DNA synthesis in isolated mitochondria of Paracentrotus lividus
Gadaleta, M.N.; Del-Prete, G.; Nicotra, A.; Saccone, C.
Bollettino della Societa Italiana di Biologia Sperimentale 51(19): 1328-1334
1975
ISSN/ISBN: 0037-8771 PMID: 1220756 Document Number: 91475
Incorporation of 3H-TTP was studied in isolated mitochondrial DNA from P. lividus unfertilized eggs and mesenchymal blastula stage embryos. After preparation the mitochondria were frozen until time of analysis. DNA polymerase activity was studied by incubating mitochondrial protein in a mixture containing 3H-TTP. Examining the kinetics of 3H-TTP incorporation into the insoluble acid material showed that the rate of the reaction remained linear for 10-15 min and then declined reaching a plateau in 30 min. The amount of incorporation was greater in the blastula stage embryos. In vitro DNA synthesis in unfertilized eggs was inhibited 64% by the presence of DNAase and pancreatic RNAase. Ethidium bromide inhibited incorporation 80%. Inhibition by rifampicin, a known inhibitor of transcription, indicated that the reaction observed was related to actual DNA synthesis and not simply repair synthesis. Mercaptoethanol and dithiotreitol, at concentrations of 1 mM, also inhibited DNA synthesis in vitro, 40 and 53%, respectively. Analysis of the product obtained from incubation of the mesenchymal blastula stage mitochondria showed radioactivity in low, medium and high MW sediments. This was another proof that results reflected true DNA synthesis and not just repair synthesis.