Although there is much interest in Cryptococcal research, tools for the genetic analysis in C. neoformans are less well developed than in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Since genomic sequencing of C. neoformans has been completed, functional genomics can now be accomplished on a large scale by the forward genetics approach. In order to effectively identify the genes disrupted randomly and to elucidate their functions, a tagging system for the deletion constructs will be needed. There are several tagging systems that have been successfully used in bacteria as well as in fungi which include the transposon or the signature tagging methods (STM). These methods have been less than successful for use in C. neoformans due to the lack of a controlable transposon in C. neoformans as well as the lack of randomness in the STM system. In 2003,we developed the Agrobacterium mediated transformation system (ATMT) in a serotype D strain of C. neoformans. This method was superior to either the electroprative or the biolistic transformation method with respect to both the transformation frequency and the rate of single copy random insertions of the tagged sequence into the geneome. ? During 2004-2005, we applied the ATMT method to all four serotypes of C. neoformans and studied the effect of pyrimidine or purine starvation on the transformation efficacy. Unlike in Saccharomyces cereviciae where adenine mutants transform at a significantly higher rate by ATMT, adenine or uracil auxotophs of C. neoformans did not show any increases in transformation frequency by ATMT. Although the transformation frequencies were higher in pyrimidine auxotrophs of certain strains which indicated that such increases may be strain dependent. During 2005-2006, we have screened 30,000 insertional mutant clones for various identifyable phenotypes such as growth under low oxygen, capsule formation etc.
