Abstract
The effect of neighboring AT-rich sequences on the right-handed B to left-handed Z transition was investigated in plasmids. The supercoil stabilized Z-DNA structure in (CG) tracts 36 and 40 base pairs (bp) in length revealed an unexpected conformational aberration at defined C residues proximal to one end (colL) when the inserts were bilaterally flanked by an 80% AT-rich segment (90 bp on one side and 331 bp on the other). The presence of the perturbed Z-conformation required (CG) stretches longer than 32 bp and bilateral flanking by the AT-rich tracts, since plasmids with the (CG) tracts unilaterally flanked had an orthodox Z-structure. The thermodynamics of the negative super-coil-induced transitions were influenced only slightly by the neighboring AT-rich regions. Hence, the nature of Z-conformations in plasmids is markedly influenced by intrinsic structural features of the (Pur-Pyr) tract and by seemingly modest changes in the properties of neighboring sequences over a distance of several helical turns.
Highlights
Z-DNA Distortion Caused by Flanking AT-rich Sequences
No reactivities were detected on the other (CG) tract
No hyper-reactive sites were seen for the (CG) blocks closer to the colR sequence, and the reactivities detected were independent of the symmetry or orientation of the (CG) blocks
Summary
Plasmids-Inserts of varying (CG) lengths were cloned into theBglII site of the vectors pRW1560 and pRW1852. pRW1560 is a pBR322 derivative with an 8-bp’ BglII linker (GAGATCTC)ligated into the filled-in EcoRI site as described [6]. pRW1852 was constructed by ligating an 8-bp BgnI linker (CAGATCTG)into the filled-in XbaI site of pColIRAXba [10] Plasmids-Inserts of varying (CG) lengths were cloned into the. BglII site of the vectors pRW1560 and pRW1852. PRW1560 is a pBR322 derivative with an 8-bp’ BglII linker (GAGATCTC). Ligated into the filled-in EcoRI site as described [6]. PRW1852 was constructed by ligating an 8-bp BgnI linker (CAGATCTG). Into the filled-in XbaI site of pColIRAXba [10] PColIRAXba is a derivative of pBR322 with a deletion of 622 bp and the replacement of the 375-bp EcoRI-BamHI fragment by 421 bp of AT-rich (80%). The correct sequences and lengths of the inserts for all the plasmids were confirmed by sequencing both of the strands using the primer extension method [9,11,12,13]
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