Add time:07/17/2019         Source:sciencedirect.com

Modification of DNA-cytosine by a 5-methyl group is thought to he an important mechanism which regulates the expression of eukaryotic genes. This modification takes place after semiconservative replication. There is very little evidence, if any. that 5MeCyt could be naturally incorporated into mammalian DNA in semiconservative replication. We have clarified the possibility of incorporating 5MedCyd pharmacologically into human leukemic cells in vitro. To this end. we developed a novel small-scale synthesis method for 14C-labeled 5MedCyd starting from commercially available [14C]dThd derivatives. Particular attention was focused upon possible incorporation of radioactive 5MedCyd derivatives into the acid-soluble cellular fraction as well as into nucleic acids and protein in human cells. The results showed that [2-14C]- and [methyl-14C]5MedCyd were incorporated into human leukemic cells to a similar extent. The radioactivity originating from these compounds was incorporated mainly into the acid-soluble pool and nucleic acids. The exact nature of the intracellular radioactive molecules in RNA is not known, but the radioactive label in DNA hydrolyzate co-chromatographed exclusively with thymine. Hence. 5MedCyd is deaminated to thymidinc before incorporating into DNA. This deamination had taken place already (partially) in the culture medium. Human leukemic cells do effectively protect their DNA from incorporation of exogenous 5MedCyd.

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