Sequence specific visualization of DNA in live mammalian cells

Abstract

In the first part of this work, the fate of plasmid DNA labeled with PNA was investigated in living cells. It was shown that foreign DNA rapidly undergoes interaction with intranuclear structural sites that strongly reduce its mobility and restrict the DNA to regions excluding nucleoli and nuclear bodies as PML bodies. The labeled plasmids partially colocalize with SAF-A, a well characterized marker of the nuclear matrix, and are resistant towards extraction by detergent and, in part, elevated salt concentration. Importantly the localization and low mobility are independent of plasmid sequence and do not require the presence of either a S/MAR DNA element or a functional promoter. In the second part of this work, the attention was focused on chromatin mobility in living mammalian cells by using the lac operator/repressor system. The experiments show that the labeled chromatin loci are highly dynamic, moving around by random walk, but are locally constrained in a defined nuclear volume. Interestingly, the mobility of the analyzed loci depends on their location and partially on the transcriptional status of the cell.