We discovered that sequences essential for replication origin function are frequently conserved in sensu stricto Saccharomyces species. Here we use analysis of phylogenetic conservation to identify replication origin sequences throughout the Saccharomyces cerevisiae genome at base pair resolution. Origin activity was confirmed for each of 228 predicted sites-representing 86% of apparent origin regions. This is the first study to determine the genome-wide location of replication origins at a resolution sufficient to identify the sequence elements bound by replication proteins. Our results demonstrate that phylogenetic conservation can be used to identify the origin sequences responsible for replicating a eukaryotic genome.Supplemental material is available at http://www.genesdev.org.

We report the development of a simple model for assessing the ability of the fungal pathogen Candida albicans to invade the chorioallantoic membrane (CAM) of fertilized hens' eggs. Wild-type and mutant strains of C. albicans were inoculated onto CAM surfaces either as a liquid suspension or on a sterile filter disc. Invasion of the membrane led to death of the embryo due to damage of the CAM, which could be examined histologically to show cell distribution and morphology, and by RT-PCR for assessment of patterns of fungal gene expression in vivo. Prophylactic or co-administration of fluconazole with the inoculum protected the embryo from infection. Secretory aspartyl protease (Sap) mutant strains with reported attenuation of virulence were virulent in the CAM model. However, a C. albicans strain with mutations in two transcription factors Efg1 and Cph1 was unable to form hyphae on the CAM or to penetrate it. The chick CAM, therefore, represents an experimentally tractable and inexpensive alternative to rodent or tissue culture-based invasion models, and can be used to investigate fungal pathogenesis and the genetic regulation of infection and membrane penetration of C. albicans.

Phenotypic variability in pathogenic fungi has long been correlated with virulence, but specific genetic and molecular mechanisms are only recently being unraveled. Fungal morphogenesis, reflecting the expression of several regulated genes, and the capacity of the rising forms or phases to cause disease has been focused on at the XIVth Congress of the International Society for Human and Animal Mycology. Three experimental models of pathogenic fungi have been discussed. In Cryptococcus neoformans, phenotypic variability or switching represents controlled and programmed changes rather than random mutations. Evaluated phenotypic traits were the capsular polysaccharide, cell and colony morphology and virulence. In the dimorphic Paracoccidioides brasiliensis, the serine-thiol proteinase from the yeast phase cleaves the main components of the basal membrane, thus being potentially relevant in fungal dissemination. In Candida albicans, relationships between adhesion proteins and those of lymphocytes and neutrophils are related to fungal pathogenicity. Regulation of the directional growth of hyphae and its tropic responses are correlated with the invasive potential of C. albicans.

The essential eukaryote release factor eRF1, encoded by the yeast SUP45 gene, recognizes stop codons during ribosomal translation. SUP45 nonsense alleles are, however, viable due to the establishment of feedback-regulated readthrough of the premature termination codon; reductions in full-length eRF1 promote tRNA-mediated stop codon readthrough, which, in turn, drives partial production of full-length eRF1. A deterministic mathematical model of this eRF1 feedback loop was developed using a staged increase in model complexity. Model predictions matched the experimental observation that strains carrying the mutant SUQ5 tRNA (a weak UAA suppressor) in combination with any of the tested sup45 UAA nonsense alleles exhibit threefold more stop codon readthrough than that of an SUQ5 yeast strain. The model also successfully predicted that eRF1 feedback control in an SUQ5 sup45 UAA mutant would resist, but not completely prevent, imposed changes in eRF1 expression. In these experiments, the introduction of a plasmid-borne SUQ5 copy into a sup45 UAA SUQ5 mutant directed additional readthrough and full-length eRF1 expression, despite feedback. Secondly, induction of additional sup45 UAA mRNA expression in a sup45 UAA SUQ5 strain also directed increased full-length eRF1 expression. The autogenous sup45 control mechanism therefore acts not to precisely control eRF1 expression, but rather as a damping mechanism that only partially resists changes in release factor expression level. The validated model predicts that the degree of feedback damping (i.e., control precision) is proportional to eRF1 affinity for the premature stop codon. The validated model represents an important tool to analyze this and other translational negative feedback loops.