Aquaporin in Candida: characterization of a functional water channel protein.

The Candida albicans genome database contains one ORF with homology to aquaporins, AQY1. Xenopus oocytes injected with cRNA encoding C. albicans Aqy1p displayed a coefficient of water permeability (P(f)) that was equivalent to the P(f) for oocytes injected with the cRNA of S. cerevisiae Aqy1p. In addition, as seen in Saccharomyces for Aqy1p and Aqy2p, deletion of AQY1 in C. albicans resulted in cells that were less sensitive than wild-type to osmotic shock. In Saccharomyces, aquaporin null cells also have a cell surface that is more hydrophobic. However, unlike Saccharomyces, there was no effect on the cell surface hydrophobicity, flocculation or cell aggregation in aqy1 null C. albicans cells. Perhaps as a result, there was no difference between the virulence of C. albicans wild-type and aqy1 null strains in a murine model for systemic candidiasis.

An adhesin of the yeast pathogen Candida glabrata mediating adherence to human epithelial cells.

Candida glabrata is an important fungal pathogen of humans that is responsible for about 15 percent of mucosal and systemic candidiasis. Candida glabrata adhered avidly to human epithelial cells in culture. By means of a genetic approach and a strategy allowing parallel screening of mutants, it was possible to clone a lectin from a Candida species. Deletion of this adhesin reduced adherence of C. glabrata to human epithelial cells by 95 percent. The adhesin, encoded by the EPA1 gene, is likely a glucan-cross-linked cell-wall protein and binds to host-cell carbohydrate, specifically recognizing asialo-lactosyl-containing carbohydrates.

Efficient homologous and illegitimate recombination in the opportunistic yeast pathogen Candida glabrata.

The opportunistic pathogen Candida glabrata causes significant disease in humans. To develop genetic tools to investigate the pathogenicity of this organism, we have constructed ura3 and his3 auxotrophic strains by deleting the relevant coding regions in a C. glabrata clinical isolate. Linearized plasmids carrying a Saccharomyces cerevisiae URA3 gene efficiently transformed the ura3 auxotroph to prototrophy. Homologous recombination events were observed when the linearized plasmid carried short terminal regions homologous with the chromosome. In contrast, in the absence of any chromosomal homology, the plasmid integrated by illegitimate recombination into random sites in the genome. Sequence analysis of the target sites revealed that for the majority of illegitimate transformants there was no microhomology with the integration site. Approximately 0.25% of the insertions resulted in amino acid auxotrophy, suggesting that insertion was random at a gross level. Sequence analysis suggested that illegitimate recombination is nonrandom at the single-gene level and that the integrating plasmid has a preference for inserting into noncoding regions of the genome. Analysis of the relative numbers of homologous and illegitimate recombination events suggests that C. glabrata possesses efficient systems for both homologous and nonhomologous recombination.

Advances in molecular genetics of Candida albicans and Candida glabrata.

Ten years ago, when molecular genetic methods were being applied vigorously to viruses, bacterial pathogens and eukaryotic parasites, there seemed to be a partial paralysis in applying them to infectious fungi; this state of affairs was more than apparent in the composition of the symposia at the ISHAM conference in 1987. Since then, however, things have changed. The ISHAM conference held in Italy in 1997 was replete with studies utilizing molecular genetic techniques to answer questions related to epidemiology, pathogenesis, drug development and typing. In the symposium Advances in Molecular Genetics of Fungal Pathogens, several new applications of molecular biology to fungal pathogenesis were reviewed. Although the presentations in this symposium covered only a fraction of the molecular methods now being applied to Candida pathogenesis, they nevertheless provided an intriguing view of what is in store for us in the coming years.

FACS-optimized mutants of the green fluorescent protein (GFP).

We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly regulated inducible promoter. We introduced random amino acid (aa) substitutions in the twenty aa flanking the chromophore Ser-Tyr-Gly sequence at aa 65-67. We then used fluorescence-activated cell sorting (FACS) to select variants of GFP that fluoresce between 20-and 35-fold more intensely than wild type (wt), when excited at 488 nm. Sequence analysis reveals three classes of aa substitutions in GFP. All three classes of mutant proteins have highly shifted excitation maxima. In addition, when produced in E. coli, the folding of the mutant proteins is more efficient than folding of wt GFP. These two properties contribute to a greatly increased (100-fold) fluorescence intensity, making the mutants useful for a number of applications.

Conserved and nonconserved functions of the yeast and human TATA-binding proteins.

Although the TATA-binding protein (TBP) is highly conserved throughout the eukaryotic kingdom, human TBP cannot functionally replace yeast TBP for cell viability. To investigate the basis of this species specificity, we examine the in vivo transcriptional activity of human TBP at different classes of yeast promoters. Consistent with previous results, analysis of yeast/human hybrid TBPs indicates that growth defects are not correlated with the ability to promote TATA-dependent polymerase II (Pol II) transcription or to respond to acidic activator proteins. Human TBP partially complements the growth defects of a yeast TBP mutant with altered TATA element-binding specificity, suggesting that it carries out sufficient Pol II function to support viability. However, human TBP does not complement the defects of yeast TBP mutants that are specifically defective in transcription by RNA polymerase III. Three independently isolated derivatives of human TBP that permit yeast cell growth replace arginine 231 with lysine; the corresponding amino acid in yeast TBP (lysine 133) has been implicated in RNA polymerase III transcription. Transcriptional analysis indicates that human TBP functions poorly at promoters recognized by RNA polymerases I and III and at RNA Pol II promoters lacking a conventional TATA element. These observations suggest that species specificity of TBP primarily reflects evolutionarily diverged interactions with TBP-associated factors (TAFs) that are necessary for recruitment to promoters lacking TATA elements.

Regional codon randomization: defining a TATA-binding protein surface required for RNA polymerase III transcription.

The TATA-binding protein (TBP) is required for transcription by all three nuclear RNA polymerases. TBP was subjected to regional codon randomization, a codon-based mutagenesis method that generates complex yet compact protein libraries. Analysis of 186 temperature-sensitive TBP mutants yielded 65 specifically defective in transcription by RNA polymerase III (Pol III). These mutants map to a limited TBP surface that may interact with Tds4, a component of the Pol III transcription factor TFIIIB. Strains that contain the Pol III-defective derivatives have increased amounts of messenger RNA, which suggests that competition among TBP-interacting factors for limiting quantities of TBP determines the ratio of Pol II and Pol III transcription in vivo.

The TATA-binding protein is required for transcription by all three nuclear RNA polymerases in yeast cells.

Using temperature- and proteolytically sensitive derivatives to inactivate the function of the yeast TATA-binding protein (TBP) in vivo, we investigated the requirement of TBP for transcription by the three nuclear RNA polymerases in yeast cells. TBP is required for RNA polymerase II (pol II) transcription from promoters containing conventional TATA elements as well as functionally distinct promoters that lack TATA-like sequences. TBP is also required for transcription of the U6 snRNA and two different tRNA genes mediated by RNA pol III as well as transcription of ribosomal RNA mediated by RNA pol I. For all promoters tested, transcription decreases rapidly and specifically upon inactivation of TBP, strongly suggesting that TBP is directly involved in the transcription process. These observations suggest that TBP is required for transcription of all nuclearly encoded genes in yeast, although distinct molecular mechanisms are probably involved for the three RNA polymerase transcription machineries.

Functional differences between yeast and human TFIID are localized to the highly conserved region.

TFIID, the general transcription factor that binds TATA promoter elements, is highly conserved throughout the eukaryotic kingdom. TFIIDs from different organisms contain C-terminal core domains that are at least 80% identical and display similar biochemical properties. Despite these similarities, yeast cells containing human TFIID instead of the endogenous yeast protein grow extremely poorly. Surprisingly, this functional distinction reflects differences in the core domains, not the divergent N-terminal regions. The N-terminal region is unimportant for the essential function(s) of yeast TFIID because expression of the core domain permits efficient cell growth. Analysis of yeast-human hybrid TFIIDs indicates that several regions within the conserved core account for the phenotypic difference, with some regions being more important than others. This species specificity might reflect differences in DNA-binding properties and/or interactions with activator proteins or other components of the RNA polymerase II transcription machinery.

RNA structure, not sequence, determines the 5' splice-site specificity of a group I intron.

The group I self-splicing introns act at exon-intron junctions without recognizing a particular sequence. In order to understand splice-site selection, we have developed an assay system based on the Tetrahymena ribozyme to allow the study of numerous 5'-splice-site variants. Cleavage at the correct site requires formation of the correct secondary structure and occurs most efficiently within a 3-base-pair window centered on base pair 5 from the bottom of the P1 stem. Within this window the ribozyme recognizes and cleaves at a "wobble" base pair; the base pair above the cleavage site also influences splicing efficiency. The recognition of RNA structure rather than sequence explains the ability of these transposable introns to splice out of a variety of sequence contexts.