Our lab has developed a DNA vector, pR-NEO, and methods which allow the reproducible stable introduction of this DNA into the human parasite Leishmania major following selection with the aminoglycoside G418, a process termed transfection. Preliminary data show that this DNA exists extra-chromosomally, replicates, expresses a correctly processed mRNA bearing the mini-exon sequence attached to a hybrid gene in which the coding region of the normal DHFR-TS mRNA is replaced by the coding region of neomycin phosphotransferase gene, and expresses this enzyme at a level sufficient to confer resistance to the drug G418 (an aminoglycoside related to neomycin). These features permit the isolation of transfected cells, in liquid culture of on plates. Moreover, this plasmid construct can increase in copy number in response to increased drug pressure, and can be reisolated from the transfected Leishmania and reintroduced into E. coli for further analysis. These findings allow us to propose to use this DNA to initiate functional genetic studies of Leishmania. Four basic areas of experimentation are proposed: 1) optimization and further development of the methods of transfection, including the development of transient assays. 2) analysis of the fate and structure of the transfected DNAs, and the structure and processing of hybrid RNAs encoded therein. 3) dissection of the pR-NEO vector in an effort to delineate minimal genetic elements such as origins, promoters, processing sites and elements conferring faithful segregation. Attention will be given to testing current molecular models such as the occurrence of poly-cistronic pre-mRNAs expressed from a distant 5' promoter. 4) manipulation of the pR-NEO vector in a way that will allow the expression of passenger DNA molecules, such as cDNAs or genomic DNA segments. This approach may ultimately permit the isolation and functional testing of other Leishmania genes.
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