Development Of Tools For Molecular Study Of Cryptococcus

Niaid Extramural Activities

Investigators

Linked publications & trials

Abstract

Molecular genetic investigations necessitate the availability of diverse tools to introduce desired genetic information into cells and the subsequent ability to detect their expression within the cells. We have in the past developed several plasmid vectors harboring desired genetic information which either integrate into the genome of the host cell or are maintained extrachromosomally. Such approaches employ either the presence of auxotrophic mutations in the host strains or drug selection markers such as hygromycin and cycloheximide which detect the presence of introduced vectors in the host strains. However, strains with spontaneous auxotrophic mutations are not readily available and the variations in the sensitivity of strains to the available drugs used as markers often present technical hurdles. Furthermore, there is a lack of a good reporter system to monitor and assess the fidelity of expression of introduced genes and their components such as promoters etc. Cryptococcus neoformans has the ability to utilize complex sugars such as sucrose, maltose etc as carbon sources by secreting an alpha-glucosidase to degrade the alpha linkages to generate glucose. We are attempting to develop a reporter system for C. neoformans in which the expression of the maltase gene when introduced on an episomal vector in host containing a disrupted copy of the native gene can be colorimetrically assayed using substrates such as X-Glucose. We have cloned and characterized the genomic DNA as well as cDNA of the C. neoformans maltase gene. Plasmid constructs containing the maltase gene disrupted with the ADE2 gene have been made. We intend to disrupt the native copy of the maltase gene of a C. neoformans strain to be subsequently used as the host strain for further investigations. Such a host strain and an episomal plasmid construct will enable us to develop a reliable reporter system which will allow the detection and assays of the desired C. neoformans genes and their regulatory components to further facilitate the molecular genetic analysis of the organism.