Nucleic Acids Research, Vol 25, Issue 21 4278-4286, Copyright © 1997 by Oxford University Press
B Papadopoulou and C Dumas
In this study we investigated the role of several parameters governing the
efficiency of gene targeting mediated by homologous recombination in the
protozoan parasite Leishmania. We evaluated the relative targeting
frequencies of different replacement vectors designed to target several
sequences within the parasite genome. We found that a decrease in the
length of homologous sequences <1 kb on one arm of the vector linearly
influences the targeting frequency. No homologous recombination was
detected, however, when the flanking homologous regions were <180 bp. A
requirement for a very high degree of homology between donor and target
sequences was found necessary for efficient gene targeting in Leishmania ,
as targeted recombination was strongly affected by base pair mismatches.
Targeting frequency increased proportionally with copy number of the target
only when the target was part of a linear amplicon, but remained unchanged
when it was present on circles. Different chromosomal locations were found
to be targeted with significantly variable levels of efficiency. Finally,
different strains of the same species showed differences in gene targeting
frequency. Overall, gene targeting mediated by homologous recombination in
Leishmania shares similarities to both the yeast and the mammalian
recombination systems.
ARTICLES
Parameters controlling the rate of gene targeting frequency in the protozoan parasite Leishmania
Centre de Recherche en Infectiologie, Centre Hospitalier de l'Universite Laval and Departement de Microbiologie, Faculte de Medecine, Universite Laval, Quebec, Canada.
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