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Fungi are ubiquitous in nature where they reside as free living organisms. Virtually all of the fungi that have been implicated in infections reside as saprobes in nature, except for the normal symbionts of humans, Malasezzia furfur and opportunistic species of Candida. While we are constantly exposed to fungi, healthy immunological competent individuals have a high degree of natural resistance against fungi. This depends on the degree of exposure to the fungal pathogen since most infections in the immunologically competent host are subclinical or asymptomatic when they occur. Characteristics that appear to be common among the emerging fungi that have been implicated in the opportunistic mycoses of humans are as follow: 1. Trauma precedes appearance of the disease or 2. An immunosuppressed state exists in the host.

The purity and integrity of isolated RNA are essential prerequisites for a successful analysis of differential gene expression by DDRT-PCR. Thus extreme care should be taken during all the steps of RNA preparation.

After transformation, recombinant plasmids are routinely checked for the presence of the insert by digesting them with appropriate restriction enzymes. However, this procedure is time-consuming, since an additional day is required for plasmid minipreparation and restriction analysis.

Once proved to be differentially expressed, a cDNA fragment is sequenced to search for homology with other cloned genes and/ or putative gene functions. The sequence of a deoxyribonucleic acid molecule can be determined by chemical (Maxam and Gilbert 1977, 1980) or enzymatic (Sanger et al. 1977) methods. The enzymatic sequencing method is based on the ability of DNA polymerase to extend a primer hybridized to the template to be sequenced until a chain-terminating nucleotide (2’,3’-di-deoxyribonucleoside 5’-triphosphate, ddNTP) is incorporated. Because the ddNTP lacks the necessary 3’-OH group required for chain elongation, the growing oligonucleotide is terminated selectively at G, A, T, or C when a set of four separate reactions, each with a different ddNTP, are performed. When a radionucleotide is included in the reaction, the resulting labeled fragments, with the same origin but ending at a different nucleotide, are visualized by autoradiography after separation by denaturing gel electrophoresis.

After separation on acrylamide gel, differentially expressed bands are identified by autoradiography and cut out from the gel; cDNA samples are then eluted from the dried gel for further analysis. The accuracy of the cut-out depends on proper alignment of the gel and autoradiograph. Several tricks, such as marking the profile of the gel onto the film, making pinholes through the gel and film, or dotting the gel with fluorescent or radioactive dyes, may be used. However, none of them ensure 100 % reliability for accurate cutting of specific bands from the gel. We suggest following the procedure described by Kim et al. (1995) and reported below. This technique is more reliable than others, since guidelines exist on how to overlap the gel and the X-ray film.

The original procedure of DDRT-PCR employed denaturing Polyacrylamide gels, commonly used for DNA sequencing, to separate a large number (50–100) of amplified cDNAs (Fig. 1). Nondenaturing gels can also be used to display differentially expressed bands. However, nondenaturing gels have not replaced denaturing ones, since both incompletely annealed single-stranded cDNAs and heteroduplexes (produced by errors of Taq polymerase) from the same gene may still be resolved as different bands by nondenaturing gel.

Although the general strategy of DDRT-PCR seems to be straightforward, the procedure is technically challenging.

Once reamplified, cDNA fragments can be subcloned directly into a suitable vector and used to transform competent cells of an appropriate Escherichia coli strain. However, if smearing or multiple bands appear in PCR products, it is necessary to purify the reamplified cDNA prior to subcloning. Many commercial kits (l.e., QIAquick Gel extraction Kit, Qiagen) allow rapid purification in only a few steps and are relatively inexpensive.

Northern blotting analysis is the standard procedure used to confirm that a cDNA band isolated from a differential display gel is a true positive and represents a gene differentially expressed in the system under study. Slot blot analysis may also be used for preliminary screening of a large number of putative positive cDNAs, but results need to be reconfirmed on Northern blot.

Histoplasma capsulatum is the causative agent of histoplasmosis, a systemic fungal disease worldwide in occurrence, and the most common respiratory mycotic infection affecting humans and animals (Schwarz 1981). Several fungi, in particular those pathogenic such as H.capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis etc., can assume a filamentous or unicellular morphology in response to changes in the environmental conditions (Maresca and Kobayashi 1989). H.capsulatum grows as mycelia in soil while the yeast phase is the only form present in patients. In laboratory conditions, dimorphism is directly and reversibly controlled by temperature changes. The temperature-induced transition and the events in establishment of infection seem to be intimately correlated. In fact, the temperature works not only as a signal for adaptation through the induction of a heat shock-like phenomenon, but also in triggering the phase transition. The role that the heat shock response plays during the differentiation process and in the adaptation to high temperature in H.capsulatum will be discussed here.