Many mRNAs in trypanosomes bear an identical 35 base sequence on their 5' termini. The sequences encoding this 35 base RNA leader element (LE) are not linked to the sequences encoding the mRNA bodies. Two mechanisms have been forwarded to explain these observations: 1) the LE is required as a primer to transcribe most trypanosome genes; and 2) the LE is joined to the rest of the mRNA in an intermolecular splicing reaction. We will study nascent transcripts of cloned trypanosome genes which utilize the 35 base LE by S1 nuclease protection, RNAse H digestion and RNase T1 fingerprinting to discriminate between the two models of LE joining. Furthermore, we will attempt to define the biological significance of the LE by defining its distribution among mRNA and non-mRNA transcripts and by identifying and comparing trypanosome proteins encoded by LE-bearing and non-bearing mRNAs. Finally, because DNA transformation systems have not been forthcoming for trypanosomes, in vitro transcription and RNA processing (LE-joining) systems will be developed from trypanosome extracts. Cloned trypanosome genes will be modified in vitro and fed into these systems to identify DNA or RNA sequences that are important for transcription or LE-joining. Protein factors that are required for the joining reaction will be partially isolated from the cell extracts and characterized.
| Lu, H Y; Buck, G A (1991) Expression of an exogenous gene in Trypanosoma cruzi epimastigotes. Mol Biochem Parasitol 44:109-14 |
| Zwierzynski, T A; Buck, G A (1990) In vitro capping in Trypanosoma cruzi identifies and shows specificity for the spliced leader RNA and U-RNAs. Nucleic Acids Res 18:4197-206 |
