A sequence insertion targeting vector for Leishmania enriettii.
We have demonstrated previously that Leishmania enriettii contains the enzymatic machinery to mediate efficient interplasmidic homologous recombination. In this report we show that a sequence insertion targeting vector, pALT-Neo-Tub, can be inserted into the genome of L. enriettii by homologous recombination between alpha-tubulin sequences found in the plasmid and their homologs in the genome. pALT-Neo-Tub, a pBluescript-derived vector containing the neor gene flanked by the alpha-tubulin intergenic and alpha-tubulin coding sequences, was used to transfect cells to G418 resistance. Analysis of the DNA from the drug resistant clones indicates that all of the insertion events are restricted to the alpha-tubulin gene repeats. As little as 200 base pairs of sequence homology between the plasmid and the genome is required for integration. Nonhomologous recombination events are not detected. These results indicate that exogenous DNA sequences can be integrated into the L. enriettii genome provided that they are flanked by homologous DNA sequences.
Tobin
J F
JF
Department of Tropical Public Health, Harvard School of Public Health, Boston, Massachusetts 02115.
Wirth
D F
DF
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D007894:Leishmania mexicana / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014404:Tubulin / Q000235:genetics
D012333:RNA, Messenger; D014404:Tubulin; D004247:DNA