Nucleosome positioning pattern derived from oligonucleotide compositions of genomic sequences.

Availability of nucleosome positioning pattern(s) is crucial for chromatin studies. The matrix form of the pattern has been recently derived (I. Gabdank, D. Barash, E. N. Trifonov. J Biomol Struct Dyn 26, 403-412 (2009), and E. N. Trifonov. J Biomol Struct Dyn 27, 741-746 (2010)). In its simplified linear form it is described by the motif CGRAAATTTYCG. Oligonucleotide components of the motif (say, triplets GRA, RAA, AAA, etc.) would be expected to appear in eukaryotic sequences more frequently. In this work we attempted the reconstruction of the bendability patterns for 13 genomes by a novel approach-extension of highest frequency trinucleotides. The consensus of the patterns reconstructed on the basis of trinucleotide frequencies in 13 eukaryotic genomes is derived: CRAAAATTTTYG. It conforms to the earlier established sequence motif. The reconstruction, thus, attests to the universality of the nucleosome DNA bendability pattern.

[The nature of the phosphorylation of retinoblastoma gene product in stable mouse and human cell lines]. / Kharakter fosforilirovaniia produkta gena retinoblastomy v stabil'nykh liniiakh kletok myshi i cheloveka.

The retinoblastoma gene product mediates the interaction between transcriptional factors and cyclin-kinase complexes, which perform a regulatory and effector function in the process of cell division. The activity of the retinoblastoma gene product is regulated by phosphorylation, which results in the appearance of additional protein molecules migrating in the region of 105--116 kDa during electrophoresis. Stereochemical analysis has established a direct correspondence between the extent of phosphorylation of the retinoblastoma gene product and its electrophoretic mobility. The results obtained permit an estimate of the phosphorylation of this protein in cells of different tissues by immunoblotting analysis of their lysates. The results of this study demonstrate that the degree of phosphorylation of retinoblastoma gene product in a series of stable cell lines increases as we go from monolayer to multilayer cultures, and further to cell cultures in suspension. Human cells produce more phosphorylated proteins compared to homologous mouse cells. The phosphorylation pattern of retinoblastoma gene product is probably tissue-specific. Much like mouse fibroblasts, HeLa cells may contain a hypophosphorylated protein with a molecular weight of 105 kDa. Phosphorylation of the retinoblastoma gene product in cells of embryonic mouse adenocarcinoma (line p19) changes in response to cloning or stimulation of differentiation by retinoic acid. The formation of all forms of retinoblastoma gene product, which pre-exist during asynchronous growth, is increased in the course of cloning. Differentiation is associated with the synthesis of an increased amount of hypophosphorylated protein. The results obtained lead to the hypothesis that even though the phosphorylation pattern of the retinoblastoma gene product is tissue-specific, it can show significant variation under the conditions of either cloning or differentiation and can be maintained for extended period of time at a new level that was not characteristic of the initial cell population.

[Growth of stable clones of mouse fibroblast cell line C3H10T1/2 expressing the human retinoblastoma gene product]. / Izuchenie rosta stabil'nykh klonov myshinykh fibroblastov linii C3H10T1/2, ékspressiruiushchikh produkt gena retinoblastomy cheloveka]

Phosphorylation and dephosphorylation of the retinoblastoma gene product (pRB) are recognized as necessary events in the cell cycle progression. To study the role of pRB in regulation of cell proliferation, the stable cell lines with constitutive expression of the exogenous RB gene can be employed. In order to obtain such cell lines in this work C3H10T1/2 mouse fibroblasts were infected with defective retrovirus encompassing the RB and Neo gene conferring resistance to geniticine (G418). The pRB production and its phosphorylation pattern were analyzed by immunoblotting in cell lysates considering well known data on correlation between pRB phosphorylation pattern and its electrophoretic mobility. Cell lines subjected to G418 selection with the following cloning procedure were identical to the control cells expressing beta-galactosidase, when compared for pRB production and phosphorylation in the cell cycle stages characterized by hyperphosphorylated pRB. However, cells of the experimental cell lines hypophosphorylated pRB much faster and accumulated much more underphosphorylated protein compared to the control cell lines. The doubling time of the cells was not affected either by changes in the pRB phosphorylation pattern or by its overproduction during separate cell cycle stages. These results suggest that maintaining of the physiological level of pRB phosphorylation in cycling cells is strictly controlled and is considered to be a more important condition of the cell cycle progression than pRB dephosphorylation.