Cloning and characterization of the suc1 cell cycle control gene from Pneumocystis carinii

Purpose: Perhaps because of its unusual cell cycle, Pneumocystis carinii (PC) has never been successfully cultured in vitro. This has hampered research into therapy for PC pneumonia. PC is now known to be a fungus with a cell cycle controlled by the activity of a serine-threonine kinase, Cdc2. The molecule Suc1 couples with and modulates the activity of Cdc2. In this study, we report the cloning and characterization of the suc1 cell cycle control gene from PC. Methods: Suc1-Sepharose^r beads and anti-Suc1 antibody were used to precipitate Cdc2 kinase from PC lysates and then phosphorylate histone. Degenerate polymerase chain reaction (dPCR) with codon bias was used to generate a partial clone. Then 5′ and 3′ rapid amplification of cDNA ends (RACE) was used to complete the mRNA and predicted protein sequence. Genomic sequence was confirmed by screening PC phage libraries. A synthetic peptide from the amino terminal was used to generate an anti-Suc1 antibody in rabbits. This was used in Western blots and kinase assays. Results: A 700 base-pair gene encodes Suc1, a 114 amino acid protein of 14 kD weight. Four introns share conserved splicing recognition sites of other PC genes. Like other PC genes, it is A+T rich. Suc1 binds to Cdc2. Conclusions: The PC suc1 has been cloned and its function demonstrated. Clinical Implications: This gene gives us further insight into the cell cycle of PC. This should help us in culturing the organism and developing therapies for PC pneumonia.