Differential regulation of p53-dependent and -independent proliferating cell nuclear antigen gene transcription by 12 S E1A oncoprotein requires CBP.

The tumor suppressor protein p53 and the adenoviral 12 S E1A oncoprotein are both known to elicit their biological effects mainly by regulating the transcription of important cellular genes. The human proliferating cell nuclear antigen (PCNA) gene is a transcriptional target of both p53 and E1A. We have analyzed the effects of p53 and 12 S E1A, separately as well as together, on PCNA gene transcription. Our results showed that whereas both p53 and 12 S E1A separately activated PCNA transcription, 12 S E1A repressed p53-mediated transcriptional activation. Thus, 12 S E1A uses a dual strategy of transcriptional activation and repression to take control of the cellular PCNA gene regulation. The cyclic AMP-response element in the PCNA core promoter, besides being crucial for basal transcription, synergizes with p53 to activate transcription. The cyclic AMP response element-binding protein (CREB)-binding protein (CBP) is an essential component of both the transcriptional activation and repression by E1A. Our data demonstrate for the first time that E1A can modulate CBP function to activate PCNA transcription, while at the same time repressing p53-mediated activation by disrupting CBP interaction with p53, thereby uncoupling PCNA transcription from the regulatory effects of p53.

Importance of calcium to the regulation of polymorphism in Wangiella (Exophiala) dermatitidis.

Critical steps implicated in the polymorphism of Wangiella dermatitidis were found to be sensitive to calcium ion availability. When grown in a defined, synthetic medium under various pH and temperature conditions, two thresholds of calcium ion concentrations were identified: a lower concentration favouring non-polarized growth leading to multicellular form development and a higher concentration promoting polarized growth characterized by yeast budding or pseudo/true hyphal growth. The phenotypic transition of yeasts to multicellular forms or to hyphae was induced at both 25 and 37 degrees C in the wild-type strain by the addition of calcium to the synthetic medium adjusted to pH 2.5, which was otherwise not conducive to the production of either growth form. However, the calcium additions did not allow maintenance of polarized growth of yeasts or hyphae in a temperature-sensitive, cell-division-cycle mutant (wdcdc2) derived from the same strain and grown at 37 degrees C in the same medium adjusted to either pH 2.5 or 6.5. Instead these conditions allowed only the nonpolarized, multicellular form development associated with this conditional mutant cultured in rich media at the 37 degree C restrictive temperature for yeast bud formation. Results from experiments using the calcium chelator EGTA added to the synthetic medium supported these conclusions at neutral pH with both the wild type and the wdcdc2 mutant cultured at 37 degrees C. The results suggested that during infection different concentrations of calcium may be encountered by W. dermatitidis in different tissues, which might directly regulate its growth and polymorphism and indirectly its virulence depending on host conditions.

Identification of the conserved coding sequences of three chitin synthase genes in Fonsecaea pedrosoi.

Primers having designs based on highly conserved stretches in the deduced amino acid sequences of chitin synthase (CHS) genes were used in PCR reactions to amplify 600 bp and 366 bp products from the genomic DNA of three major causal agents of chromoblastomycosis. Cloning and sequencing of the PCR products of one of these fungi, Fonsecaea pedrosoi, identified three CHS sequences designated as FpCHS1, FpCHS2 and FpCHS3. FpCHS1 and FpCHS2 were homologous to regions of CHS1 and CHS2 of Saccharomyces cerevisiae, and their derived amino acid sequences fell into chitin synthase classes I and II, respectively. FpCHS3 was homologous to a region of the CAL1/CSD2 gene of S. cerevisiae, which codes for the chitin synthase three (Chs3) enzyme in that fungus. Phylogenetic trees constructed using the deduced amino acid sequences of PCR-amplified CHS products from many fungi clustered F. pedrosoi with other dematiaceous fungi, providing new molecular evidence for the genetic relatedness of these organisms. The identification of these CHS genes in F. pedrosoi will facilitate future studies of the functional roles of chitin synthases in the unique in vivo dimorphism exhibited by chromoblastomycotic fungi.

Use of the polymerase chain reaction to identify coding sequences for chitin synthase isozymes in Phialophora verrucosa.

Based on conserved amino-acid regions predicted for the chitin synthases (Chs) of Saccharomyces cerevisiae, two different primer sets were synthesized and used in polymerase chain reactions (PCRs) to amplify 614-bp and 366-bp sequences from genomic DNA of the zoopathogenic fungus Phialophora verrucosa. DNA-sequencing and Southern-blotting analyses of the 614-bp DNA amplification products suggested that portions of two distinct P. verrucosa chitin synthase genes (PvCHS1, PvCHS2), coding for two different zymogenic-type PvChs isozymes, had been identified. The deduced amino-acid sequence of each fell into different Chs classes, namely class I and class II. In addition, the 366-bp DNA segment was shown to code for a conserved region having homology with the CSD2/CAL1 gene of S. cerevisiae, which encodes a nonzymogenic-type enzyme, Chs3, in that fungus. The amino-acid sequence derived from PvCHS3 exhibits 88.2% similarity and 78.4% identity to the same amino-acid region of the S. cerevisiae enzyme. These results provide a critical first step toward investigating the molecular and pathogenic importance of CHS gene regulation in this fungus and for exploring steps leading to Chs function as potential targets for the design of new therapeutic agents.

Calcium regulates in vitro dimorphism in chromoblastomycotic fungi.

Cladosporium carrionii, Fonsecaea pedrosoi and Phialophora verrucosa, the three most important agents of chromoblastomycosis, produced large numbers of sclerotic bodies at 25 degrees C, and greater numbers at 37 degrees C, after inoculation into a defined pH 2.5 medium containing 0.1 mmol l-1 Ca2+. Higher concentrations of Ca2+ reversed this tendency and promoted maintenance of hyphal growth. Addition of the Ca2+ chelator EGTA to the same medium buffered at pH 6.5 also induced sclerotic bodies, but in a more concentration-dependent fashion. EGTA at 0.5-1.0 mmol l-1 induced maximum numbers of sclerotic bodies in Cl. carrionii, whereas 2 and 8 mmol l-1 concentrations were required for the same results with F. pedrosoi and P. verrucosa, respectively. These findings suggest that Ca2+ concentrations in human tissue may play a paramount role in the dimorphic switching between hyphae and sclerotic bodies among chromoblastomycotic agents during infection.

Cell cycle regulation of polymorphism in Wangiella dermatitidis.

Phenotypic switching in Wangiella dermatitidis between polarized growth processes leading to yeast budding or hyphal apical extension and nonpolarized processes leading to isotropically enlarged forms that may become multicellular is a cell-cycle related phenomenon. Exit of yeasts from the budding growth cycle by this polymorphic agent of phaeohyphomycosis can be induced by incubation of wild-type (wt) cells in pH 2.5 medium at 25 degrees C, or of certain temperature-sensitive, cell-division-cycle (cdc) mutants at pH 6.5 at 37 degrees C. Continued incubation under these semirestrictive conditions causes yeasts to form unbudded, multinucleate, multicellular phenotypes that resemble the sclerotic bodies of chromoblastomycotic fungi. However, new results with Ca2+ indicate that at pH 2.5 critical, but low, concentrations of this ion are crucial for regulating multicellular form development, higher concentrations allow maintenance of polarized growth, and that switching between polarized and nonpolarized growth may involve a Ca2+/proton exchange mechanism. Support for this important role for Ca2+ is provided by experiments with EGTA in media buffered against pH change at near neutrality. Under these conditions, withholding Ca2+ with high EGTA concentrations caused yeasts to arrest in a terminal phenotype characterized by the presence of a bud initial that tended not to enlarge. In contrast, lower concentrations of EGTA often induced yeast-to-multicellular-form conversion. Other new results have suggested that even brief culture of yeasts under conditions that arrest their budding growth cycles may induce commitment to phenotypic transitions.(ABSTRACT TRUNCATED AT 250 WORDS)