Tag Archives: comet assay

DNA loop domain rearrangements in blast transformed human lymphocytes and lymphoid leukaemic Jurkat T cells

K. Afanasieva1, V. Olefirenko1, A. Martyniak1,
L. Lukash2, A. Sivolob1*

1Taras Shevchenko National University of Kyiv, Ukraine;
2Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv;
*e-mail: sivolob@univ.kiev.ua

Received: 06 April 2020; Accepted: 25 June 2020

Chromatin loops are important elements of both chromatin higher-order structure and transcription regulation system. Our previous works have shown that several features of the loop domain organization could be investigated by single cell gel electrophoresis (the comet assay) using the kinetic approach. In this study we applied this technique to study DNA loop domain organization in lymphoid cells: human lymphocytes, lymphoblasts cultivated during 24 h and 44 h, and T cells of Jurkat cell line. Two features of the loop domain organization were found to depend on the cell functional state. First, DNA fraction in the loops of large sizes (more than ~200 kb) was essentially increased in proliferating (de-differentiated) cells in comparison with terminally differentiated lymphocytes. Second, the linear density of the loops not larger than ~200 kb was decreased in transcriptionally active cells and was increased upon their inactivation.

DNA loops after cell lysis resemble chromatin loops in an intact nucleus

K. S. Afanasieva, V. V. Olefirenko, A. V. Sivolob

ESC Institute of Biology and Medicine, Taras Shevchenko National University of Kyiv, Ukraine;
e-mail: aphon@ukr.net

The comet assay has proved itself to be not only a method of detection of DNA damages at the level of individual cells but also an approach for investigation of spatial organization of DNA loop domains in nucleoids. Usually, those nucleoids are obtained after cell lysis in high-salt buffer (e. g. 2.5 M NaCl) with a detergent: these conditions ensure the removal of cell membranes and most of the chromatin proteins, while supercoiled DNA loop domains remain untouched. In this work, we tested the comet assay applied to nucleoids obtained in low-salt solution (1 M NaCl). These nucleoids keep most of the histones and thus contain the loops resembling the chromatin loops to a greater extent. It was shown that, despite some quantitative differences, the most general features of the kinetics of DNA exit are about the same for nucleoids obtained in high- and low-salt conditions. It can be concluded that the DNA loops in high-salt nucleoids can be efficiently used to investigate the spatial DNA organization in chromatin.

Redistribution of DNA loop domains in human lymphocytes under blast transformation with interleukin 2

K. S. Afanasieva, M. I. Chopei, A. V. Lozovik,
S. R. Rushkovsky, A. V. Sivolob

Educational and Scientific Centre Institute of Biology and Medicine,
Taras Shevchenko National University of Kyiv, Ukraine;
e-mail: aphon@ukr.net

At higher order levels chromatin fibers in interphase nuclei are organized into loop domains. Gene regulatory elements (promoters and enhancers) are often located near the sites of loop attachments. Therefore, loop domains play a key role in regulation of cell transcriptional activity. We investigated the kinetics of DNA loop exit during single cell gel electrophoresis (the comet assay) of nucleoids obtained from two cell types that differ in their synthetic activity – human lymphocytes and lymphoblasts. Lymphocyte activation and transformation into lymphoblasts (blast transformation) was performed with interleukin 2. The results obtained suggest that a rearrangement of the loops occurs after lymphocyte activation. After blast transformation we observed an increase of the amount of loop domains on the surface of nucleoids against a decrease of the inner loop fraction. Therefore, the comet assay can be used for detection of large-scale changes in the cell nucleus that follow changes in cell functional state.

Protein intercalation in DNA as one of main modes of fixation of the most stable chromatin loop domains

М. I. Chopei, K. S. Afanasieva, А. V. Sivolob

Taras Shevchenko National University of Kyiv, Ukraine;
e-mail: aphon@ukr.net; sivolob@univ.kiev.ua

The main mechanism of DNA track formation during comet assay of nucleoids, obtained after removal of cell membranes and most of proteins, is the extension to anode of negatively supercoiled DNA loops attached to proteins, remaining in nucleoid after lysis treatment. The composition of these residual protein structures and the nature of their strong interaction with the loop ends remain poorly studied. In this work we investigated the influence of chloroquine intercalation and denaturation of nucleoid proteins on the efficiency of electrophoretic track formation during comet assay. The results obtained suggest that even gentle protein denaturation is sufficient to reduce considerably the effectiveness of the DNA loop migration due to an increase in the loops size. The same effect was observed under local DNA unwinding upon chloroquine intercalation around the sites of the attachment of DNA to proteins. The topological interaction (protein intercalation into the double helix) between DNA loop ends and nucleoid proteins is discussed.

C(60) fullerene prevents genotoxic effects of doxorubicin in human lymphocytes in vitro

K. S. Afanasieva1, S. V. Prylutska1, A. V. Lozovik1, K. I. Bogutska1, A. V. Sivolob1,
Yu. I. Prylutskyy1, U. Ritter2, P. Scharff2

1Taras Shevchenko National University of Kyiv, Ukraine;
е-mail: prylut@ukr.net;
2Technical University of Ilmenau, Institute of Chemistry
and Biotechnology, Ilmenau, Germany 

The self-ordering of C60  fullerene, doxorubicin and their mixture precipitated from aqueous solutions was investigated using atomic-force microscopy. The results suggest the complexation between the two compounds. The genotoxicity of doxorubicin in complex with C60  fullerene (С60+Dox) was evaluated in vitro with comet assay using human lymphocytes. The obtained results show that the C60  fullerene prevents the toxic effect of Dox in normal cells and, thus, С60+Dox complex might be proposed for biomedical application.