IDENTIFICATION AND CHARACTERIZATION OF A PHASE-SPECIFIC, NUCLEAR-DNA BINDING-PROTEIN FROM THE DIMORPHIC PATHOGENIC FUNGUS HISTOPLASMA-CAPSULATUM

Genes expressed in the parasitic yeast (Y) phase of the dimorphic fung al pathogen Histoplasma capsulatum which are transcriptionally silent in the mycelial (M) phase have recently been cloned and analyzed. To u nderstand the molecular regulation of genes involved in the transition to and maintenance of the Y phase, the presumptive 5' regulatory regi ons of two Y phase specific genes (yps-3 and yps 21:E-9) were PCR ampl ified as labelled probes to identify nuclear DNA binding proteins whic h may influence phase-specific gene transcription. Protein-DNA interac tions were assessed by Southwestern blot analysis in which sodium dode cyl sulfate-polyacrylamide gel electrophoresis-separated protein extra cts from Y and M phases of the virulent G217B strain of H. capsulatum were visualized by their capability for in situ binding to the labelle d 517-bp (G217B yps-3) or the 395-bp (G217B yps 21:E-9) putative 5' re gulatory regions. A 30-kDa nuclear protein unique to the M-phase extra cts of the highly virulent G217B strain, but absent in the Y phase of the same organism, was identified. In contrast, the low-virulence, the rmal-sensitive Downs strain of H.. capsulatum lacked detectable p30 bi nding activity in either yeast- or mycelial phase extracts, regardless of the source of labelled probe (395-bp G217B yps 21:E-9 probe or 512 -bp HindIII-EcoRI-labelled Downs yps21:E-9), A decanucleotide motif, T CCTT TTTTT, was identified in the upstream regulatory regions of these yps genes, as well as in the putative alpha-tubulin promoter, and was conserved with 70 to 100% homology, This recognition sequence was suf ficient for p30M binding with P-32-labelled ligated oligonucleotides w hen used in the Southwestern assay, These findings describe the first nuclear DNA binding factor identified in H. capsulatum which binds to target sequences in a phase-specific manner, suggesting that p30M may govern aspects of gene transcription in this pathogenic fungus, in whi ch a temperature-sensitive switch influences morphology and virulence.