Development of forward genetics in apicomplexan parasites.  There has been extraordinary progress in the development of genetic tools for Toxoplasma and Plasmodium with the exception of a robust forward genetic strategy.  Our efforts to develop forward genetics have followed two lines of experimentation.  We first generated a panel of temperature-sensitive cell cycle mutants by chemical mutagenesis (Radke et al., 2000), in order to establish the feasibility of generating cell cycle mutants in Toxoplasma.  Secondly, in collaboration with Boris Streipen (U. Georgia) we developed a new complementation strategy that exploits recent advances in plasmid recombination.  Toxoplasma expression libraries have been constructed (genomic or cDNA fragments) where each insert is flanked by lambda phage recombinational sites (att sites).  The integration of library plasmids into the Toxoplasma genome occurs at a high frequency due to enhancer sequences also contained within the vector.  By means of att site recombination, integrated sequences in parasite genomic DNA may be accurately shuttled back into plasmid via a simple overnight procedure (Streipen, White et al., 2001).  A second recombination reaction shuttles the cDNA or genomic inserts back into the original expression vector, and thus, repeated selection to eliminate false positives is a straightforward process.  This model has been extensively tested using a Toxoplasma cDNA library to select for HXGPRT, and as noted above, Cryptosporidium IMPDH and HXGPRT have also been isolated via this approach..  We believe that this system has tremendous potential to uncover novel cell cycle mechanisms of apicomplexan parasites and efforts to complement selected ts-mutants are now underway.