Molecular evolution of a malaria resistance gene (DARC) in primates.

Genes involved in host-pathogen interactions are often strongly affected by positive natural selection. The Duffy antigen, coded by the Duffy antigen receptor for chemokines (DARC) gene, serves as a receptor for Plasmodium vivax in humans and for Plasmodium knowlesi in some nonhuman primates. In the majority of sub-Saharan Africans, a nucleic acid variant in GATA-1 of the gene promoter is responsible for the nonexpression of the Duffy antigen on red blood cells and consequently resistance to invasion by P. vivax. The Duffy antigen also acts as a receptor for chemokines and is expressed in red blood cells and many other tissues of the body. Because of this dual role, we sequenced a ~3,000-bp region encompassing the entire DARC gene as well as part of its 5' and 3' flanking regions in a phylogenetic sample of primates and used statistical methods to evaluate the nature of selection pressures acting on the gene during its evolution. We analyzed both coding and regulatory regions of the DARC gene. The regulatory analysis showed accelerated rates of substitution at several sites near known motifs. Our tests of positive selection in the coding region using maximum likelihood by branch sites and maximum likelihood by codon sites did not yield statistically significant evidence for the action of positive selection. However, the maximum likelihood test in which the gene was subdivided into different structural regions showed that the known binding region for P. vivax/P. knowlesi is under very different selective pressures than the remainder of the gene. In fact, most of the gene appears to be under strong purifying selection, but this is not evident in the binding region. We suggest that the binding region is under the influence of two opposing selective pressures, positive selection possibly exerted by the parasite and purifying selection exerted by chemokines.

A screen for deficiencies in GPI-anchorage of wall glycoproteins in yeast.

Many of the genes and enzymes critical for assembly and biogenesis of yeast cell walls remain unidentified or poorly characterized. Therefore, we designed a high throughput genomic screen for defects in anchoring of GPI-cell wall proteins (GPI-CWPs), based on quantification of a secreted GFP-Sag1p fusion protein. Saccharomyces cerevisiae diploid deletion strains were transformed with a plasmid expressing the fusion protein under a GPD promoter, then GFP fluorescence was determined in culture supernatants after mid-exponential growth. Variability in the amount of fluorescent marker secreted into the medium was reduced by growth at 18 degrees C in buffered defined medium in the presence of sorbitol. Secondary screens included immunoblotting for GFP, fluorescence emission spectra, cell surface fluorescence, and cell integrity. Of 167 mutants deleted for genes affecting cell wall biogenesis or structure, eight showed consistent hyper-secretion of GFP relative to parental strain BY4743: tdh3 (glyceraldehyde-3-phosphate dehydrogenase), gda1 (guanosine diphosphatase), gpi13 and mcd4 (both ethanolamine phosphate-GPI-transferases), kre5 and kre1 (involved in synthesis of beta1,6 glucan), dcw1(implicated in GPI-CWP cross-linking to cell wall glucan), and cwp1 (a major cell wall protein). In addition, deletion of a number of genes caused decreased secretion of GFP. These results elucidate specific roles for specific genes in cell wall biogenesis, including differentiating among paralogous genes.

Yeast cell adhesion molecules have functional amyloid-forming sequences.

The occurrence of highly conserved amyloid-forming sequences in Candida albicans Als proteins (H. N. Otoo et al., Eukaryot. Cell 7:776-782, 2008) led us to search for similar sequences in other adhesins from C. albicans and Saccharomyces cerevisiae. The beta-aggregation predictor TANGO found highly beta-aggregation-prone sequences in almost all yeast adhesins. These sequences had an unusual amino acid composition: 77% of their residues were beta-branched aliphatic amino acids Ile, Thr, and Val, which is more than 4-fold greater than their prevalence in the S. cerevisiae proteome. High beta-aggregation potential peptides from S. cerevisiae Flo1p and C. albicans Eap1p rapidly formed insoluble amyloids, as determined by Congo red absorbance, thioflavin T fluorescence, and fiber morphology. As examples of the amyloid-forming ability of the native proteins, soluble glycosylphosphatidylinositol (GPI)-less fragments of C. albicans Als5p and S. cerevisiae Muc1p also formed amyloids within a few days under native conditions at nM concentrations. There was also evidence of amyloid formation in vivo: the surfaces of cells expressing wall-bound Als1p, Als5p, Muc1p, or Flo1p were birefringent and bound the fluorescent amyloid-reporting dye thioflavin T. Both of these properties increased upon aggregation of the cells. In addition, amyloid binding dyes strongly inhibited aggregation and flocculation. The results imply that amyloid formation is an intrinsic property of yeast cell adhesion proteins from many gene families and that amyloid formation is an important component of cellular aggregation mediated by these proteins.

Role of Fig2p in agglutination in Saccharomyces cerevisiae.

In W303-derived strains, disruption of FIG2 increased agglutinability of alpha cells, but not a cells, and did not alter expression of alpha-agglutinin, binding of 125I-labeled alpha-agglutinin, or mating efficiency. Fig2p overexpression led to alpha-cell-specfic suppression of agglutinability. These results imply that Fig2p is an indirect masker of the active sites in alpha-agglutinin.