MOLECULAR ANALYSES OF LINEAR CHLOROPLAST PLASMIDS FROM ERNODESMIS VERTICILLATA

Ernodesmis verticillata contains novel, linear plasmid‐like DNA molecules in its chloroplasts, whose function remains unclear. Their molecular architecture is putatively a “hairpin,” wherein every molecule consists of a long inverted repeat folded back on itself. Thus, each molecule is composed of a terminal (telomeric) domain, a central inverted repeat, and a “loop” domain. Cloning strategies have been devised for characterizing the terminal and loop regions, since they might contain landmark features like replication origins. Polymerase chain reaction (PCR) was used to amplify loop domains of native molecules, and ligation of the PCR products with commercial cloning vectors initially yielded 11 clones. So far, no recognizable sequences have turned up in the loop domains of the molecules. Unlike what has been reported for most linear plasmids, we have been unable to verify that any proteins are associated with either the 5′‐ or 3′‐ends of the Ernodesmis plasmids. In fact, the 5′‐end of each molecule contains a terminal phosphate that is accessible to alkaline phosphatase and subsequently to T4 polynucleotide kinase in vitro. It is also possible to modify the 3'‐end with terminal deoxynucleotidyl transferase (TdT) for homopolymeric tailing. Poly‐(C) tailing of native molecules promotes their annealing to poly‐(G) tailed vectors, for cloning of the terminal domains. An initial library of 14 TdT clones (10 unique) indicates that short (11–28 bp) direct repeats occur near the termini of the plasmids. Shorter (4–6 bp) inverted repeats at the very ends may lead to terminal foldbacks that might serve to protect the termini.