Development of molecular tools for cryptococcal research: Cryptococcus neoformans is a basidiomycetous yeast-like pathogen affecting primarily immunocompromised patients particularly those with AIDS. It is phylogenetically remote from ascomycetous fungi and molecular tools successfully used for Saccharomyces cerevisiae or other commonly studied fungi such as Neurospora crassa or Aspergillus nidulans are of limited use in C. neoformans. Furthermore, to obtain high frequency of transformation in C. neoformans, transforming vectors have to be linearized prior to transformation. The tranforming plasmids more often are episomally maintained when selection pressure is exerted. The structural integrity of the episomally maintained transforming plasmid is unpredictable since the ends of the linear plasmid are not protected. Integration into genome occurs randomly. Most currently developed transformation vector contains repeats of telomeric sequences at both ends to protect the plasmid. However, the plasmids are lost once the selection pressure is removed. In the previous year we have reported on the construction of stably maintained episomal shuttle vector which contained 4.5 kb DNA fragment isolated from artificially created, stably maintained minichromosome in C. neoformans. Further tests showed that the plasmid can be transformed without linearization and the 4.5 kb insert allowed the maintenance of two selection markers, ADE2 and URA5 regardless of the media used. Transformation systems in C. neoformans depend on auxotrophic strains because using heterologous expression of the bacterial hygromycin B phosphotransferase gene or any other antibiotic resistance gene as a selection marker have not been successful. Isolation of a strong native promoter to facilitate expression of heterologous genes is therefore essential. We have cloned glyceraldehyde-3-phosphate dehydrogenase gene(GPD) from C. neoformans in order to isolate its promoter. Partial sequence of the GPD gene showed 68.7 % homology with yeast GPD. ofS.c S. cerevisiae and a higher homology with that of the mouse (73.5%). The GPD gene encodes a key glycolytic enzyme which is constitutively expressed and may account for 2-5% of the mRNA and GPD protein constitute 5% of the cellular protein. PCR primers were designed according to the published GPD sequence of Schizophyllum communne, a basidiomycete phylogenetically closer to C. neoformans than other fungal species from which the GPD genes were cloned.
