The Candida albicans HOG1 gene (HOG1 Ca ) was cloned by functional complementation of the osmosensitive phenotype associated with Saccharomyces cerevisiae hog1⌬ mutants. HOG1 Ca codes for a 377-amino-acid protein, 78% identical to S. cerevisiae Hog1p. A C. albicans hog1 null mutant was found to be sensitive to osmotic stress and failed to accumulate glycerol on high-osmolarity media.Fungi, like other microorganisms, are able to respond to changes in extracellular osmolarity, adjusting their intracellular composition to prevent the dehydration that could impair normal cell growth (4). Recently, different signal transduction pathways involving members of the mitogen-activated protein (MAP) kinase family have been shown to regulate different aspects of cell physiology (2, 7, 10, 18), one of them being responsible for adaptation to high osmolarity in Saccharomyces cerevisiae (5). The HOG1 gene (5) (high-osmolarity glycerol response pathway) encodes a MAP kinase which plays an essential role in osmoadaptation. Candida albicans is a commensal dimorphic pathogenic yeast which under situations that diminish the host immune response is able to colonize the human body and seriously compromise human health (20). Interest in this organism as a model pathogen has increased in view of the relevance of the fungal infections that it causes, especially in industrialized countries (12). The identification of signal transduction pathways in pathogenic fungi is essential for understanding fungal mechanisms of adaptation to a complex and changing environment like the human body and also for identifying potential novel targets in antifungal therapy. In this work, we have addressed the existence of a HOG pathway in C. albicans.A C. albicans gene library (19) was used to screen S. cerevisiae JBY10 (MATa ura3 leu2 his3 trp1 lys2 ade2 hog1-⌬1:: TRP1) (obtained from M. Gustin) by electroporation. Of more than 200,000 transformant clones, 121 were initially selected for an osmoresistant phenotype on 1 M sorbitol-minimal SD medium (0.67% yeast nitrogen base without amino acids, 2% glucose) plates. Three of these positive transformants were further characterized. Sequencing of approximately 1.6 kb from the insert present in one of them, pHOG1c24.2 (Fig. 1), allowed us to identify an open reading frame which encoded a putative protein, CaHog1p, of 377 amino acids (42.3 kDa), with an overall 78% identity (86% similarity) to S. cerevisiae Hog1p, this similarity not being restricted to the 11 MAP kinase subdomains but also evident within the carboxy-terminal nonkinase domain. CaHog1p was 55.7% identical to S. cerevisiae Fus3p, 53.9% identical to Homo sapiens Erk1p, 52% identical to C. albicans Mkc1p, and 48.8% identical to S. cerevisiae Slt2p. A TGY motif, characteristic of hyperosmolarityactivated MAP kinases (6), is found in subdomain VIII in C. albicans Hog1p, similar to Xenopus laevis Mpk2 (21) and mammalian p38 (14) and CSBP kinases (17), which have been shown to complement S. cerevisiae hog1⌬ mutants. HOG1 Ca did not complement S. cerevisi...

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The LUC gene coding for Photinus pyralis firefly luciferase was cloned in different yeast episomal plasmids in order to assess its possibilities as an in vivo reporter gene. Activity of the enzyme in transformed cells in vivo was measured by following light emission and assay conditions optimized in intact cells, with regard to oxygen concentration, temperature, cell concentration in assay mixtures and external ATP concentration. Among the factors tested, light emission was drastically influenced by the external pH in the assay (which resulted in a ten-fold amplification signal) and by substrate permeability. The growth phase of the cells was also important for the level of activity detected. Cloning of firefly luciferase gene under the control of different yeast-regulated promoters (ADH1, GAL1-10) enabled us to measure their strength which correlated well with previously described data. We conclude that firefly luciferase is an adequate gene reporter for the in vivo sensitive determination of gene expression and promoter strength in yeast.

Within the field of Saccharomyces cerevisiae functional genomics, DNA microarrays have become a very useful tool to study genome-wide gene-expression changes under diverse experimental conditions. Here, the design and production of a gene microarray, called the ‘yeast cell wall chip’, specifically tailored to investigate cell wall functions, is described. This array has been validated and shown to be useful to address gene involvement in the regulation of the response to cell wall damage in yeast. The advantages of this tailored gene microarray, which contains 390 genes, in terms of reproducibility, accuracy, versatility and ease of use are reported. Importantly, the microarray design permits the performance of a double hybridization process (two experiments) on the same slide. Cell wall stress leads to the transcriptional activation of a set of genes involved in cell wall remodelling. This response has been shown to be strongly controlled by the MAP kinase (MAPK) Slt2p, but other signalling pathways have also been suggested to be involved in this process. Here, using the tailored microarray, the role of the HOG1 pathway in the regulation of the transcriptional compensatory response to cell wall damage was evaluated by comparing the transcriptional profiles of a hog1 mutant and a wild-type strain in the presence of Congo red. Two genes, YFL014W (HSP12) and YLR414C, were found to be dependent on the Hog1p MAPK for their induction, indicating that an additional level of regulation of cell wall functions is mediated by this MAPK.