An approach to functional complementation by introduction of large DNA fragments into Trypanosoma cruzi and Leishmania donovani using a cosmid shuttle vector.
To extend the range of genetic tools available for the functional analysis of trypanosomatid genes we have constructed a cosmid shuttle vector (pcosTL) which facilitates the introduction of large DNA fragments into Trypanosoma cruzi and Leishmania donovani. The vector contains several features to simplify library construction and insert mapping and transformed cells can be selected on the basis of G418 resistance. To evaluate the vector and to determine the fidelity of replication we first constructed cosmid libraries and isolated clones containing the T. cruzi major cysteine protease genes (a tandemly repeated array) and the L. donovani trypanothione reductase gene (a single copy gene). T. cruzi and L. donovani cells transfected with their respective cosmids were characterised by the presence of multiple copies of cosmid DNA and by a considerable over-expression of the corresponding enzyme activity. Rearrangements or deletions of insert sequences were not detected. These findings and the observation that cosmid DNA can be rescued unaltered from transformed parasites suggest that the pcosTL vector will be ideally suited for studies involving functional complementation.
Kelly
J M
JM
Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, UK.
Das
P
P
Tomás
A M
AM
D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D003360:Cosmids; D003546:Cysteine Endopeptidases / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics*; D015723:Gene Library; D017125:Genes, Protozoan; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D007893:Leishmania donovani / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics; D014162:Transfection; D014349:Trypanosoma cruzi / Q000201:enzymology / Q000235:genetics*
D017931:DNA Primers; D016054:DNA, Protozoan; D004274:DNA, Recombinant; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D003546:Cysteine Endopeptidases