Sequence‐dependent bending in plasmid pUC19

Abstract

The circular permutation assay has been used to characterize sequence-dependent bending in plasmid pUC19 (2686 bp). Linear, permuted sequence isomers of pUC19 exhibit different mobilities in large-pore polyacrylamide gels, suggesting that the plasmid contains two sequence-dependent bends, one located at approximately 806 bp, close to the start site of transcription, and the other at approximately 2617 bp, near the promoter of the ampicillin resistance gene. The mobility patterns are independent of the buffer in which the gels are cast and run, independent of pH over the range 6.5-8.6, and independent of the presence or absence of various monovalent ions added to the buffer, suggesting that the maxima in the mobility patterns correspond to stable bends in the helix backbone, not anisometric flexibility. Although the mobility patterns are unchanged when Ba(++) or Zn(++) ions are added to the buffer, several of the sequence isomers exhibit multiple sharp sub-bands in solutions containing Mg(++) or Ca(++) ions, indicating the presence of multiple conformational isomers. In addition, the mobility maximum near the start site of transcription is shifted by approximately 100 bp in the presence of Ca(++) ions, suggesting that the location of the actual bend center is somewhat variable, depending on the ionic composition of the surrounding medium. The number of maxima observed in the mobility pattern can be increased by adding a bend from another plasmid, such as Litmus 28; the number of maxima can be decreased by removing one of the bends in pUC19 by restriction enzyme digestion and religation. Hence, the number of maxima in the mobility pattern is an accurate indication of the number of stable, sequence-dependent bends present in the plasmid. The combined results indicate that the circular permutation assay in large-pore polyacrylamide gels is a reliable method of detecting and analyzing sequence-dependent bends in kilobase-sized DNA molecules.

Curved DNA molecules migrate anomalously slowly in polyacrylamide gels even at zero gel concentration

“…Since A-tracts are inherently bent [4][5][6], multiple A-tracts that occur approximately in phase with the helix repeat cause macroscopic curvature of the helix backbone. Curved DNA molecules have shorter end-to-end lengths and larger cross-sectional areas than normal DNAs containing the same number of bp, and therefore require require larger pores to migrate through the gel matrix [7][8][9][10]. Hence, curved DNA molecules behave as though they were larger than their true sizes when electrophoresed in polyacrylamide gels [7][8][9][10][11] or solutions containing entangled linear polyacrylamides [12][13][14].…”

“…Curved DNA molecules have shorter end-to-end lengths and larger cross-sectional areas than normal DNAs containing the same number of bp, and therefore require require larger pores to migrate through the gel matrix [7][8][9][10]. Hence, curved DNA molecules behave as though they were larger than their true sizes when electrophoresed in polyacrylamide gels [7][8][9][10][11] or solutions containing entangled linear polyacrylamides [12][13][14]. Recently it has been observed, using CE, that curved DNA molecules migrate anomalously slowly in free solution as well as in polyacrylamide gels [15].…”

Characterization of a novel endoribonuclease capable of degrading c-myc mRNA in vitro.

“…To test samples for endoribonuclease activity, a radiolabelled RNA probe was first generated from the CRD c-myc DNA sequence (nts 1705-1792) contained within a pUC19 plasmid. pUC19 plasmids, which are isolated from E. coli, are 2686 base pairs in length, resistant to ampicillin, and include a multiple cloning site (MCS) (Figure 2) (Stellwagen 2003).…”

“…These plasmids are double-stranded circular DNA molecules, of which >90% is in supercoiled form (Stellwagen 2003). The pUC19 plasmid that was provided already had the CRD c-myc DNA sequence (nts 1705-1792) incorporated into its MCS, just upstream of an EcoR l restriction site at nt 396.…”

“…When these uncut circular DNA structures are run on a 1% agarose gel they normally appear as three bands. The lower bands usually represent various forms of double stranded super-coiled plasmid DNA, whereas the higher bands usually represents relaxed circular, or nicked single-stranded DNA (Stellwagen 2003). The Proteinase K-treated plasmid was successfully digested using the restriction enzyme E coR \, as compared to the uncut control ( Figure 6, lane 2 compared to 3).…”