MOLECULAR GENETIC ANALYSIS OF PNEUMOCYSTIS

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Our long term goal is to understand ultimately control the pathogenicity of pneumocystis infections in humans. The approach we propose here is to use cloned pc DNA fragments to characterize gene structure and organization, and to isolate DNA probes with which to explore the relationship between pneumocystis of rats and man. A principal tool in such analysis will be chromosomal mapping using pulsed field electrophoresis. We have selected cloning targets based on two criteria, accessibility, and relevance to the long term goal. Initially, we will pursue genes which by virtue of their conservation among eucaryotes, are most readily accessible. The genes encoding ribosomal RNAs and the calmodulin gene will be our primary initial cloning targets. These initial studies will define the basic molecular genetic scheme in pc, and provide the tools needed to initiate molecular genetic characterization of the pathogen both in laboratory rats and in patients. In concert with these experiments, we will isolate a panel of chromosome-specific probes. Such experiments should also yield dispersed repetitive DNA elements, if any are present in the pc genome. Clones of rRNA genes, chromosome markers and repetitive elements will be used as hybridization probes to examine genomic variation among isolates of pc form both rat and human hosts. Finally, we will pursue genes that we anticipate will be more difficult to clone, but are most pertinent to the problem of chemotherapy of pneumocystis infections. These will include genes encoding cation translocation ATPAses, ornithine decarboxylase, and dihydrofolate reductase, The latter two genes will be used as hybridzation probes with which to explore the possible role of genetic variation as a response to drug exposure.