The proposed studies are aimed at identifying and characterizing the Plasmodium falciparum ribosomal RNA precursor (pre-rRNA) and its transcription unit. The precursor to the 25S and 17S ribosomal RNA species will be identified by in vivo labelling using (32P) orthophosphoric acid or (3H) nucleosides. The fate of the putative precursor will be determined in pulse-chase experiments. Two-dimensional oligonucleotide fingerprint analyses of the T1-resistant fragments, labelled in vivo or in vitro, will determine the relationship of this putative precursor to the 25S and 17S rRNA species. Genomic libraries of plasmodial DNA will be established using the phage lambda L47 DNA or E. coli cosmid cloning vector pJB8. Recombinant molecules, after in vitro packaging, will be used to analyze the ribosomal RNA transcription unit. Southern blot analyses, single and multiple restriction endonuclease digests, BAL 31 digestion, S1 nuclease mapping, nucleic acid sequencing and R loop analyses will be employed to determine the order of transcription of the 25S and 17S rRNAs. Nuclease S1 digestion and DNA sequence analyses of restriction fragments of cloned plasmodial DNA will be used to identify and characterize the initiation and termination regions of the plasmodial rRNA gene.
