Differential display: A whole-organism approach to studying phenol metabolism in Trichosporon cutaneum

Currently, little is known about the mechanisms by which fungi respond to and degrade toxic chemicals. Their responses are defined, in part, by which genes are turned on or off. Trichosporon cutaneum catabolizes a variety of carbon sources including phenol and was selected for this study. In this study, differential display, a genomic based technique, is used to define the response of the filamentous fungus, Trichosporon cutaneum, to the aromatic compound, phenol. Messenger RNA (mRNA) was purified from cells grown in phenol or succinate medium, reverse transcribed using various primers and amplified via polymerase chain reaction (PCR) using arbitrary primers and radioactive nucleotides. PCR products generated from each mRNA population were electrophoresed on denaturing polyacrylamide gels. The population of radioactive species from the two samples was compared and PCR products differing in intensity between the two growth conditions were isolated, reamplified, cloned and sequenced. Northern blot and dot blot analyses enabled confirmation of differences detected. Using this approach, genes defined by sequence similarity as a potential transcription factor and the F subunit of vacuolar ATPase were shown to be upregulated while those genes encoding a basic amino acid permease, KDEL ER lumen retention receptor were shown to be down regulated in cells grown in phenol media. A number of genes of unknown function also were shown to be differentially expressed. In the cases tested, differential expression was confirmed by Northern blot or dot blot analyses.