FIG. 8.

PcG repression is not associated with altered DNA supercoiling. Circular episomes produced from the FLP cassettes were subject to electrophoresis through agarose gels supplemented with 2 μg of chloroquine per ml. Under these conditions, more negatively supercoiled DNA has a faster mobility. The gels were Southern blotted and probed with lacZ sequence. (A) Total genomic DNA was prepared from adult fly heads, either immediately following a 1-h heat shock treatment to induce FLP activity (60′) or following 1 h of heat shock plus 2 h of recovery at room temperature (180′). All of the circles produced in the p[bx], p[iab-4], p[Mcp], and p[Abd-B] lines come from PcG-repressed DNA. The control is the 3rd chromosome insertion, which is not PcG repressed. The average linking number is approximately the same for all lanes. The locations of the unexcised DNA remaining in the chromosome (ch) and of nicked circles (nc) are marked with arrows. The average linking number for the sample in the first lane is indicated with an arrowhead (see Materials and Methods). (B) Total genomic DNA was prepared from whole embryos following 1 h of heat shock plus a half-hour of recovery at room temperature to induce FLP activity. This DNA was subjected to chloroquine gel and Southern blot analyses as described above. Circles produced from the p[Mcp] line with wild-type PcG activity are compared to circles produced from p[Mcp] and p[Abd-B] lines, which are null for the Polycomb protein. The loss of PcG repression does not alter the topology of the DNA in these regions.