The effect of location and direction of an episomal gene on the restoration of a phenotype by functional complementation in Leishmania.
The feasibility of genetic manipulation in trypanosomatids has allowed the introduction of molecular approaches for the investigation of gene function. The development of cosmids that carry approximately 40 kb of insert and can be easily introduced in trypanosomatids greatly increased the possibility of gene rescue by functional complementation in these parasites. Although functional complementation is widely used, some of its aspects, such as differential levels of expression along the insert clone, are not clear. We have used the DHFRTS gene as a tool to better understand the mechanisms of transcription of genes present in episomes and the results obtained via functional complementation in Leishmania. This gene was chosen not only because its inactivation in the parasite generates an easily recoverable phenotype, auxotrophy for thymidine (TdR), but also because null mutants are already available. The null mutant available contained two resistance markers (neomycin phosphotransferase-NEO and hygromycin phosphotransferase-HYG), and the loss of heterozygosity (LOH) was induced to recover clones sensitive to hygromycin B, which were necessary for the rescue of transfectants. Analyses of the Leishmania clones confirmed the loss of the HYG gene associated with unanticipated genomic rearrangements. A LOH clone was transfected with cosmids containing the DHFRTS gene in several distinct contexts in order to evaluate the levels of expression of the complementing gene. Results presented here show that the lost phenotype is rescued, irrespective to the DHFRTS location or direction of transcription indicating that functional complementation can be achieved without concern for the position of the complementing gene in a cosmid.
Pedrosa
André L
AL
Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, Monte Alegre, SP, Brazil.
Cruz
Angela K
AK
D000818:Animals; D003360:Cosmids; D005816:Genetic Complementation Test; D007891:Leishmania / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D019656:Loss of Heterozygosity; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D009097:Multienzyme Complexes; C056930:thymidylate synthase-dihydrofolate reductase; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase