The Roles of Whole-Genome and Small-Scale Duplications in the Functional Specialization of Saccharomyces cerevisiae Genes

Researchers have long been enthralled with the idea that gene duplication can generate novel functions, crediting this process with great evolutionary importance. Empirical data shows that whole-genome duplications (WGDs) are more likely to be retained than small-scale duplications (SSDs), though their relative contribution to the functional fate of duplicates remains unexplored. Using the map of genetic interactions and the re-sequencing of 27 Saccharomyces cerevisiae genomes evolving for 2,200 generations we show that SSD-duplicates lead to neo-functionalization while WGD-duplicates partition ancestral functions. This conclusion is supported by: (a) SSD-duplicates establish more genetic interactions than singletons and WGD-duplicates; (b) SSD-duplicates copies share more interaction-partners than WGD-duplicates copies; (c) WGD-duplicates interaction partners are more functionally related than SSD-duplicates partners; (d) SSD-duplicates gene copies are more functionally divergent from one another, while keeping more overlapping functions, and diverge in their sub-cellular locations more than WGD-duplicates copies; and (e) SSD-duplicates complement their functions to a greater extent than WGD–duplicates. We propose a novel model that uncovers the complexity of evolution after gene duplication.     

Synergistic repression of anaerobic genes by Mot3 and Rox1 in Saccharomyces cerevisiae

Two groups of anaerobic genes (genes induced in anaerobic cells and repressed in aerobic cells) are negatively regulated by heme, a metabolite present only in aerobic cells. Members of both groups, the hypoxic genes and the DAN/TIR/ERG genes, are jointly repressed under aerobic conditions by two factors. One is Rox1, an HMG protein, and the second, originally designated Rox7, is shown here to be Mot3, a global C2H2 zinc finger regulator. Repression of anaerobic genes results from co-induction of Mot3 and Rox1 in aerobic cells. Repressor synthesis is triggered by heme, which de-represses a mechanism controlling expression of both MOT3 and ROX1 in anaerobic cells; it includes Hap1, Tup1, Ssn6 and a fourth unidentified factor. The constitutive expression of various anaerobic genes in aerobic rox1Δ or mot3Δ cells directly implies that neither factor can repress by itself at endogenous levels and that stringent aerobic repression results from the concerted action of both. Mot3 and Rox1 are not essential components of a single complex, since each can repress independently in the absence of the other, when artificially induced at high levels. Moreover, the two repression mechanisms appear to be distinct: as shown here repression of ANB1 by Rox1 alone requires Tup1–Ssn6, whereas repression by Mot3 does not. Though artificially high levels of either factor can repress well, the absolute efficiency observed in normal cells when both are present—at much lower levels—demonstrates a novel inhibitory synergy. Evidently, expression levels for the two mutually dependent repressors are calibrated to permit a range of variation in basal aerobic expression at different promoters with differing operator site combinations.     

Multiple Elements and Auto-Repression Regulate Rox1, a Repressor of Hypoxic Genes in Saccharomyces Cerevisiae

The ROX1 gene encodes a heme-induced repressor of hypoxic genes in yeast. Using RNA blot analysis and a ROX1/lacZ fusion construct that included the ROX1 upstream region and only the first codon, we discovered that Rox1 represses its own expression. Gel-retardation experiments indicated that Rox1 was capable of binding to its own upstream region. Overexpression of Rox1 from the inducible GAL1 promoter was found to be inhibitory to cell growth. Also, we found that, as reported previously, Hap1 is partially responsible for heme-induction of ROX1, but, in addition, it also may play a role in ROX1 repression in the absence of heme. There is a second repressor of anaerobic ROX1 expression that requires the general repressor Tup1/Ssn6 for its function.     

Functional attributes of the Saccharomyces cerevisiae meiotic recombinase Dmc1

The role of Dmc1 as a meiosis-specific general recombinase was first demonstrated in Saccharomyces cerevisiae. Progress in understanding the biochemical mechanism of ScDmc1 has been hampered by its tendency to form inactive aggregates. We have found that the inclusion of ATP during protein purification prevents Dmc1 aggregation. ScDmc1 so prepared is capable of forming D-loops and responsive to its accessory factors Rad54 and Rdh54. Negative staining electron microscopy and iterative helical real-space reconstruction revealed that the ScDmc1-ssDNA nucleoprotein filament harbors 6.5 protomers per turn with a pitch of ～106 Å. The ScDmc1 purification procedure and companion molecular analyses should facilitate future studies on this recombinase.     

Biosynthesis of Riboflavine in Saccharomyces cerevisiae: the Role of Genes rib1 and rib7

Haploid strains of Saccharomyces cerevisiae with mutations in two different rib genes were constructed. These strains were studied by tetrad analysis and by quantitative determination of accumulation products. The genes rib(1), rib(7), and rib(2) are not linked to each other. rib(1)-rib(7) strains and rib(1)-rib(2) strains exhibit the phenotypic properties of rib(1) strains. rib(7)-rib(2) strains show the phenotypic properties of rib(7) strains. The results support the conclusion that the genes rib(1) and rib(7) code for the first and second enzyme of the riboflavine pathway, respectively.     

The Rox1 repressor of the Saccharomyces cerevisiae hypoxic genes is a specific DNA-binding protein with a high-mobility-group motif.

The ROX1 gene encodes a repressor of the hypoxic functions of the yeast Saccharomyces cerevisiae. The DNA sequence of the gene was determined and found to encode a protein of 368 amino acids. The amino-terminal third of the protein contains a high-mobility-group motif characteristic of DNA-binding proteins. To determine whether the Rox1 repressor bound DNA, the gene was expressed in Escherichia coli cells as a fusion to the maltose-binding protein and this fusion was partially purified by amylose affinity chromatography. By using a gel retardation assay, both the fusion protein and Rox1 itself were found to bind specifically to a synthetic 32-bp DNA containing the hypoxic consensus sequence. We assessed the role of the general repressor Ssn6 in ANB1 repression. An ANB1-lacZ fusion was expressed constitutively in an ssn6 deletion strain, and deletion of the Rox1 binding sites in the ANB1 upstream region did not increase the level of derepression, suggesting that Ssn6 exerts its effect through Rox1. Finally, ROX1 was mapped to yeast chromosome XVI, near the ARO7-OSM2 locus.     

Bioinformatic and Expression Analyses of Genes Mediating Zinc Homeostasis in Nostoc punctiforme

Zinc homeostasis was investigated in Nostoc punctiforme. Cell tolerance to Zn²⁺ over 14 days showed that ZnCl₂ levels above 22 μM significantly reduced cell viability. After 3 days in 22 μM ZnCl₂, ca. 12% of the Zn²⁺ was in an EDTA-resistant component, suggesting an intracellular localization. Zinquin fluorescence was detected within cells exposed to concentrations up to 37 μM relative to 0 μM treatment. Radiolabeled ⁶⁵Zn showed Zn²⁺ uptake increased over a 3-day period, while efflux occurred more rapidly within a 3-h time period. Four putative genes involved in Zn²⁺ uptake and efflux in N. punctiforme were identified: (i) the predicted Co/Zn/Cd cation transporter, putative CDF; (ii) the predicted divalent heavy-metal cation transporter, putative Zip; (iii) the ATPase component and Fe/Zn uptake regulation protein, putative Fur; and (iv) an ABC-type Mn/Zn transport system, putative zinc ZnuC, ZnuABC system component. Quantitative real-time PCR indicated the responsiveness of all four genes to 22 μM ZnCl₂ within 3 h, followed by a reduction to below basal levels after 24 h by putative ZIP, ZnuC, and Fur and a reduction to below basal level after 72 h by putative CDF efflux gene. These results demonstrate differential regulation of zinc transporters over time, indicating a role for them in zinc homeostasis in N. punctiforme.     

Clustering of the Genes for Allantoin Degradation in Saccharomyces cerevisiae

We have shown that allantoin degradation in Saccharomyces cerevisiae proceeds exclusively through the intermediate formation of allantoic acid, urea, and allophanic acid. The number of reactions between allantoic acid and urea, however, remains obscure owing to our inability to isolate a mutant defective in ureidoglycolate hydrolase. Structural genes for the enzymes, allantoinase (dal1) and allantoicase (dal2) are located on chromosome IX promixal to the centromere in the order dal1-dal2-lysl.     

Genetic Order of the Galactose Structural Genes in Saccharomyces cerevisiae

The galactose structural genes of Saccharomyces cerevisiae were ordered by determining the genotypes of mitotic and meiotic recombinants from crosses heterozygous for the three genes. The most probable order is centromere-gal7-gal10-gal1. Nonreciprocal recombination was more frequent than reciprocal exchange, and both mitotic and meiotic co-conversions involving mutant sites in all three genes were observed.     

Roles of the Snf1-Activating Kinases during Nitrogen Limitation and Pseudohyphal Differentiation in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Snf1 protein kinase is important for growth on carbon sources that are less preferred than glucose. When glucose becomes limiting, Snf1 undergoes catalytic activation, which requires phosphorylation of its T-loop threonine (Thr210). Thr210 phosphorylation can be performed by any of three Snf1-activating kinases: Sak1, Tos3, and Elm1. These kinases are redundant in that all three must be eliminated to confer snf1Δ-like growth defects on nonpreferred carbon sources. We previously showed that in addition to glucose signaling, Snf1 also participates in nitrogen signaling and is required for diploid pseudohyphal differentiation, a filamentous-growth response to nitrogen limitation. Here, we addressed the roles of the Snf1-activating kinases in this process. Loss of Sak1 caused a defect in pseudohyphal differentiation, whereas Tos3 and Elm1 were dispensable. Sak1 was also required for increased Thr210 phosphorylation of Snf1 under nitrogen-limiting conditions. Expression of a catalytically hyperactive version of Snf1 restored pseudohyphal differentiation in the sak1Δ/sak1Δ mutant. Thus, while the Snf1-activating kinases exhibit redundancy for growth on nonpreferred carbon sources, the loss of Sak1 alone produced a significant defect in a nitrogen-regulated phenotype, and this defect resulted from deficient Snf1 activation rather than from disruption of another pathway. Our results suggest that Sak1 is involved in nitrogen signaling upstream of Snf1.Snf1 protein kinase of the yeast Saccharomyces cerevisiae belongs to the conserved Snf1/AMP-activated protein kinase (AMPK) family; members of this family play central roles in responses to metabolic stress in eukaryotes (reviewed in references 17 and 18). Interest in Snf1/AMPK pathways is high due to their important functions. Deregulation of AMPK signaling in humans has been linked to type 2 diabetes, heart disease, and cancer (for a review, see reference 16). Snf1 homologs of pathogenic fungi have been implicated in virulence and drug resistance (23, 63, 64).Yeast Snf1 (Cat1, Ccr1) was first identified by its requirement for growth on carbon sources that are less preferred than glucose (5, 7, 65). Subsequent evidence indicated that Snf1 protein kinase (6) is directly involved in glucose signaling, since its activity is stimulated in response to glucose limitation (62). Catalytic activation of Snf1 occurs through phosphorylation of its conserved T-loop threonine (Thr210) (12) by upstream kinases (40, 62). Three protein kinases—Sak1, Tos3, and Elm1—have been identified that can phosphorylate Thr210 of Snf1 (22, 41, 55). These kinases are related to the mammalian kinases that activate AMPK by phosphorylating the equivalent T-loop threonine (Thr172) (reviewed in references 17 and 18). We recently presented evidence that Snf1 homologs of two pathogenic Candida species, Candida albicans and C. glabrata, also undergo T-loop phosphorylation (42).It is not entirely clear why S. cerevisiae has three different kinases that can activate Snf1. Judging by assays of Snf1 kinase activity, Sak1 makes the largest individual contribution to Snf1 activation in the cell (19, 22). However, deletion of SAK1 alone does not result in growth defects on alternative carbon sources, and all three Snf1-activating kinases must be eliminated to produce a phenotypic defect comparable to that of the snf1Δ mutant (22, 39, 55). Deletion of TOS3 was reported to moderately affect growth on nonfermentable carbon sources; this correlated with a reduction in Snf1 activity, although effects on another pathway(s) cannot be excluded (25). Mutation of ELM1 affects cell cycle progression and cell morphology, but this effect is unrelated to Elm1''s role as a Snf1-activating kinase and pertains to its role in the activation of Nim1-related protein kinases involved in morphogenesis checkpoint control (1, 56).While showing significant redundancy for growth on nonpreferred carbon sources, the Snf1-activating kinases could exhibit specialization in Snf1 signaling in response to stresses other than carbon stress. Evidence indicates that Snf1 is important for adaptation to a number of stress conditions (reviewed in reference 18). In some cases, such as genotoxic stress or exposure to hygromycin B, weak activity of unphosphorylated Snf1 appears to be sufficient for resistance (10, 48). In others, such as sodium ion stress and alkaline stress, Thr210 phosphorylation of Snf1 is required for adaptation, and Snf1 becomes activated upon stress exposure (21, 40). As with glucose limitation, however, in these latter cases Sak1 makes the largest contribution to Snf1 activation judging by biochemical assays, and yet it remains dispensable for wild-type levels of stress-resistant growth in phenotypic tests; loss of all three Snf1-activating kinases results in growth defects comparable to those of cells lacking Snf1 (21). Thus, investigation of these stresses provided no evidence for phenotypically relevant specialization of Sak1, Tos3, or Elm1 in Snf1 signaling.Diploid pseudohyphal differentiation is a developmental response to nitrogen limitation (15). When nitrogen becomes limiting, diploid cells adopt elongated morphology, alter their budding pattern, and generate filaments (pseudohyphae) consisting of chains of cells attached to one another. One of the key events in this process is activation of the FLO11 (MUC1) gene, which encodes a cell surface glycoprotein involved in cell-cell adhesion (29, 33, 34). Following up on an observation that Snf1 is important for FLO11 expression on low glucose, we previously found that diploids lacking Snf1 fail to undergo pseudohyphal differentiation on low nitrogen (27, 28). The requirement of Snf1 for a nitrogen-regulated process raised the possibility that Snf1 is directly involved in nitrogen signaling. In support of this notion, we subsequently showed that weak activity of nonphosphorylatable Snf1-T210A is not sufficient for pseudohyphal differentiation and that Thr210 phosphorylation of Snf1 increases in response to nitrogen limitation (43).Here, we have examined the roles of Sak1, Tos3, and Elm1 in pseudohyphal differentiation and Snf1 activation on low nitrogen. We show that elimination of Sak1 leads to a significant defect in nitrogen-regulated pseudohyphal differentiation, whereas Tos3 and Elm1 are dispensable. Sak1 is also required for normal Thr210 phosphorylation of Snf1 under nitrogen-limiting conditions. Our data strongly suggest that the loss of Sak1 affects pseudohyphal differentiation by affecting Snf1 activation and not by disruption of another pathway. Collectively, our findings implicate Sak1 in nitrogen signaling upstream of Snf1.     

Identification of Aerobically and Anaerobically Induced Genes in Enterococcus faecalis by Random Arbitrarily Primed PCR

Enterococci have emerged among the leading causes of nosocomial infection. With the goal of analyzing enterococcal genes differentially expressed in environments related to commensal or environmental colonization and infection sites, we adapted and optimized a method more commonly used in the study of eukaryotic gene expression, random arbitrarily primed PCR (RAP-PCR). The RAP-PCR method was systematically optimized, allowing the technique to be used in a highly reproducible manner with gram-positive bacterial RNA. In the present study, aerobiosis was chosen as a variable for the induction of changes in gene expression by Enterococcus faecalis. Aerobically and anaerobically induced genes were detected and identified to the sequence level, and differential gene expression was confirmed by quantitative, specifically primed RT-PCR. Differentially expressed genes included several sharing identity with those of other organisms related to oxygen metabolism, as well as hypothetical genes lacking identity to known genes.     

Functional expression and characterization of the myrosinase MYR1 from Brassica napus in Saccharomyces cerevisiae

Myrosinases are thioglucosidases that hydrolyze the natural plant products glucosinolates. We have expressed the myrosinase MYR1 from Brassica napus in Saccharomyces cerevisiae. The recombinant myrosinase was enzymatically active which shows that the MYR1, which in the plant is complex bound with myrosinase-binding proteins and myrosinase-associated proteins, is functional in its free form. Characterization of the recombinant MYR1 with respect to pH optimum, substrate specificity, activation by ascorbic acid, and inhibitors showed similar characteristics as previously observed for other plant myrosinases. The indolizidine alkaloid castanospermine, an inhibitor of O-glycosidases, inhibited the hydrolysis of p-hydroxybenzylglucosinolate with a K(i) value of 0.3 microM and 2-deoxy-2-fluoroglucotropaeolin, a specific inhibitor of thioglucosidases, inhibited the enzyme with a K(i) value of 1 mM. The expression of the myrosinase in yeast was transient and the growth of the yeast cells was significantly reduced during the period of expression of the myrosinase. Immunoblot analysis showed that the highest level of expression of MYR1 was obtained 24 h after induction with galactose. The amount of myrosinase protein correlated with the level of enzyme activity. The transient expression of myrosinase indicates that myrosinase is toxic to the cells. This is the first report on successful heterologous expression of a myrosinase and provides an important tool for, e.g., further characterization of myrosinase by site-directed mutagenesis and for studying the interaction between myrosinase and myrosinase-binding proteins, myrosinase-associated proteins, and epithiospecifier proteins.     

Functional characterization of the Saccharomyces cerevisiae ABC-transporter Yor1p overexpressed in plasma membranes

Yor1p, a Saccharomyces cerevisiae plasma membrane ABC-transporter, is associated to oligomycin resistance and to rhodamine B transport. Here, by using the overexpressing strain Superyor [A. Decottignies, A.M. Grant, J.W. Nichols, H. de Wet, D.B. McIntosh, A. Goffeau, ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p, J. Biol. Chem. 273 (1998) 12612-12622], we show that Yor1p also confers resistance to rhodamine 6G and to doxorubicin. In addition, Yor1p protects cells, although weakly, against tetracycline, verapamil, eosin Y and ethidium bromide. The basal ATPase activity of the overexpressed form of Yor1p was studied in membrane preparations. This activity is quenched upon addition of micromolar amounts of vanadate. Vmax and Km values of approximately 0.8 s(-1) and 50+/-8 microM are measured. Mutations of essential residues in the nucleotide binding domain 2 reduces the activity to that measured with a Deltayor1 strain. ATP hydrolysis is strongly inhibited by the addition of potential substrates of the transporter. Covalent reaction of 8-azido-[alpha-(32)P]ATP with Yor1p is not sensitive to the presence of excess oligomycin. Thus, competition of the drug with ATP binding is unlikely. Finally, we inspect possible hypotheses accounting for substrate inhibition, rather than stimulation, of ATP hydrolysis by the membrane preparation.