Differential immunoreactivity for Z-DNA in rat myoblast nuclei during their terminal differentiation

L6 myoblasts in vitro accomplish the process of terminal differentiation from dividing mononucleated cells to quiescent plurinucleated myotubes, synthesizing muscle-specific proteins. They have been tested, using paraformaldehyde and acetic acid fixations and immunocytochemical techniques, for the presence of Z-DNA at different stages: namely after 3, 5 and 8-9 days of culture. The nuclei of the actively dividing 3-day myoblasts were strongly Z-DNA and B-DNA-positive. The inhibition of replication by araC did not diminish the reaction. In the myotubes, the nuclei became Z-DNA-negative but were still B-DNA-positive. In contrast, the nuclei of a non-fusing alpha-amanitin-resistant mutant (Ama102) stayed Z-DNA-positive. These results tend to show that during the process of terminal differentiation Z-DNA either becomes less accessible or is present in undetectable amounts. In circular DNAs, it has been shown that the presence of Z-DNA depends on their negative supercoiling. In addition, the presence of closed superhelical loops of nuclear DNA has been demonstrated in several mammalian cell types; moreover, the density of DNA topological turns in these loops varies during cellular differentiation and malignant transformation. The relationship between these results and ours is discussed.