More than half of Acquired Immunodeficiency Syndrome (AIDS) patients contract Pneumocystis carinii pneumonia (PCP) and 40- 50% die of it. Prior to the advent of AIDS, PCP occurred only rarely in malnourished or immunosuppressed individuals and responded will to chemotherapy. As a result, P. carinii has been little studied and little is known of its basic biology. The long range objective of this project is to study the molecular biology and genetics of this parasite with the identification of new therapeutic approaches as the ultimate aim. Specifically, we will construct genomic and cDNA libraries from RNA and DNA isolated from rat-grown and/or tissue culture-grown parasites. We will screen these libraries for genes from each of the three major classes; i.e., the 28S, 18S, rRNA gene cluster (Class I); protein encoding genes (Class II), and the 5S rRNA gene (Class III). The structures of these genes will be examined, their promoters identified, RNA processing mechanisms defined and the polymerases responsible for their transcription characterized. Major cytoplasmic, membrane, and stage specific proteins will also be identified and their genes cloned and characterized. Selected stage-specific and membrane proteins will be expressed in E. coli and antibodies will be induced and used to probe the locations and functions of the proteins. Antibodies will also be used in attempts to block attachment and cytopathic effect of P. carinii in in vitro culture. Different isolates of P. carinii will be examined by pulse-field gradient electrophoresis (PFGE) in epidemiological studies. Pre-existing human clinical material will also be examined by PFGE and chromosomes will be visualized by Southern Hybridization using recombinant gcDNA containing highly repeated P. carinii DNA as probe. Finally, the highly repeated sequences and the rDNA clones will be assessed for specifically and sensitivity as potential diagnostic tools.
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