Figure 3

Role of the 3'-UTR of the HSP83 gene in the temperature-dependent accumulation of transcripts. (A) Schematic representation of a portion of the L. infantum HSP83 gene cluster, showing the two most 5' genes of the cluster. The Sal I+Bgl II genomic fragment was cloned in both orientations into pXcat plasmid to yield constructs pXCATIRs (sense) and pXCATIRas (antisense). Construct pXCAT3H was obtained by insertion of the 3'-UTR of HSP83 gene downstream of the cat ORF (see Materials and methods for cloning details). The position of the L. major DST sequence (5'-DST) [25], providing the miniexon addition site for the cat transcripts, is shown. Both pXCATIRs-transfected (panel B) and pXCATIRas-transfected (panel C) promastigotes were incubated either at 26°C or 37°C for 30 min, 1 h, 2 h, and 4 h; afterwards, RNA was extracted and used to prepare Northern blots that were hybridized with a cat probe. (D) RNA was extracted from pXCAT3H-transfected promastigotes after incubation at 37°C for various time points (0, 2, and 4 h). The Northern blot was hybridized with the cat probe. At the bottom, panel rRNA shows an ethidium bromide staining of the RNA samples loaded on the agarose gel.