Characterization of repetitive sequence families in mouse heart small polydisperse circular DNAs: age-related studies

Using alkaline denaturation-renaturation, exonuclease III digestion and density gradient centrifugations, we have isolated covalently closed circular DNA (cccDNA) molecules from 1-, 8-, 16-, and 24-month C57BL/6 mouse heart tissues. Electron microscopic analyses demonstrated that all these preparations contained small polydisperse circular DNAs (spcDNAs). spcDNAs showed similar size distributions at all ages, but more discrete size classes and slightly larger circles were observed in the 24-month heart spcDNA preparations. Based upon the final yields of spcDNAs, there appeared to be no age-related changes in the quantity of these circular molecules in vivo. Furthermore, [3H]-pBR322 recovery studies revealed no endogenous factors that might have affected the yield of spcDNAs from young and old tissues. To determine if there were any age-related changes in the quantity of repetitive sequences in spcDNAs, we probed heart spcDNAs with B1, B2, IAP, L1 and satellite sequences of the mouse genome. The hybridization results showed that these sequence families were differentially represented at all ages in spcDNAs. B2 sequences were the highest across all the age groups while L1 sequences were the lowest. The quantity of B1-, B2-, IAP-, and L1-spcDNAs appeared to decrease at 24-months. Satellite sequences appeared to decrease from 1-month to 8-months, but no change beyond 8-months.