Shuttle cosmid vectors for the trypanosomatid parasite Leishmania.
We have developed two shuttle cosmid vectors for the trypanosomatid protozoan parasite Leishmania. Cosmids cLHYG and cLNEO contain hyg and neo markers, conferring resistance to hygromycin B and G418, respectively, replicate extrachromosomally after transfection into promastigotes, and bear a unique BamHI cloning site. To ensure the representation of telomeric sequences, which represent about 5% of the Leishmania genome, random insert DNAs were prepared by shearing followed by blunt-end ligation with BamHI adapters. Representative genomic libraries from Leishmania species representing the four major pathogenic complexes were prepared using cosmid cLHYG. The cosmid libraries were efficiently transfected into Leishmania, and individual cosmids were readily recovered by transformation back into Escherichia coli. The relatively small size of the Leishmania genome (50 Mb) combined with the capacity and transfection efficiency of these cosmid libraries (> 1000 Leishmania transfectants/plate) suggests the feasibility of functional genetic complementation in this parasite.
Ryan
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
Dasgupta
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Beverley
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D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003360:Cosmids; D016054:DNA, Protozoan / Q000235:genetics; D004274:DNA, Recombinant; D015245:Deoxyribonuclease BamHI; D004351:Drug Resistance / Q000235:genetics; D004926:Escherichia coli; D017125:Genes, Protozoan; D005816:Genetic Complementation Test; D005819:Genetic Markers; D015698:Genomic Library; D005839:Gentamicins / Q000494:pharmacology; D006921:Hygromycin B / Q000494:pharmacology; D007891:Leishmania / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D013045:Species Specificity; D016615:Telomere; D014162:Transfection / Q000379:methods*
D016054:DNA, Protozoan; D004274:DNA, Recombinant; D005819:Genetic Markers; D005839:Gentamicins; D006921:Hygromycin B; C010680:antibiotic G 418; D015245:Deoxyribonuclease BamHI