Molecular biological approaches have contributed significantly towards understanding the biology and genomic structure of the family or organisms known as Pneumocystis carinii. One of the more dramatic findings was the identification of a large gene family that encodes at least 100 proteins expressed on the surface of all forms of the organism, the Major Surface Glycoproteins (MSG) (also called gpA). These genes are tandemly arrayed at the ends of each of the 13-17 linear chromosomes of P. carinii and regulation of gene copies is mediated by a single subtelomeric expression site. The MSG proteins play a role in organism adherence to host cells and are recognized by the host immune system. Dr. Wakefield and colleagues have shown that protease genes (PRT1) are associated with tandem arrays of MSG genes in subtelomeric regions of the chromosomes. These protease genes are present in multiple copies and encode subtilisin-like serine proteases. The protease proteins are also expressed on the surface of the P. carinii organisms. A third gene family, encoding the major surface glycoproteins related antigens (MSR), has also been identified in the subtelomeric regions. The Pneumocystis Genome Project was initiated in April of 1999 and will physically map and sequence the genomes of Pneumocystis carinii from rats and humans. A pWEB cosmid library used for the Project contains approximately 8X coverage of the genome size with an average insert size of approximately 38kb. Clones containing MSG are in approximately 2 per cent abundance, those containing PRT1 genes in 2 per cent, both less than the expected 8 to 10 percent. Thus, the pWEB cosmid library is under-represented in subtelomeric sequences and specialized techniques will be needed to clone these regions. The goals of the present proposal are to generate clones of the P. carinii chromosome ends, essential for completion of the genome project and for the analysis of the structure of the ends and identification of the gene families therein. These regions will be cloned as yeast artificial chromosomes (YACs) using transformation-associated recombination (TAR) in Saccharomyces cerevisiae. Linear YACs will be converted to circular cosmid-sized plasmids and transferred to E. coli for characterization. Determination of the structural linkages of these regions will be used to evaluate the heterogeneity of this gene family and its differential expression. Execution of these studies will likely lead to identification of novel genetic systems used by these lower eukaryotes; aid in our understanding of the evolution of these phylogenetically unique microbes; and suggest possible interdiction strategies for their control.
