title: A dual luciferase system for analysis of post-transcriptional regulation of gene expression in Leishmania.
abstract: Gene expression in kinetoplastid parasites is regulated via post-transcriptional mechanisms that modulate mRNA turnover, translation rate, and/or post-translational protein stability. To facilitate the analysis of post-transcriptional regulation, a dual luciferase system was developed in which firefly and Renilla luciferase reporters genetically fused to compatible drug resistance genes are integrated in place of one allele of the gene of interest and of an internal control gene, respectively, in a manner that preserves the cognate pre-mRNA processing signals. The sensitivity and reproducibility of the assay coupled with the ability to rapidly assemble reporter integration constructs render the dual luciferase system suitable for analysis of multiple candidates derived from global expression analysis platforms. To demonstrate the utility of the system, regulation of three genes in response to purine starvation was examined in Leishmania donovani promastigotes. This dual luciferase system should be directly applicable to the analysis of post-transcriptional regulation in other kinetoplastids.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239, United States.
authors.forename: Radika
authors.initials: R
authors.lastname: Soysa
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239, United States.
authors.forename: Nicola S
authors.initials: NS
authors.lastname: Carter
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239, United States. Electronic address: yatesp@ohsu.edu.
authors.forename: Phillip A
authors.initials: PA
authors.lastname: Yates
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D007891:Leishmania / Q000235:genetics* / Q000378:metabolism; D049409:Luciferases, Firefly / Q000235:genetics* / Q000378:metabolism; D049410:Luciferases, Renilla / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D049410:Luciferases, Renilla; D049409:Luciferases, Firefly
keywords: Firefly luciferase; Gene regulation; Leishmania; Nutrient stress response; Renilla luciferase; Translational regulation