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Initial characterization of the terminal domains of linear plasmid‐like dna's from ernodesmis verticillata (chlorophyta)
Author(s) -
La Claire J.W.,
Wang J.
Publication year - 2003
Publication title -
journal of phycology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.85
H-Index - 127
eISSN - 1529-8817
pISSN - 0022-3646
DOI - 10.1111/j.0022-3646.2003.03906001_90.x
Subject(s) - biology , exonuclease , exonuclease iii , plasmid , microbiology and biotechnology , dna , recombinant dna , molecular cloning , inverted repeat , genetics , biochemistry , genome , peptide sequence , escherichia coli , gene , dna polymerase
Terminal regions of linear plasmid‐like DNAs from the chloroplasts of Ernodesmis verticillata (Kützing) Bøergesen were characterized. The 2.2‐kb molecules are believed to consist of a single strand of DNA folded back on itself in a hairpin configuration. The 5′ end of isolated molecules is capable of being phosphorylated with polynucleotide kinase, and the 3′ end can be homopolymeric tailed with terminal deoxynucleotidyl transferase. Thus, both ends are accessible for further manipulation. Exonuclease III and lambda exonuclease digest native molecules only after heat denaturation and certain cooling regimes, suggesting that conformational changes brought about by de‐ and renaturation make the 3′and 5′ ends (respectively) vulnerable to exonuclease activity. The cloning strategy involved poly‐G tailing of native molecules, and hybridizing them to a poly‐C‐tailed vector. Two rounds of cloning the 3′ ends yielded a total of 14 clones. Molecular sequencing revealed that two distinct clones have extensive terminal inverted repeats (TIRs). Short TIRs are also evident in most clones, possibly indicating that molecular foldbacks protect the 3′ ends. Multiple tandem and direct repeats are also prevalent near the 3′ termini. Despite overall sequence heterogeneity among most clones, a consensus sequence found at the 5′ end of these clones probably has structural and/or functional significance. Pairwise sequence comparisons and the presence of extensive repeats suggest that these molecules may be highly recombinant.