Figure 5.

Enrichment of plasmids in their cohesed form from metaphase cells by velocity sedimentation: test of the topological model for cohesion. (A) The experimental regimen for enriching metaphase cells from the appropriate [cir⁰] host strain harboring pSG6 and going through the cell cycle in glucose or galactose is schematically indicated. At 45 min for the cell cycle in glucose and at 75 min for that in galactose, the predominant population consisted of large budded cells with a single DAPI staining mass at the bud neck. (B) The sedimentation patterns of pSG6 in 12.5–37.5% sucrose gradient were followed under conditions where the plasmid-borne CEN alone or STB alone or neither of the two was active. Samples were run in agarose gels in the cold (4°C) and probed by pSG6-specific radio-labeled DNA. C, cohesed plasmids; NC, non-cohesed plasmids. (C) Representative fast (F), intermediate (I) and slow (S) sedimenting fractions from the gradient were reanalyzed by agarose gel electrophoresis with or without EcoRI digestion, followed by SDS treatment. For reference, S fractions treated or untreated with EcoRI but without subsequent SDS addition (left panel) and DNA prepared form the lysate by phenol extraction and ethanol precipitation (rightmost lane) were included in the run. (D and E) Representative fractions from the sucrose gradient (fast, slow and intermediate) were treated with EcoRI (D) or with TEV protease (E), and subjected to agarose gel electrophoresis under native conditions.