxonomic structure of the epiphytic yeast communities formed on Sphagnum mosses and on the leaves of vascular plants. On mosses, low abundance of red yeasts was found (the most typical epiphytes on vascular plant leaves), along with a relatively high content and diversity of nonpigmented dimorphic basidiomycetes related to the order Leucosporidiales. The species composition of epiphytic yeasts from mosses is different from that of both forest and meadow grasses and of the parts of vascular plants submerged in the turf. The specific composition of the Sphagnum mosses yeast community is probably determined by the biochemical characteristics of this environment, rather than by the hydrothermal regime in the turf.