Molecular and Cellular Biology, April 1999, p. 2895-2902, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Department of Pathology,
Received 25 September 1998/Returned for modification 1 December
1998/Accepted 28 December 1998
The recent advent of gene-targeting techniques in malaria
(Plasmodium) parasites provides the means for introducing
subtle mutations into their genome. Here, we used the TRAP
gene of Plasmodium berghei as a target to
test whether an ends-in strategy, i.e., targeting plasmids of the
insertion type, may be suitable for subtle mutagenesis.
We analyzed the recombinant loci generated by insertion of linear
plasmids containing either base-pair substitutions, insertions, or deletions in their targeting sequence. We show that
plasmid integration occurs via a double-strand gap repair mechanism.
Although sequence heterologies located close (less than 450 bp) to the
initial double-strand break (DSB) were often lost during plasmid
integration, mutations located 600 bp and farther from the DSB were
frequently maintained in the recombinant loci. The short lengths of
gene conversion tracts associated with plasmid integration into
TRAP suggests that an ends-in strategy may be widely
applicable to modify plasmodial genes and perform structure-function
analyses of their important products.
*
Corresponding author. Mailing address: Department of
Pathology, Division of Immunology, New York University Medical Center, 550 First Ave., New York, NY 10016. Phone: (212) 263-5346. Fax: (212)
263-8179. E-mail: menarr01{at}mcrcr6.med.nyu.edu.
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