Integration of DNA copies in a host genome is a necessary stage in the life cycle of retroviruses and LTR-retrotransposons. There is still no clear understanding of integration specificity of retroelements into a target site. The selection of the target DNA is believed to potentially affect a number of factors such as transcriptional status, association with histones and other DNA-binding proteins, and DNA bending. The authors performed a comprehensive computer analysis of the integration specificity of Drosophila melanogaster LTR-retrotransposons and retroviruses including an analysis of the nucleotide composition of targets, terminal sequences of LTRs, and integrase sequences. A classification of LTR-retrotransposons based on the integration specificity was developed. All the LTR-retrotransposons of the gypsy group with three open frames (errantiviruses) and their derivatives with two open frames demonstrate strict specificity to a target DNA selection. Such specificity correlates with the structural features of the target DNA: bendability, A-philicity, or protein-induced deformability. The remaining LTR-retrotransposons (copia and BEL groups, blastopia and 412 subgroups of the gypsy group) do not show specificity of integration. Chromodomain is present in the integrase structures of blastopia and 412 subgroup LTR-retrotransposons and may facilitate the process of non-specific integration.

Using random (combinatorial) DNA-libraries with various degrees of diversity, it was shown that their amplification by polymerase chain reaction in real time resulted in appearance of a maximum on amplification curves. The relative decrease of fluorescence after passing the maximum was directly proportional to the logarithm of the number of oligonucleotide sequence variants in the random DNA-library provided that this number was within in the interval from 1 to 104 and remained practically unaltered when the number of variants was in the interval from 105 to 108. The obtained dependence was used in the course of SELEX to evaluate changes in the diversity of random DNA-libraries from round to round in selection of DNA-aptamers to the recombinant SMAD4 protein. As a result, oligonucleotides containing sequences able to form a site of SMAD4-DNA interactions known as SBE (SMAD-binding element) have been selected thus indicating that the SMAD4-SBE interaction dominates the aptamer selection.

Selected reaction monitoring (SRM) is a mass spectrometric technique characterized by the exceptionally high selectivity and sensitivity of protein detection. However, even with this technique, the quantitative detection of low- and ultralow-abundance proteins in blood plasma, which is of great importance for the search and verification of novel protein disease markers, is a challenging task due to the immense dynamic range of protein abundance levels. One approach used to overcome this problem is the immunoaffinity enrichment of target proteins for SRM analysis, employing monoclonal antibodies. Aptamers appear as a promising alternative to antibodies for affinity enrichment. Here, using recombinant protein SMAD4 as a model target added at known concentrations to human blood plasma and SRM as a detection method, we investigated a relationship between the initial amount of the target protein and its amount in the fraction enriched with SMAD4 by an anti-SMAD4 DNA-aptamer immobilized on magnetic beads. It was found that the aptamer-based enrichment provided a 30-fold increase in the sensitivity of SRM detection of SMAD4. These results indicate that the aptamer-based affinity enrichment of target proteins can be successfully employed to improve quantitative detection of low-abundance proteins by SRM in undepleted human blood plasma.

Introduction of retrotransposone gypsy in the genome of Drosophila melanogaster occurs specifi cally and is performed by integrase of this mobile element. Gypsy integrase is capable not only of introduction but also of precise excision of retrotransposon DNA from the target site. In the present work, we study the endonuclease activity of recombinant integrase gypsy in vitro. It is indicated that the enzyme hydrolyzes the substrate, forming single chain and double chain breaks. Conformation of the substrate greatly influences hydrolysis: integrase conducts double chain breaks only in a supertwisted circular DNA molecule. The data make it possible to understanding the integration mechanism of retroviruses and transposition of mobile ele ments.

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