The genome of the human malaria parasite Plasmodium falciparum contains 5 classes of large rRNA transcription units. These genes encode the 25S, 17S and 5.8S rRNAs components. However, only one gene class is expressed during the asexual erythrocytic life cycle of the parasite. The plasmodial genome also contains three 5S rRNA genes which are linked. This is the lowest number reported for any organism. The present studies are aimed at determining what transcriptional control mechanims regulate the expression of both the large and 5S rRNA genes. The goals are to identify promoter and enhancer elements associated with these rRNA genes. These studies are aimed at elucidating the mechanism of gene transcription and differentiation, and will assist in identifying specific factors, either cell, host or stage specific, that regulate transcription in plasmodia by their interaction with promoter and/or enhancer regions. It is anticipated that the approach outlined may provide data which will elucidate the strategy of gene switching in plamsodia. If such a mechanism can be identified, it may be possible to halt the growth or development of the parasite within its vertebrate or invertebrate host. The methodology will involve the construction of a series of deletion mutants which lack varying amounts of the upstream non-transcribed spacer region, which contains the control regions. The ability of these mutants to initiate transcription, and successfully transcribe a marker plasmodial rRNA """"""""mini-gene,"""""""" will be determined using a plasmodial in vitro transcription assay system. The development of the system is one of our specific aims. Enhancer activity will be measured by the ability of putative enhancer regions to affect the transcription of the """"""""mini- gene"""""""". Important domains will be identified by DNA """"""""footprint"""""""" analyses to detect binding domains wthin the non-transcribed spacer regions. They will be sequenced to determine their interrelationship with one another and the rRNA promoter regions. These analyses will be undertaken, not only for the cloned large rRNA genes and 5S rRNA genes currently available in the laboratory, but will be expanded to include additional transcription units. Future goals are to identify host, tissue or stage specific factors which play a role in gene differentiation in P. falciparum.
