Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver

• Published in: Journal of the Serbian Chemical Society 2005, Vol.70 (5), p.705-712
• Main Authors: Vujcic, Miroslava, Terzic, Natasa, Ristic-Fira, Aleksandra, Kanazir, Dusan, Ruzdijic, Sabera
• Format: Article
• Language: English
• Subjects: ageing; emsa; glucocorticoid receptor; gre; liver
• ISSN: 0352-5139; 1820-7421
• DOI: 10.2298/JSC0505705V

In order to contribute to the understanding of mechanisms by which regulatory proteins recognize genetic information stored in DNA, analyses of their interaction with specific nucleotides are usually performed. In this study, the electrophoretic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), middle- aged (12-month-old) and aged (24-month-old), with radioactively labelled synthetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradiograms. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p < 0.05). The specificity of the nuclear proteins-GRE interaction was demonstrated by competition experiments with unlabelled GRE. In a supershift assay, using the antibody BuGR2, it was shown that the GR proteins present in nuclear extracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing. . U cilju doprinosa razumevanju mehanizama pomocu kojih regulatorni proteini prepoznaju geneticku informaciju koju nosi DNK, analiziraju se njihove interakcije sa specificnim nukleotidima. U ovom radu je metodom EMSA analizirana interakcija jedarnih proteina iz jetri pacova iz tri starosne grupe (mladi - 3 meseca, srednje doba ? 12 meseci i stari ? 24 meseca) sa sintetickim, radioaktivno obelezenim, oligonukleotidnim analogom GRE. Nivo vezujuce aktivnosti GRE je odredjivan kvantitativno denzitometrijskom autoradiografijom. Rezultati su pokazali da postoji statisticki znacajan pad vrednosti GRE-vezujuce aktivnosti do 78% kod zivotinja srednjeg starosnog doba i do 49 % kod starih zivotinja, u poredjenju sa vrednostima dobijenim za mlade zivotinje (p < 0.05). Specificnost interakcije jedarnih proteina i GRE je odredjena eksperimentima kompeticije sa neobelezenim GRE. Koriscenjem antitela BuGR2 pokazano je da je glukokortikoidni receptor protein koji u jedarnom ekstraktu ima najveci afinitet za GRE probu. Analizirana je stabilnost kompleksa protein-DNK i zakljuceno je da se menja tokom starenja.