In the course of an analysis of the functions and assembly of the cell wall of Candida albicans, we have cloned and characterized a gene, which we designated CSP37 (cell surface protein), encoding a 37-kDa polypeptide which is a membrane-associated protein. Candida albicans is an imperfect fungus capable of causing life-threatening infections in immunocompromised patients as well as a variety of mucosal infections in generally healthy individuals (50). Due to its importance as a human pathogen and the limited number of safe drugs to control deep-rooted infections, many laboratories have undertaken cellular, molecular, and genetic studies to understand the mechanisms governing C. albicans biology. However, the diploid nature of this fungus and the failure to identify a sexual cycle have hampered many classical genetic approaches (59).A number of factors are thought to contribute to the virulence of C. albicans, but their relative importance during pathogenesis remains unclear (14). Potential virulence determinants include the ability to switch from a yeast to a mycelial form (31,62) and also between different colonial morphologies (64), the production of extracellular hydrolytic enzymes (28, 36), synthesis of receptor-ligand molecules required for recognition, and adhesion to the host tissues (7, 8), etc. Since the cell wall is involved in these processes, some groups have attempted to elucidate the mechanism of synthesis of its different constituents, their assembly, and the modifications that occur during the morphological transition. Thus, a number of cell surface antigens have been reported to be preferentially expressed in hyphal or blastoconidial cell morphologies (10,52,66,67), and several genes related to cell wall architecture (assembly and functions) have been cloned. These genes have been identified by various methods, including differential hybridization screening (27), complementation in Saccharomyces cerevisiae (3), cross-hybridization with genes cloned from other organisms (heterologous genes) (20, 65), hybridization with sequence-specific oligonucleotides (9), and PCR amplification (11,12). We have used another approach to clone morphologyspecific C. albicans genes related to the cell wall. We have isolated cDNA clones by screening with polyclonal antibodies raised against isolated cell walls of blastoconidia and mycelial cells (17,61).In this paper, a cDNA clone that reacted with polyclonal antibodies specific for mycelial cell walls was studied. It encodes a novel protein with no significant homology to known sequences and absent from the S. cerevisiae genome. ⌬csp37 null mutants were constructed and subsequently phenotypic analysis and virulence testing were conducted. Location of the protein codified and potential functions are discussed. MATERIALS AND METHODSMicroorganisms and growth conditions. The C. albicans strains used in this study are listed in Table 1. Cells were routinely grown in YPD (2% glucose, 1% yeast extract, 2% Bacto Peptone [Difco, Detroit, Mich.]) with shaking at the selecte...

A complete nucleotide sequence of two ras-related yeast genes (c- rassc -1 and c- rassc -2) isolated from the yeast strain Saccharomyces cerevisiae is reported. They encode predicted polypeptides of 40,000 and 41,000 daltons, respectively. The N-terminal 170 amino acids from both genes show extensive amino acid homology to other ras genes from vertebrates, whereas their C-termini have diverged. These genes should be useful in the elucidation of a normal biological function of ras-related genes in a simple system like yeast.

The Aspergillus oryzae alpha-amylase cDNA was placed under the control of the Saccharomyces cerevisiae actin promoter (pACT1) and introduced into the ribosomal DNA locus of an industrial baker's yeast strain. To obtain a strain eligible for commercial use, we constructed an integrative cassette lacking bacterial DNA sequences but containing the alpha-amylase cDNA and ribosomal DNA sequences to target the integration to this locus. High-copy-number integrants were obtained including a defective TRP1d promoter in the integrative cassette. We selected one transformant, Rib-AMY (CECT10872), in which the multi-integrated sequences were stable even after 200 generations of growth in nonselective medium. This transformant also expressed and secreted high levels of alpha-amylase. Bread made with this strain had a higher volume, lower density, and softer crumbs than bread made with a control strain. The Rib-AMY transformant also was useful in retarding bread firming. This new strain fulfills all the requirements for commercial utilization and should reduce or eliminate the requirement for addition of exogenous alpha-amylase to the flour, reducing allergenic work-related symptoms due to this enzyme.

Adding glucose to Saccharomyces cerevisiae cells growing among nonfermentable carbon sources leads to glucose repression. This process may be resolved into several steps. An early repression response requires any one of the three glucose kinases present in S. cerevisiae (HXK1, HXK2, or GLK1). A late response is only achieved when Hxk2p is present.