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Unbudded yeast cells from exponentially growing Sporothrix schenckiiwere harvested, selected by filtration and allowed to form buds in basal medium with glucose at pH 7··2. These cells undergo bud formation and cell duplication at 25°°C earlier than at 35°°C. The time of cell duplication at 25°°C was dependent upon the initial cell concentration. These cells carried out RNA and protein synthesis before cell evagination as early as 10 min after inoculation, and DNA synthesis during the 6 to 9 h period after inoculation, prior to nuclear division and bud formation. Nuclear division and bud formation seemed to occur concomitantly during the 9 to 12 h period after inoculation and were inhibited by the addition of hydroxyurea (0··1 m) to the medium. The preferred site for bud formation was the pole opposite the birth scar. Septum formation was evidenced at the mother cell-bud junction. A light septum first appeared in medium sized buds followed by a dark septum in large buds.

Sporothrix schenckiiconidia were induced to germinate in varying concentrations of added calcium. Calcium had a stimulatory effect on the process and a calcium concentration of 1 mmwas found to be optimal. Measurements of radioactive calcium uptake confirmed that uptake was taking place during the germination of conidia to the mycelial form of the fungus. Three calcium uptake peaks were observed, the first occurred 10 min after inoculation and the other two peaks coincided with the first 2 cycles of DNA synthesis in these cells. Ionophore A23187 and cobalt sulfate inhibited germination of S. schenckiicondidia and altered the normal calcium uptake pattern in these cells. On the other hand, phorbol 12-myristate-13-acetate, a stimulator of protein kinase C (PKC) activity, stimulated germination in medium without calcium. This suggests that PKC activation might account for the germination of conidia in the absence of added calcium. The effects of increasing the calcium concentration on RNA and protein synthesis were also studied. A stimulation of RNA synthesis was found at all times tested with maximum stimulation 6 h after inoculation. A stimulation in protein synthesis was also observed but only after 12 h incubation.

As an initial step in the study of the role of G proteins in signal transduction in Sporothrix schenckii, we identified a Gai subunit using different experimental approaches. Western blots of fungal membrane preparations using anti-Gacommon and anti-Gai1-Gai2 antibodies identified a band of approximately 41 kDa. Pertussis toxincatalyzed adenosine diphosphate (ADP)-ribosylation of these membrane fractions confirmed the presence of a protein substrate of 41 kDa. A 357 bp polymerase chain reaction (PCR) product obtained using fungal DNA as template and primers targeted to conserved Gai sequences, was used as a probe to isolate a clone from an S. schenckii genomic library. A partial sequence for a Gai subunit was obtained from this clone. The sequence was completed using the rapid amplification of cDNA ends (RACE) technique with mycelium and yeast cDNA. The cDNA sequence revealed a 1059 bp open reading frame encoding a 353 amino acid Gai subunit of 41 kDa, more than 90% identical to the CPG-1 of Cryphonectria parasitica, and GNA-1 of Neurospora crassa. The genomic sequence was obtained by PCR using fungal DNA, and revealed a 1250 bp sequence and the presence of three introns. These results provide evidence for the first time of the presence and expression of a Gai homolog in a pathogenic dimorphic fungus.

Sporothrix schenckii conidia were induced to germinate in varying concentrations of added calcium. Calcium had a stimulatory effect on the process and a calcium concentration of 1 mm was found to be optimal. Measurements of radioactive calcium uptake confirmed that uptake was taking place during the germination of conidia to the mycelial form of the fungus. Three calcium uptake peaks were observed, the first occurred 10 min after inoculation and the other two peaks coincided with the first 2 cycles of DNA synthesis in these cells. Ionophore A23187 and cobalt sulfate inhibited germination of S. schenckii condidia and altered the normal calcium uptake pattern in these cells. On the other hand, phorbol 12-myristate-13-acetate, a stimulator of protein kinase C (PKC) activity, stimulated germination in medium without calcium. This suggests that PKC activation might account for the germination of conidia in the absence of added calcium. The effects of increasing the calcium concentration on RNA and protein synthesis were also studied. A stimulation of RNA synthesis was found at all times tested with maximum stimulation 6 h after inoculation. A stimulation in protein synthesis was also observed but only after 12 h incubation.

Calcium ions (Ca2+) have been identified as mediators of proliferative and morphogenetic processes in many eukaryotic cells. The effects of these ions on the cellular and macromolecular processes that accompany the dimorphic transition from the yeast-to-mycelial form of Sporothrix schenckii have been studied. Ca2+ were found to stimulate germ tube formation and growth in these cells at an optimal concentration of 1·0 mm. Studies concerning the effects of this cation on the molecular processes that precede germ tube formation revealed that the earliest molecular event which was stimulated by 1·0 mm Ca2+ was RNA synthesis. An increased incorporation of radioactivity into RNA in the presence of 1·0 mm Ca2+ was first observed at 0–3h, and subsequently at all other times tested, following inoculation. A stimulation in rRNA and tRNA synthesis was detected in the presence of 1·0mm Ca2+. The incorporation of radioactivity into proteins was stimulated 3–5h following induction in the presence of Ca2+ suggesting a specific effect of Ca2+ on protein synthesis. This increased incorporation takes place prior to the start of DNA synthesis. Incorporation of radioactivity into DNA was also stimulated in the presence of Ca2+, 6 and 9 h after inoculation. This stimulation resulted in nuclear division taking place with a shorter lag period and proceeding with increased kinetics. The results reported here are evidence that Ca2+ plays a role in the control of the early molecular and cellular processes that accompany the yeast-to-mycelium transition in S. schenckii and offer an explanation of how Ca2+ can control the expression of the dimorphic potential of this fungus.

Unbudded yeast cells from exponentially growing Sporothrix schenckii were harvested, selected by filtration and allowed to form buds in basal medium with glucose at pH 7·2. These cells undergo bud formation and cell duplication at 25°C earlier than at 35°C. The time of cell duplication at 25°C was dependent upon the initial cell concentration. These cells carried out RNA and protein synthesis before cell evagination as early as 10 min after inoculation, and DNA synthesis during the 6 to 9 h period after inoculation, prior to nuclear division and bud formation. Nuclear division and bud formation seemed to occur concomitantly during the 9 to 12 h period after inoculation and were inhibited by the addition of hydroxyurea (0·1 m) to the medium. The preferred site for bud formation was the pole opposite the birth scar. Septum formation was evidenced at the mother cell-bud junction. A light septum first appeared in medium sized buds followed by a dark septum in large buds.