Leah D. Fletcher, Ph.D.

Dissertation research performed under the direction of Christine C. Dykstra and Richard R. Tidwell

ABSTRACT
Pneumocystis carinii pneumonia (PCP) is a leading-cause of death among AIDS patients in the United States. The lack of a reliable in vitro cultivation system has limited both drug testing and genetic analysis of this pathogen. Our analysis of P. carinii protein-coding genes has revealed a significant A + T codon bias. Polymerase chain reaction (PCR) was utilized to isolate and identify the genes encoding calmodulin, beta-tubulin, DNA polymerase II, and RNA polymerase I, II, III, actin I and centractin from P. carinii. Primer pairs were designed to incorporate P. carinii codon preference and known conserved protein regions from other organisms. This strategy should be used for a large variety of P. carinii genes and assist the comprehensive analysis of the genomic structure of this important pathogen. The complete nucleotide sequences of the P. carinii calmodulin, actin I and centractin genes were determined. Phylogenetic analysis of the evolutionarily conserved calmodulin and actin I genes using the neighbor-joining and protein parsimony methods, confirmed the recent suggestion that P. carinii is more closely related to the fungi. In addition, amino acid homology, bacterial expression of an actin II cDNA clone and Western blotting with an affinity purified anti-centractin antibody indicated that an unusual actin isoform called centractin exists in P. carinii. This research has increased the genomic database on P. carinii and will lead to a better understanding of the basic biology of this unusual organism.