By means of differential hybridization techniques, several yeast-phase-specific DNA sequences were identified in the dimorphic pathogenic fungus Histoplasma capsulatum. A 1.85-kilobase (kb) HindIIl fragment from one genomic clone, yps-3, hybridized to at least three distinct yeast poly(A)+ RNAs of 1.3, 1.05, and 0,95 kb from the virulent strain G217B. These mRNAs were not detected in mycelia. When mycelia from G217B were induced to become yeast by transfer from 25 to 37°C, a process requiring

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... proximately 9 days, expression of yps-3 was detected within 24 h, although not in the initial 2 h following the temperature shift. In contrast, a low-virulence strain (Downs) which completes the transition in approximately 2 weeks failed to express the yps-3 gene during phase transitions. A third isolate, G186B, intermediate in its virulence properties and in the time required for the transition (11 days), expressed a single 1.25-kb mRNA but only at low levels in the yeast phase and only after 3 days following the 25-to-37°C temperature shift. Although yps-3 expression does not appear to be essential for the transformation to the yeast phase, it may facilitate the early adaptive processes which permit the mycelium-to-yeast transition and survival of the yeast phase of H. capsulatum at elevated host temperatures. The phase-specific yps-3 nuclear gene is carried on highly polymorphic restriction fragments in all three strains, suggesting that this probe may provide a sensitive diagnostic tool for the classification of H. capsulatum isolates. Histoplasma capsulatum is a human fungal pathogen which is the etiologic agent of histoplasmosis. H. capsulatum is dimorphic, existing as a mycelium at 25°C and as a yeast at 37°C in infected tissues. The mycelium-to-yeast transition in vitro can be induced by shifting the temperature of incubation from 25 to 37°C. Major alterations in cellular metabolism occur during this transition, as assessed by changes in respiration (14, 23) and in the steady-state levels of many mRNAs and proteins (7, 11). It is thus likely that the dimorphic transition involves selective gene expression which follows an ordered developmental program, including changes in nutritional requirements (22, 24, 25) and adaptation to heat shock (12). The relation between virulence and the phase transition is of special interest. Conversion to the yeast phase is probably required for progressive infection, since agents which inhibit the mycelium-to-yeast conversion, such as p-chloromercuriphenylsulfonic acid, render the virulent H. capsulatum strains nonpathogenic for mice (17). Although the transition to the yeast phase is not required for the survival of H. capsulatum at elevated temperatures (15) , thermal tolerance appears to be a key determinant of pathogenicity for three strains of H. capsulatum which differ in virulence (16). Therefore, it is likely that genes required for early adaptation to elevated temperatures and for the transition to and maintenance of the yeast state may specify important virulence determinants. One way to study such regulatory processes in H. capsulatum is to isolate and analyze phase-specific genes. By means of differential hybridization techniques, we have isolated a series of yeast-phase-specific clones from thermotolerant H. capsulatum G217B. One genomic phage clone, yps-3, was selected for further study. Features of its expression in three strains of H. capsulatum with different levels of * Corresponding author. virulence for mice and different tolerances to elevated temperatures are reported here. MATERIALS AND METHODS Organism and culture conditions. The temperature-sensitive Downs strain of H. capsulatum which has low virulence for mice was isolated at the Washington University School of Medicine and is a part of our permanent culture collection (9). H. capsulatum G186B and G217B are Central and North American isolates which are intermediate and high level, respectively, in terms of their thermotolerances and pathogenic properties, and were obtained from the American Type Culture Collection (4, 5). Cultures were maintained in liquid medium containing 2% glucose and 1% yeast extract at 25 and 37°C for mycelia and yeast, respectively. Isolation of nucleic acids. High-molecular-weight DNA from H. capsulatum strains was prepared by breaking cells under liquid nitrogen with a mortar and pestle as described by Akins and Lambowitz (1). Total-cell RNA was isolated from mycelium, yeast, or transforming cultures by gentle homogenization in UNSET buffer (8 M urea, 0.15 M NaCl, 0.1 M Tris hydrochloride, pH 7.5, 1 mM EDTA, 2% sodium dodecyl sulfate), followed by extraction with phenol-chloroform-isoamyl alcohol. Following precipitation, the RNA was suspended in NSET buffer (UNSET base lacking urea), digested with 100 ,ug of proteinase K per ml for 30 min, reextracted, and precipitated. Polyadenylated RNA was obtained by oligo(dT) cellulose chromotography as described by Ono et al. (18) . Mitochondria from strain G217B were prepared by gentle Braun homogenization in 0.33 M sucrose-1 mM EDTA-0.1% bovine serum albumin followed by isolation on 25 to 50% sucrose step gradients (21, 30). The mitochondrial band was removed and lysed in 100 mM EDTA-200 mM-NaCl-100 mM Tris hydrochloride, pH 8.0, containing 100 ,ug of proteinase K per ml at 37°C. The mitochondrial DNA was 1384 on May 9, 2020 by guest http://iai.asm.org/ Downloaded from