Qualitative and quantitative characteristics of the extracellular DNA delivered to the nucleus of a living cell

BACKGROUND: The blood plasma and other intertissue fluids usually contain a certain amount of DNA, getting there due to a natural cell death in the organism. Cells of this organism can capture the extracellular DNA, whereupon it is delivered to various cell compartments. It is hypothesized that the extracellular DNA is involved in the transfer of genetic information and its fixation in the genome of recipient cell. RESULTS: The existence of an active flow of extracellular DNA into the cell is demonstrated using human breast adenocarcinoma (MCF-7) cells as a recipient culture. The qualitative state of the DNA fragments delivered to the main cell compartments (cytoplasm and interchromosomal fraction) was assessed. The extracellular DNA delivered to the cell is characterized quantitatively. CONCLUSION: It is demonstrated that the extracellular DNA fragments in several minutes reach the nuclear space, where they are processed so that their linear size increases from about 500 bp to 10,000 bp. The amount of free extracellular DNA fragments simultaneously present in the nuclear space may reach up to 2% of the haploid genome. Using individual DNA fragments with a known molecular weight and sequence as an extracellular DNA, it is found that these fragments degrade instantly in the culture liquid in the absence of a competitor DNA and are delivered into the cell as degradants. When adding a sufficient amount of competitor DNA, the initial undegraded molecules of the DNA fragments with the known molecular weight and sequence are detectable both in the cytoplasm and nuclear space only at the zero point of experiments. The labeled precursor α-dNTP*, added to culture medium, was undetectable inside the cell in all the experiments

Tissue-Specific Alternative Splicing Analysis Reveals the Diversity of Chromosome 18 Transcriptome

The Chromosome-centric Human Proteome Project (C-HPP) is aimed to identify the variety of protein products and transcripts of the number of chromosomes. The Russian part of C-HPP is devoted to the study of the human chromosome 18. Using widely accepted Tophat and SpliceGrapher, a tool for accurate splice sites and alternative mRNA isoforms prediction, we performed the extensive mining of the splice variants of chromosome 18 transcripts and encoded protein products in liver, brain, lung, kidney, blood, testis, derma, and skeletal muscles. About 6.1 billion of the reads represented by 450 billion of the bases have been analyzed. The relative frequencies of splice events as well as gene expression profiles in normal tissues are evaluated. Using ExPASy PROSITE, the novel features and possible functional sites of previously unknown splice variants were highlighted. A set of unique proteotypic peptides enabling the identification of novel alternative protein species using mass-spectrometry is constructed. The revealed data will be integrated into the gene-centric knowledgebase of the Russian part of C-HPP available at http://kb18.ru and http://www.splicing.zz.mu/

Tissue-Specific Alternative Splicing Analysis Reveals the Diversity of Chromosome 18 Transcriptome

The Chromosome-centric Human Proteome Project (C-HPP) is aimed to identify the variety of protein products and transcripts of the number of chromosomes. The Russian part of C-HPP is devoted to the study of the human chromosome 18. Using widely accepted Tophat and SpliceGrapher, a tool for accurate splice sites and alternative mRNA isoforms prediction, we performed the extensive mining of the splice variants of chromosome 18 transcripts and encoded protein products in liver, brain, lung, kidney, blood, testis, derma, and skeletal muscles. About 6.1 billion of the reads represented by 450 billion of the bases have been analyzed. The relative frequencies of splice events as well as gene expression profiles in normal tissues are evaluated. Using ExPASy PROSITE, the novel features and possible functional sites of previously unknown splice variants were highlighted. A set of unique proteotypic peptides enabling the identification of novel alternative protein species using mass-spectrometry is constructed. The revealed data will be integrated into the gene-centric knowledgebase of the Russian part of C-HPP available at http://kb18.ru and http://www.splicing.zz.mu/