Perfect Palindromic Lac Operator DNA Sequence Exists as a Stable Cruciform Structure in Supercoiled DNA in vitro but not in vivo

A perfect palindromic 66-base pair (bp) DNA sequence derived from the lac operator and cloned into plasmid pMB9 [Betz, J. L. & Sadler, J. R. (1981) Gene 13, 1-12] can exist in a 66-bp linear form or as two 33-bp cruciform arms. The fraction of the sequence in the cruciform depends on the superhe...

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Veröffentlicht in:Proceedings of the National Academy of Sciences - PNAS 1983-04, Vol.80 (7), p.1797-1801
Hauptverfasser: Sinden, Richard R., Broyles, Steven S., Pettijohn, David E.
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container_issue 7
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container_title Proceedings of the National Academy of Sciences - PNAS
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creator Sinden, Richard R.
Broyles, Steven S.
Pettijohn, David E.
description A perfect palindromic 66-base pair (bp) DNA sequence derived from the lac operator and cloned into plasmid pMB9 [Betz, J. L. & Sadler, J. R. (1981) Gene 13, 1-12] can exist in a 66-bp linear form or as two 33-bp cruciform arms. The fraction of the sequence in the cruciform depends on the superhelical density of the plasmid DNA. Relaxed DNA contains no cruciforms. The palindrome in the cruciform structure is cut by EcoRI endonuclease at the base of the cruciform arms, releasing 33-bp fragments; when in the linear form only 66-bp fragments are produced. The cruciform structure is fixed by trimethylpsoralen crosslinks in the cruciform arms. This together with the EcoRI cutting provides an assay for the cruciform structures in the DNA of living cells. Using this assay we show that the cruciform structure rarely if ever exists in vivo, but after DNA isolation >90% of the sequence is in cruciforms. Results suggest that the plasmid DNA as organized in vivo either lacks sufficient torsional tension to form this cruciform or the palindrome is restrained in the linear form by other bound molecules.
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L. &amp; Sadler, J. R. (1981) Gene 13, 1-12] can exist in a 66-bp linear form or as two 33-bp cruciform arms. The fraction of the sequence in the cruciform depends on the superhelical density of the plasmid DNA. Relaxed DNA contains no cruciforms. The palindrome in the cruciform structure is cut by EcoRI endonuclease at the base of the cruciform arms, releasing 33-bp fragments; when in the linear form only 66-bp fragments are produced. The cruciform structure is fixed by trimethylpsoralen crosslinks in the cruciform arms. This together with the EcoRI cutting provides an assay for the cruciform structures in the DNA of living cells. Using this assay we show that the cruciform structure rarely if ever exists in vivo, but after DNA isolation &gt;90% of the sequence is in cruciforms. 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source MEDLINE; JSTOR Archive Collection A-Z Listing; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry
subjects Base Sequence
Bromides
Crosslinking
DNA
DNA, Bacterial
DNA, Superhelical
Electrophoresis
Enzymes
Escherichia coli
Escherichia coli - genetics
Furocoumarins
Lac Operon
Molecular structure
Molecules
Nucleic Acid Conformation
Nucleotide sequences
Operon
Palindromes
Plasmids
title Perfect Palindromic Lac Operator DNA Sequence Exists as a Stable Cruciform Structure in Supercoiled DNA in vitro but not in vivo
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