Adaptive regrowth in respiratory deficient strain of yeast Saccharomyces cerevisiae due to deletion of YKU70 gene

Yu. Rymar, S. Rushkovsky, S. Demidov, L. Velykozhon, O. Pronina, B. Morgun
Taras Shevchenko National University of Kyiv, Kyiv; Taras Shevchenko National University of Kyiv, Kyiv; Taras Shevchenko National University of Kyiv, Kyiv; Institute of Plant Physiology and Genetics, National Academy of Sciences of Ukraine, Kyiv; Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Kyiv; Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Kyiv

Abstract


It is known that significant causes of malignant tumors are destabilization of the nuclear genome and mitochondrial dysfunction. Adaptive regrowth in yeast colonies (the appearance of cell subpopulations more adapted to unfavorable conditions under conditions of the death of the original culture) is used as a model of the initial stages of carcinogenesis. To study the features of the formation of adaptive regrowth, a reparationdefective and respiratory-deficient yeast strain of Saccharomyces cerevisiae was created. The thermosensitive mutation in the yku70 gene was used as an inducer of nuclear genome instability (at 37 оC it causes cell cycle arrest due to a reduction of the length of telomeric regions of chromosomes). Damage to the mitochondrial DNA of the ∆yku70 strain led to its respiratory deficiency (petite mutation). The isolated petite mutant ∆yku70 strain was cultured at optimal 28 оC and restrictive 37 оC temperatures, the state of the cell suspension was evaluated by light and fluorescence microscopy, to determine the viability of cells was used the analysis of microcolonies growth. Isolation of adaptive regrowth clones and analysis of their properties by the method of serial dilutions were conducted. To assess the genome stability of selected clones of adaptive regrowth, PCR analysis of the microsatellite sequences YOR267C, SC8132X, SCPTSY7 was conducted. When culturing the petite mutant of the strain ∆yku70 at a restrictive temperature of 37 оC for 7 days, the formation of viable subpopulations was detected, which can overcome the arrest of the cell cycle in the G2 / M phase. Further analysis of the isolated clones of adaptive regrowth showed that they differ in cell survival at restrictive temperature, resistance to UV radiation and the ability to form adaptive regrowth on colonies. In the analysis of microsatellite repeats in adaptive regrowth clones, no manifestations of instability of the studied sequences were detected.

Keywords


Saccharomyces cerevisiae, adaptive regrowth, YKU70, microsatellite instability

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References


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Received: 09.09.2020

Revised: 09.10.2020

Signed for the press: 09.10.2020




DOI: http://dx.doi.org/10.17721/1728_2748.2020.82.53-58

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