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Stress‐induced cruciform formation in a cloned d(CATG)10 sequence.
Author(s) -
Naylor L.H.,
Lilley D.M.,
Sande J.H.
Publication year - 1986
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1986.tb04511.x
Subject(s) - puc19 , pbr322 , cruciform , biology , microbiology and biotechnology , plasmid , dna , stereochemistry , chemistry , genetics , history , archaeology
The synthetic alternating purine‐pyrimidine sequence, d(CATG)10.d(CATG)10, has been cloned into a 2.079‐kb pBR322‐derived plasmid (pLN1) and its conformation studied under torsional stress. The resultant plasmid, pLNc40, is hypersensitive to cleavage by the single strand‐specific nucleases, S1 nuclease and Bal31 nuclease, and to modification by the single strand‐selective reagent, osmium tetroxide. The S1‐hypersensitive site of this plasmid predominates over those previously mapped in pBR322. Site‐specific cleavage of pLNc40 with the resolvase T4 endonuclease VII demonstrates that this alternating purine‐pyrimidine tract selectively forms a cruciform structure when stably integrated into a negatively supercoiled plasmid. Quantitative measurements of the twist change (‐4.3 +/‐ 0.2) and free energy of formation (16.2 +/‐ 0.5 kcal/mol) of this cruciform have been made from two‐dimensional gel electrophoresis experiments, and correspond well with the predicted values of cruciform formation for this sequence. We conclude that cruciform extrusion versus the B‐Z transition is the favoured conformation of this insert under torsional stress.