Figure 3.

Insertion of Form X in a full length plasmid. Plasmid pE10 was cut so as to obtain two fragments, a short 120 bp fragment containing the poly (CA) · poly (TG) tract, and a large 2264 bp fragment. The small fragment was ³²P end labelled and part of it was converted to Form X. After reinsertion of the short fragment by ligation into the large 2264 bp fragment, the original plasmid pE10 was reconstituted either in its regular form, or as a Form X containing plasmid. To compare the linking numbers of both plasmids, they were analyzed by two dimension agarose gel electrophoresis with the first dimension without chloroquine and the second dimension in the presence of 1.3 μM chloroquine. The markers consisted of a series of topoisomers of plasmid pE10 obtained by recircularization of the linear plasmid in the presence of variable amounts of ethidium bromide. After electrophoresis, the gels were first stained with ethidium bromide and photographed to detect the markers, then dried and exposed to detect the radioactivity of the reformed plasmid. a. The experiment was performed with the 120 bp fragment in its regular linear form. b. Same experiment with Form X of the 120 bp fragment, c. Scheme of the different species present on the gels: o.c. open circles; lin. linear fragment; di. dimeric circles; +4 to -7: number of supercoils, positive or negative, in the marker topoisomers which were separated on the gel. Beyond 7 negative supercoils, all topoisomers migrate together under the conditions used. d. Analysis of the products on a 4% polyacrylamide gel, to show that Form X has remained stable after insertion in plasmid pE10. Lanes 1 and 2: pE10 containing Form X or the regular fragment, respectively. Lane 3: pE10 containing Form X was redigested with EcoRI + ClaI, the 120 bp fragment is recovered as Form X. Lane 4: pE10 containing Form X was incubated 2 min at 100°C and redigested with EcoRI + ClaI, Form X is recovered, showing that it is resistant to 100°C when inserted in a circular molecule. Lanes 5 and 6: redigestion of pE10 containing the regular linear form of the 120 bp fragment, without or with previous incubation at 100°C, respectively. The regular form of the 120 bp fragment is recovered, as expected. Lanes 7 and 8: controls showing respectively Form X and the regular 120 bp linear fragment used in these experiments.