DNA-mediated transformations in C. neoformans, regardless of the methods employed, necessitate the use of effective selection markers. The two most successfully used selection markers have been for the nutritional auxotrophies of uracil (URA5) and adenine (ADE2). However, auxotrophic mutants which could serve as hosts are not always available. In order to overcome such a requirement, it would be necessary to express a dominant selectable drug maker (such as hygromycin) in C. neoformans. To achieve such expression, we decided to make use of endogenous promoter from a constitutively expressed native gene. In the past year we have cloned and characterized the native glyceraldehyde-3-phosphate dehydrogenase (GPD) gene and identified its promotor region. Plasmid vectors were constructed for use in transformation where this region allowed expression of this heterologous E. coli hygromycin B resistance gene in C. neoformans. In order to improve efficiency of expression of this heterologous E. coli gene in C. neoformans. In order to improve efficiency of expression of this heterologous gene, its translational terminator sequence was replaced with a sequence from the capsule gene CAP59 from serotype D strain of C. neoformans.
