Abstract
We have analyzed the effect of base composition at the center of symmetry of inverted repeated DNA sequences on cruciform transitions in supercoiled DNA. For this we have constructed two series of palindromic DNA sequences: one set with differing center and one set with differing center and arm sequences. The F series consists of two 96-base pair perfect inverted repeats which are identical except for the central 10 base pairs which consist of pure AT or GC base pairs. The S series was constructed such that the overall base composition of the inverted repeats was identical but in which the positioning of blocks of AT- and GC-rich sequences varied. The rate of cruciform formation for the inverted repeats in plasmid pUC8 was dramatically influenced by the 8-10 base pairs at the center of the inverted repeat. Inverted repeats with 8-10 AT base pairs in the center were kinetically much more active in cruciform formation than inverted repeats with 8-10 GC base pairs in the center. These experiments show a dominant influence of the center sequences of inverted repeats on the rate of cruciform formation.
Highlights
The center of F1 consisted of 10 G/C base pairs, while that of F2 consisted of 10 A/T base pairs.These two sequences were designed to determine the effect of the center of an inverted repeated sequence onits cruciform transition
The results reported here demonstrate thattherate of cruciform formation is dependent on the base sequence at the center of symmetry
Thisexperiment excludes the possibility that the rateof cruciform formation is defined entirely by sequences either in the inverted repeat or in the plasmid DNA that exclude the 10 bp at the center of symmetry
Summary
A t 37 “C and showed a reduced rate of formation at Plnsmids,Cloning, and BacterialStrains-Thesequences of the. Decruciforming Treatment-We have shown previously that when a 66-bp loc operator cruciformwas heated above the calculated T,,o,f the fragment and quickly cooledthat most of the pre-existingcruciforms were converted backto the linear form [9] We have used this procedure to remove cruciforms quickly and without the use of the ethidium bromide treatment used previously. The tubes were quickly dropped into a dry ice-ethanol bath (about -76 "C) and left there for 30 s This procedure removed >95% of pre-existingcruciforms in plasmids pUC8F1 and pUC8F2 at u > -0.061.For the S series at u = -0.046 about 85% of pre-existing cruciforms were removed from pUCBS1,80% removed from pUC8S3aAn,d about 60% from pUC8S2and pUC8S3B. Where comparisons have been madDe,NA decruciformed by this procedure behaves no differently (in subsequent cruciform transition analysis) than DNA decruciformed by winding in positive supercoils, the method we have used previously [4,9]
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