Molecular Genetic and Genomic Approaches to the Study of Medically Important Fungi

Towards a molecular genetic system for the pathogenic fungus Paracoccidioides brasiliensis

“…The development of efficient genetic transformation systems for fungi has been crucial for establishing a link between in vitro analysis of DNA and its in vivo function (Magee et al, 2003). Classical genetic tools as electroporation, protoplasting, and cell permeabilization with lithium acetate were initially developed for the transformation of several non-pathogenic fungi (Ruiz-Diez, 2002).…”

Milestones in Candida albicans gene manipulation

“…Therefore, replacing one copy of URA3 was considered adequate for a reliable experiment. It was around this time that “reconstitution” of a mutant strain by introduction of a wild-type copy of the gene to rescue the mutant phenotype was considered an essential part of a C. albicans experiment (Magee et al, 2003; Brand et al, 2004). …”

“…Similar to C. albicans , gene manipulation in many other clinically important Candida , Cryptococcus and Aspergillus species continues to evolve (Magee et al, 2003; Hull and Heitman, 2002; Brookman and Denning, 2000; Brakhage and Langfelder, 2002). For example, the URA5 gene is a popular marker for gene disruption in Cryptococcus species.…”

“…First, the organism is diploid (Barnett, 2008; Jones et al, 2004) and in most cases both alleles of a gene must be manipulated. Second, compared to the highly studied yeast, Saccharomyces cerevisiae, C. albicans lacks natural plasmids, such as the 2-micron plasmid, for use in transformation and shows lower transformation and recombination frequencies making the manipulation of both alleles of a gene even more challenging (De Backer et al, 2000; Magee et al, 2003). Third, C. albicans shows non-conventional codon usage where the CUG codon is decoded as serine, not leucine (Santos and Tuite, 1995).…”

Validation and clinical application of a molecular method for the identification of Cryptococcus neoformans/Cryptococcus gattii complex DNA in human clinical specimens

“…Improvements in PCR techniques have allowed the detection of minimal amounts of DNA from the C. neoformans species; in addition, PCR can be used in association with other techniques, making it a valuable tool for molecular epidemiology studies. [22][23][24] Several target sequences have been utilized to identify the C. neoformans complex, including URA5, CAP59, M13, and ITS (18S, 5.8S, and 28S). The ITS region of rDNA has been the most frequently used region for the detection of fungal sequences because of its high degree of variation compared to that of other ribosomal DNA regions facilitates identification.…”