Nucleic Acids Research, Vol 25, Issue 18 3681-3686, Copyright © 1997 by Oxford University Press
M Kunnimalaiyaan and BL Nielsen
Using a partially purified replication complex from tobacco chloroplasts,
replication origins have been localized to minimal sequences of 82 (pKN8,
positions 137 683-137 764) and 243 bp (pKN3, positions 130 513-130 755) for
ori A and ori B respectively. Analysis of in vitro replication products by
two-dimensional agarose gel electrophoresis showed simple Y patterns for
single ori sequence- containing clones, indicative of rolling circle
replication. Double Y patterns were observed when a chloroplast DNA
template containing both ori s (pKN9) was tested. Dpn I analysis and
control assays with Escherichia coli DNA polymerase provide a clear method
to distinguish between true replication and DNA repair synthesis. These
controls also support the reliability of this in vitro chloroplast DNA
replication system. EM analysis of in vitro replicated products showed
rolling circle replication intermediates for single ori clones (ori A or
ori B), whereas D loops were observed for a clone (pKN9) containing both
ori s. The minimal ori regions contain sequences which are capable of
forming stem-loop structures with relatively high free energy and other
sequences which interact with specific protein(s) from the chloroplast
replication fraction. Apparently the minimal ori sequences reported here
contain all the necessary elements for support of chloroplast DNA
replication in vitro.
ARTICLES
Fine mapping of replication origins (ori A and ori B) in Nicotiana tabacum chloroplast DNA
Department of Botany and Microbiology, Auburn University, Auburn, AL 36849, USA.
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