Biological Limitations on the Length of Highly Repetitive DNA Sequences that May be Stably Maintained within Plasmid Replicons in Escherichia coli

Abstract

By following a logical set of selection rules, a dodecadeoxyribonucleotide with potentially unique coding properties was designed and synthesized. This dodecamer is palindromic throughout 2/3 of its length, it self–anneals to generate multimers with 4bp single–stranded extensions, and, upon treatment with DNA ligase, it can be covalently joined to any double–stranded DNA molecule previously digested with EcoRI. The presence within our polymerized dodecamer of cleavage sites for endonucleases HaeIII and Bal I facilitated the monitoring of polymerization, ligation, and cloning. The polymerized dodecamer was positioned downstream from the lac UV5 promoter in a frameshift plasmid, pPCφ1, chosen to ensure that a particular translational reading frame would be utilized.

Cloned, synthetic palindromic DNA was stable in vivo, but only when its size did not exceed 120 bp. The apparent lack of viability of cells harboring plasmids with inserts longer than 120 bp appears to be solely attributable to the palindromic character of the DNA. An alternative approach that has shown promise as a way of increasing the cellular content of a highly palindromic sequence is to interrupt the sequences with nonpalindromic DNA. This strategy was used to construct stable plasmids with 30–90 copies of a palindromic sequence that would not otherwise persist in E. coli.

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Gupta, S., Weith, H. & Somerville, R. Biological Limitations on the Length of Highly Repetitive DNA Sequences that May be Stably Maintained within Plasmid Replicons in Escherichia coli. Nat Biotechnol 1, 602–609 (1983) doi:10.1038/nbt0983-602

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