Figure 4.

Circular multimers of 5S rDNA and their rolling circle intermediates. Genomic DNA from HEK-293 (human embryonic kidney cells) digested with EcoRI and 'plasmid safe' DNase, was analysed on two-dimensional (2D)gel and hybridized with 5S rDNA probe. A series of spots forming a ladder indicate the presence of extrachromosomal circular multimers of the repeat (A). (B-D) For size analysis of the multimers, HEK-293 DNA digested with HindIII and 'plasmid safe', was mixed with plasmids of 2.7 kb, 7.76 kb and a 15.5 dimer of the latter prior to 2D-gel analysis. The blot was hybridized with 5S rDNA, revealing the circular multimers (B) and then rehybridized with a plasmid probe revealing the linear and the relaxed forms of the plasmids (D). (C) Merging the two autoradiograms verified that the eccDNA was indeed in the size of 5S rDNA multiples (see text). Arrowheads indicate multiples of 5S rDNA, and white arrows indicate plasmid markers. (E-F) Intermediates of rolling circle replication emerge from eccDNA. (E) Genomic DNA from LAN-1 cells, digested with EcoRI and 'plasmid safe', was analysed on 2D gel and hybridized with 5S rDNA probe. Long exposure reveals sigmoid arcs emerging from the 1n and 2n eccDNA (black arrows). Similar sigmoid arcs were found emerging from circular multimers of the 5 kb histone gene cluster of Drosophila (F), and were identified as rolling circle intermediates [17].