Clothespin Structure

Restriction digest analysis of pFOXC2 and pFOXC3 DNAs indicates that the plasmids have a "clothespin" structure located upstream of the plasmid open reading frame (ORF) and noncovalently linked ends with two to five copies of a pentameric repeat (5'-ATCTA) at the other terminus (Walther and Ken-nell 1999). The ability to cut pFOXC2 and pFOXC3 DNAs with restriction endonucleases that require duplexed DNA substrates, together with observa tions of the plasmid's structure by electron microscopy (Kistler and Leong 1986), indicates that the plasmids are double-stranded molecules. The (+) or coding strand terminus downstream of the plasmid ORF has an accessible 3'-hydroxyl (inferred by its ability to serve as a substrate for terminal de-oxynucleotide transferase and sensitivity to 3'^ 5' exonuclease III), whereas the 5' end of the opposing (-) strand appears to be modified in a manner that results in resistance to 5'^ 3' X-exonuclease and the inability to be endlabeled with [y32P]ATP (Walther and Kennell 1999).

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