| Abstract [eng] |
Despite a great deal of research, the functional significance of tightly bound DNA-protein complexes is not yet clear, therefore these complexes are perfect object for pioneering research. Very little is known about plant TBP-DNA complexes. In this work we investigated barley TBP-DNA complexes from different organs (first leaves, roots and coleoptiles) at different developmental stages. We characterized individual components of tightly bound DNA-proteins complexes: polypeptides (TBP) and DNA. We isolated and characterized TBP proteins from barley first leaves, roots and coleoptiles of different age and differentiation stage. Also we isolated and characterized the DNA fragments from barley first leaves and water ripe and milky ripe grain TBP-DNA complexes. We demonstrated that in different developmental stages of coleoptiles, first leaves and roots TBP-DNA complexes were identified as a group of 15-160 kDa proteins, most of TBPs are acidic. Some of barley TBPs (10, 25, 38, 40 and 55 kDa) exhibit phosphatase, maybe Ser/Thr activity. We have identified also that some of TBPs tyrosines were phosphorylated, this modification depends on organ and developmental stage. Identified barley TBPs were involved in fundamental genetic processes, as well as in chromatin rearrangement and regulation processes. Nuclear matrix proteins, enzymes, transcription factors, serpins, immunophilins, and transposon polypeptides were identified among TBPs. We demonstrated that expression of TBPs depends on organ and developmental stage. TBPs manifested high affinity to DNA fragments from the same organ TBP-DNA complexes. We established that the DNA sequences from TBP-DNA complexes are similar to S/MAR, but have peculiar traits. Most of sequences are localized in subtelomeres, telomeres, long arm of chromosomes (which chromosomes?). DNA sequences forming TBP-DNA complexes are homologous to various transposons, retrotransposons and repeat sequences. These sequences are heterogeneous, but have some motifs, that are common for all of them: ori motifs, topoisomerase II binding sites, short A+T rich stretches, curved DNA stretches. These sequences are rich in transcription factors binding sites. Our results show that examined sequences are specific. They have some characteristics of S/MAR sequences and they may be important for maintenance of DNA high-order structure and for regulation of gene expression. By means of hybridization between the DNA fragments of TBP-DNA complexes and microarray of barley Amy32b and Bmy1 genes we demonstrated that barley TBPs interact with Amy32b gene promotor and with Bmy1 gene Exon III, Intron III 1.5 kb region. In case of both genes, interaction with TBP proteins decreases in the course of grain development (during transition from water ripe to milky ripe stage). The changes in TBPs distribution in the genes were coupled to changes in their expression during water ripe and milky ripe stages. Effect of TBPs binding on gene expression are different – Amy32b expression increases, but Bmy1 expression decreases. We suppose that obtained results about tightly bound DNA-protein complexes can help to answer some important questions, for example, TBPs and nuclear matrix interaction, TBPs localization in nucleus, TBPs composition and functions, tightly bound to TBP DNA sequences composition and functions in genome. Performed research allows us to better understand plant cell nucleus and interactions between nuclear proteins and DNA. |
