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Variation among individuals arises in part from differences in DNA sequences, but the genetic basis for variation in most traits, including common diseases, remains only partly understood. Many DNA variants influence phenotypes by altering the expression level of one or several genes. The effects of such variants can be detected as expression quantitative trait loci (eQTL) (1). Traditional eQTL mapping requires large-scale genotype and gene expression data for each individual in the study sample, which limits sample sizes to hundreds of individuals in both humans and model organisms and reduces statistical power (2-6). Consequently, many eQTL are probably missed, especially those with smaller effects (7). Furthermore, most studies use messenger RNA rather than protein abundance as the measure of gene expression. Studies that have used mass-spectrometry proteomics (8-13) reported unexpected differences between eQTL and protein QTL (pQTL) for the same genes (9,10), but these studies have been even more limited in scope. Here we introduce a powerful method for identifying genetic loci that influence protein expression in the yeast Saccharomyces cerevisiae. We measure single-cell protein abundance through the use of green fluorescent protein tags in very large populations of genetically variable cells, and use pooled sequencing to compare allele frequencies across the genome in thousands of individuals with high versus low protein abundance. We applied this method to 160 genes and detected many more loci per gene than previous studies. We also observed closer correspondence between loci that influence protein abundance and loci that influence mRNA abundance of a given gene. Most loci that we detected were clustered in 'hotspots' that influence multiple proteins, and some hotspots were found to influence more than half of the proteins that we examined. The variants that underlie these hotspots have profound effects on the gene regulatory network and provide insights into genetic variation in cell physiology between yeast strains., We developed a method for detecting genetic influences on protein levels in large populations of genetically distinct individual yeast cells (Extended Data Fig. 1). The method leverages extreme QTL mapping [...]

Two DNA repair pathways are known to mediate DNA double-strand-break (DSB) repair: homologous recombination (HR) and nonhomologous end joining (NHEJ). In addition, a nonconservative backup pathway showing extensive nucleotide loss and relying on microhomologies at repair junctions was identified in NHEJ-deficient cells from a variety of organisms and found to be involved in chromosomal translocations. Here, an extrachromosomal assay was used to characterize this microhomology-mediated end-joining (MMEJ) mechanism in fission yeast. MMEJ was found to require at least five homologous nucleotides and its efficiency was decreased by the presence of nonhomologous nucleotides either within the overlapping sequences or at DSB ends. Exol exonuclease and Rad22, a Rad52 homolog, were required for repair, suggesting that MMEJ is related to the single-strand-annealing (SSA) pathway of HR. In addition, MMEJ-dependent repair of DSBs with discontinuous microhomologies was strictly dependent on Po14, a PolX DNA polymerase. Although not strictly required, Msh2 and Pms 1 mismatch repair proteins affected the pattern of MMEJ repair. Strikingly, Pku70 inhibited MMEJ and increased the minimal homology length required for efficient MMEJ. Overall, this study strongly suggests that MMEJ does not define a distinct DSB repair mechanism but reflects 'micro-SSA.'

Heterologous expression of bacterial virulence factors in Saccharomyces cerevisiae is a feasible approach to study their molecular function. The authors have previously reported that the Salmonella typhimurium SigD protein, a phosphatidylinositol phosphatase involved in invasion of the host cell, inhibits yeast growth, presumably by depleting an essential pool of phosphatidylinositol 4,5-bisphosphate, and also that a catalytically inactive version, Sig[D.sup.R468A], was able to arrest growth by a different mechanism that involved disruption of the actin cytoskeleton. This paper describes marked differences between the phenotypes elicited by expression of SigD and Sig[D.sup.R468A] in yeast. First, expression of Sig[D.sup.R468A] caused accumulation of large unbudded cells and loss of septin organization, while SigD expression caused none of these effects. Second, growth inhibition by Sig[D.sup.R468A] was mediated by a cell cycle arrest in G2 dependent on the Swel morphogenetic checkpoint, but SigD-induced growth inhibition was cell cycle independent. And third, SigD caused strong activation of the yeast MAP kinase Slt2, whereas Sig[D.sup.R488A] rather inactivated another MAP kinase, Kss1. In a screen for suppressors of Sig[D.sup.R468A]-induced growth arrest by overexpression of a yeast cDNA library, the Cdc42 GTPase was isolated. Furthermore, Sig[D.sup.R468A] was co-purified with Cdc42 from yeast lysates. It is concluded that the Salmonella SigD protein deprived of its phosphatase activity is able to disrupt yeast morphogenesis by interfering with Cdc42 function, opening the possibility that the SigD N-terminal region might directly modulate small GTPases from the host during infection.

ATP-binding cassette (ABC) proteins transport a wide variety of substrates, including sugars, amino acids, metal ions, lipids, peptides and proteins, across membranes, and most ABC proteins contain transmembrane domains (ABC transporters). Sequencing of the Schizosaccharomyces pombe genome has allowed identification of all genes encoding ABC transporters in fission yeast. To date, six such genes have been characterized, and an additional five genes encoding ABC transporters were identified from the genome sequence. In an attempt to characterize all of the ABC transporters in fission yeast, all 11 genes were disrupted. While all the genes were found to be dispensable for cell viability, some disruptants lacked apparent phenotypes. GFP-tagged ABC transporters were localized to membranes as follows: plasma membrane (2), vacuolar membrane (4), mitochondrial membrane (2), endoplasmic reticulum membrane (2), and endosome and Golgi membranes (1). Two Cluster II. 1 proteins, Abc2p (SPAC3F10.11 c) and Abc4p (SPAC30.04c), were found to be localized to vacuolar membranes, and to be responsible for accumulation of a characteristic red pigment in the vacuole of an adenine biosynthetic mutant. The doubly disrupted mutant abc2[DELTA] abc4[DELTA] exhibited drug sensitivity, and a decreased accumulation of monochlorobimane, suggesting that both of the proteins encoded by these genes are involved in detoxification of xenobiotics, and vacuolar sequestration of glutathione S-conjugates.

Pseudomonas aeruginosa secretes a number of toxins by a type III system, and these are important in virulence. One of them, ExoS, is a bifunctional toxin, with a GTPase-activating protein domain, as well as ADP ribosyltransferase (ADPRT) activity. These two domains have numerous potential cellular targets, but the overall mechanism of ExoS action remains unclear. The effects of ExoS in a simple eukaryotic system, the yeast Saccharomyces cerevisiae, using a tetracycline-regulated expression system were studied. This system allowed controlled expression of ExoS in yeast, which was not possible using a galactose-induced system. ExoS was found to be an extremely potent inhibitor of yeast growth, and to be largely dependent on the activity of its ADPRT domain. ExoS produced a dramatic alteration in actin distribution, with the appearance of large aggregates of cortical actin, and thickened disorganized cables, entirely dependent on the ADPRT domain. This phenotype is suggestive of actin stabilization, which was verified by showing that the cortical aggregates of actin induced by ExoS were resistant to treatment with latrunculin A, an agent that prevents actin polymerization. ExoS increased the numbers of mating projections produced following growth arrest with mating pheromone, and prevented subsequent DNA replication, an effect that is again dependent on the ADPRT domain. Following pheromone removal, ExoS produced altered development of the mating projections, which became elongated with a swollen bud-like tip. These results suggest alternative pathways for ExoS action in eukaryotic cells that may result from activation of small GTPases, and this yeast expression system is well suited to explore these pathways.

The Candida albicans homologues of the most studied Saccharomyces cerevisiae stationaryphase genes, SNO1 and SNZ1, were used to test the hypothesis that, within a biofilm, some cells reach stationary phase within continuously fed, as well as static, C. albicans biofilms grown on dental acrylic. The authors first studied the expression patterns of these two genes in planktonic growth conditions. Using real-time RT-PCR (RT-RTPCR), increased peak expression of both SNZ1 and SNO1 was observed at 5 and 6 days, respectively, in C. albicans grown in suspension culture. SNZ1-yellow fluorescent protein (YFP) and SNQ1-YFP were constructed to study expression at the cellular level and protein localization in C. albicans. Snz1p-YFP and Sno1p-YFP localized to the cytoplasm with maximum expression (>900%) at 5 and 6 days, respectively, in planktonic conditions. When yeast growth was reinitiated, loss of fluorescence began immediately. Germ tubes and hyphae were non-fluorescent. Pseudohyphae began appearing at 9 days in planktonic yeast culture and expressed each protein by 11 days; however, the cells budding from pseudohyphae were not fluorescent. Biofilm was formed in vitro under either static or continuously fed conditions. Increased expression of the two genes was shown by RT-RTPCR, beginning by day 3 and increasing through to day 15 (continuously fed biofilm). Only the bottommost layer of acrylic-adhered cells in the biofilm showed 25 and 40% fluorescence at 6 and 15 days, respectively. These observations suggest that only a few cells in C. albicans biofilms express genes associated with the planktonic stationary phase and that these are found at the bottom of the biofilm adhered to the surface.

The binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin are characterized to provide a molecular basis for better understanding of frataxin's function. It is observed that the metal-ligand coordination geometry of metal-binding sites is consistent with a six-coordinate iron/(oxygen/nitrogen) based ligand geometry, leading to the development of a model for how yeast frataxin may interact with iron.

A new proteomic methodology is used that purifies and identifies palmitoylated proteins to characterize the palmitoyl proteome of the yeast Saccharomyces cerevisiae. This approach identifies 12 of the 15 known palmitoyl proteins plus many new palmitoyl proteins.

770 mostly novel protein-protein interactions were identified using a stringent yeast two-hybrid screen. The protein-protein interaction network was developed for the 20 or more different inherited cerebellar ataxias characterized by Purkinje cell degeneration.

A physical association of promoter and terminator regions of the yeast BUD3 and SEN1 genes is reported. These interactions are RNA Polymerase II (RNAP II) transcription dependent, require the Ssu72 and Pta1 components of the CPF 3'-end processing complex, and require the phosphatase activity of Ssu72.

The genome-wide localization and dynamics of Htz1, the yeast histone H2A was determined. Htz1-bearing nucleosomes were deposited at repressed/basal promoters but facilitated activation through their susceptibility to loss, thereby helping to expose promoter DNA.

The nucleosome modifications across the yeast genome were studied by using chromatin immunoprecipitation coupled with DNA microarrays to produce high-resolution genome-wide maps of histone acetylation and methylation. The acetylation and methylation of histones are associated with transcriptional activity, but acetylation occurs at the beginning of genes whereas methylation occurs throughout transcribed regions.

The epigenetic map is a new and reliable genome-wide microarray study of histone modifications and gene expression in yeast. The epigenetic map is widely used in the research of epigenome.

The direct regulation of the budding-yeast homolog of Wee1, Swe1, by cyclin-dependent kinase 1 (Cdk1) is discussed. The regulation of Swe1-Cdk1 complex plays an important role in controlling the transition into mitosis.

We have examined the in vivo requirement of two recently identified nonessential components of the budding yeast anaphase-promoting complex, Swm1p and Mnd2p, as well as that of the previously identified subunit Apc9p. swm1[DELTA] mutants exhibit synthetic lethality or conditional synthetic lethality with other APC/C subunits and regulators, whereas mnd2[DELTA] mutants are less sensitive to perturbation of the APC/C. swm1[DELTA] mutants, but not mnd2[DELTA] mutants, exhibit defects in APC/C substrate turnover, both during the mitotic cell cycle and in [alpha]-factor-arrested cells. In contrast, apc9[DELTA] mutants exhibit only minor defects in substrate degradation in [alpha]-factor-arrested cells. In cycling cells, degradation of C1b2p, but not Pds1p or C1b5p, is delayed in apc9[DELTA]. Our findings suggest that Swm1p is required for full catalytic activity of the APC/C, whereas the requirement of Mnd2p for APC/C function appears to be negligible under standard laboratory conditions. Furthermore, the role of Apc9p in APC/C-dependent ubiquitination may be limited to the proteolysis of a select number of substrates.

The Sch9 protein kinase regulates Hsp90-dependent signal transduction activity in the budding yeast Saccharomyces cerevisiae. Hsp90 functions in concert with a number of cochaperones, including the Hsp110 homolog Sse1. In this report, we demonstrate a novel synthetic genetic interaction between SSE1 and SCH9. This interaction was observed specifically during growth at elevated temperature and was suppressed by decreased signaling through the protein kinase A (PKA) signal transduction pathway. Correspondingly, sse1[DELTA] sch9[DELTA] cells were shown by both genetic and biochemical approaches to have abnormally high levels of PKA activity and were less sensitive to modulation of PKA by glucose availability. Growth defects of an sse1[DELTA] mutant were corrected by reducing PKA signaling through overexpression of negative regulators or growth on nonoptimal carbon sources. Hyperactivation of the PKA pathway through expression of a constitutive RAS2 allele likewise resulted in temperature-sensitive growth, suggesting that modulation of PKA activity during thermal stress is required for adaptation and viability. Together these results demonstrate that the Sse1 chaperone and the growth control kinase Sch9 independently contribute to regulation of PKA signaling.

During meiotic recombination in the yeast Saccharomyces cerevisiae, heteroduplex DNA is formed when single-stranded DNAs from two homologs anneal as a consequence of strand invasion. If the two DNA strands differ in sequence, a mismatch will be generated. Mismatches in heteroduplex DNA are recognized and repaired efficiently by meiotic DNA mismatch repair systems. Components of two meiotic systems, mismatch repair (MMR) and large loop repair (LLR), have been identified previously, but the substrate range of these repair systems has never been defined. To determine the substrates for the MMR and LLR repair pathways, we constructed insertion mutations at HIS4 that form loops of varying sizes when complexed with wild-type HIS4 sequence during meiotic heteroduplex DNA formation. We compared the frequency of repair during meiosis in wild-type diploids and in diploids lacking components of either MMR or LLR. We find that the LLR pathway does not act on single-stranded DNA loops of 19, nucleotides in length, indicating that the two pathways overlap slightly in their substrate range during meiosis. Our data reveal differences in mitotic and meiotic MMR and LLR; these may be due to alterations in the functioning of each complex or result from subtle sequence context influences on repair of the various mismatches examined.

Flor or velum formation by certain wine strains of Saccharomyces cerevisiae is a form of cellular aggregation that manifests as an air-liquid interfacial biofilm at the end of alcoholic fermentation. Studies demonstrate that FLO11 is required for yeast biofilm formation at an air-liquid interface and that the biofilm cells are not less dense than the suspending medium.

The processing of constitutive poly(A) sites is examined in detail along with the fundamental mechanisms responsible for the regulation of alternative ploy(A) site selection. The recognition of three distinct sequence elements by CFI(sub m), CPSF, and CstF suggests that vertebrate ploy(A) site definition is mechanistically more similar to that of yeast and plants than anticipated.

The dif operon is essential for fruiting body formation, fibril (exopolysaccharide) production and social motility of Myxococcus xanthus. The dif locus contains a gene cluster homologous to chemotaxis genes such as mcp (difA), che W (difC), che Y (difD), cheA (difE) and cheC (difF), as well as an unknown ORF called difB. This study used yeast two-hybrid analysis to investigate possible interactions between Dif proteins, and determined that DifA, C, D and E interact in a similar fashion to chemotaxis proteins of Escherichia coil and Bacillus subtilis. It also showed that DifF interacted with DifD, and that the novel protein DifB did not interact with Dif proteins. Furthermore, DifA--F proteins were used to determine other possible protein--protein interactions in the M. xanthus genomic library. The authors not only confirmed the specific interactions among known Dif proteins, but also discovered two novel interactions between DifE and Nla19, and DifB and YidC, providing some new information about the Dif signalling pathway. Based on these findings, a model for the Dif signalling pathway is proposed.

Mus81 and Rhp51 are required for generating deletion mutations in fission yeast replication mutants that experience replication stress. A study provides novel insights into the way Cds1 regulates Mus81 accordingly when the cells experience different replication stress to preserve genome integrity.

The meiotic recombination hot spot ura4A (formerly ura4-aim) of Schizosaccharomyces pombe was observed at the insertion of the [ura4.sup.+] gene 15 kb centromere-proximal to ade6 on chromosome III. Crosses heterozygous for the insertion showed frequent conversion at the heterology with preferential loss of the insertion. This report concerns the characterization of 12 spontaneous ura4A mutants. A gradient of conversion ranging from 18% at the 5' end to 6% at the 3' end was detected. A novel phenomenon also was discovered: a mating-type-related bias of conversion. The allele entering with the [h.sup.+] parent acts preferentially as the acceptor for conversion (ratio of 3:2). Tetrad analysis of two-factor crosses showed that heteroduplex DNA is predominantly asymmetrical, enters from the 5' end, and more often than not covers the entire gene. Restoration repair of markers at the 5' end was inferred. Random spore analyses of two-factor crosses and normalization of prototroph-recombinant frequencies to physical distance led to the demonstration of map expansion: Crosses involving distant markers yielded recombinant frequencies higher than the sum of the frequencies measured in the subintervals. Finally, marker effects on recombination were defined for two of the ura4A mutations.

We have investigated the relative roles in vivo of Saccharomyces cerevisiae DNA polymerase [eta], DNA polymerase [zeta], Rev1 protein, and the DNA polymerase [delta] subunit, Pol32, in the bypass of an abasic site, T-T (6-4) photoadduct and T-T cis-syn cyclobutane dimer, by transforming strains deleted for RAD30, REV3, REV1, or POL32 with duplex plasmids carrying one of these DNA lesions located within a 28-nucleotide single-stranded region. DNA polymerase [eta] was found to be involved only rarely in the bypass of the T-T (6-4) photoadduct or the abasic sites in the sequence context used, although, as expected, it was solely responsible for the bypass of the T-T dimer. We argue that DNA polymerase [zeta], rather than DNA polymerase [delta] as previously suggested, is responsible for insertion in bypass events other than those in which polymerase [eta] performs this function. However, DNA polymerase [delta] is involved indirectly in mutagenesis, since the strain lacking its Pol32 subunit, known to be deficient in mutagenesis, shows as little bypass of the T-T (6-4) photoadduct or the abasic sites as those deficient in Pol [zeta] or Rev1. In contrast, bypass of the T-T dimer in the pol32[DELTA] strain occurs at the wild-type frequency.

TOR protein kinases are key regulators of cell growth in eukaryotes. TOR is also known as the target protein for the immunosuppressive and potentially anticancer drug rapamycin. The fission yeast Schizosaccharomyces pombe has two TOR homologs, [tor1.sup.+] is required under starvation and a variety of stresses, while [tor2.sup.+] is an essential gene. Surprisingly, to date no rapamycin-sensitive TOR-dependent function has been identified in S. pombe. Herein, we show that S. pombe auxotrophs, in particular leucine auxotrophs, are sensitive to rapamycin. This sensitivity is suppressed by deletion of the S. pombe FKBP12 or by introducing a rapamycin-binding defective tor1 allele, suggesting that rapamycin inhibits a tor1p-dependent function. Sensitivity of leucine auxotrophs to rapamycin is observed when ammonia is used as the nitrogen source and can be suppressed by its replacement with proline. Consistently, using radioactive labeled leucine, we show that cells treated with rapamycin or disrupted for [tor1.sup.+] are defective in leucine uptake when the nitrogen source is ammonia but not proline. Recently, it has been reported that [tsc1.sup.+] and [tsc2.sup.+], the S. pombe homologs for the mammalian TSC1 and TSC2, are also defective in leucine uptake. TSC1 and TSC2 may antagonize TOR signaling in mammalian cells and Drosophila. We show that reduction of leucine uptake in tor1 mutants is correlated with decreased expression of three putative amino acid permeases that are also downregulated in tsc1 of tsc2. These findings suggest a possible mechanism for regulation of leucine uptake by tor1p and indicate that tor1p, as well as tsc1p and tsc2p, positively regulates leucine uptake in S. pombe.

Defects in anaphase-promoting complex (APC) activity, which regulates mitotic progression and chromatin assembly, results in genomic instability, a hallmark of premature aging and cancer. We investigated whether APC-dependent genomic stability affects aging and life span in yeast. Utilizing replicative and chronological aging assays, the APC was shown to promote longevity. Multicopy expression of genes encoding Snf1p (MIG1) and PKA (PDE2) aging-pathway components suppressed [apc5.sup.CA] phenotypes, suggesting their involvement in APC-dependent longevity. While it is known that PKA inhibits APC activity and reduces life span, a link between the Snf1p-inhibited Mig1p transcriptional modulator and the APC is novel. Our mutant analysis supports a model in which Snf1p promotes extended life span by inhibiting the negative influence of Mig1p on the APC. Consistent with this, we found that increased MIG1 expression reduced replicative life span, whereas mig1[DELTA] mutations suppressed the [apc5.sup.CA] chronological aging defect. Furthermore, Mig1p and Mig2p activate APC gene transcription, particularly on glycerol, and mig2[DELTA], but not mig1[DELTA], confers a prolonged replicative life span in both APC5 and [acp5.sup.CA] cells. However, glucose repression of APC genes was Mig1p and Mig2p independent, indicating the presence of an uncharacterized factor. Therefore, we propose that APC-dependent genomic stability, is linked to prolonged longevity by the antagonistic regulation of the PKA and Snf1p pathways.

The aglZ gene of Myxococcus xanthus was identified from a yeast two-hybrid assay in which MglA was used as bait. MglA is a 22-kDa cytoplasmic GTPase required for both adventurous and social gliding motility and sporulation. Genetic studies showed that aglZ is part of the A motility system, because disruption or deletion of aglZ abolished movement of isolated cells and aglZ sglK double mutants were nonmotile. The aglZ gene encodes a 153-kDa protein that interacts with purified MglA in vitro. The N terminus of AglZ shows similarity to the receiver domain of two-component response regulator proteins, while the C terminus contains heptad repeats characteristic of coiled-coil proteins, such as myosin. Consistent with this motif, expression of AglZ in Escherichia coli resulted in production of striated lattice structures. Similar to the myosin heavy chain, the purified C-terminal coiled-coil domain of AglZ forms filament structures in vitro.

The understanding of the post-replication repair in DT40 in view of bacterial, yeast and mammalian genetics is mentioned. A genetic analysis of translesion synthesis and homologous recombination is put forth.

The attempts to expand the genetic code that progressed to nucleus containing cells are discussed. The result showed that the transformed yeast cells make an experimental elevation of code complexity possible in mammals.

The F-box protein Rcy1p is part of a non-SCF (Skp1p-cullin-F-box protein) complex involved in recycling of internalized material. Like rcy1A, cells lacking the Rab-GTPase Ypt6p or its heterodimeric GEFs Rgp1p and Ric1p are unable to recycle the v-SNARE Snc1p. Here we provide genetic evidence suggesting that Rcy1p is a positive regulator of Ypt6p. Deletion of the GAP Gyp2p restores recycling in rcy1[DELTA], while overexpression of an active form of Ypt6p partially suppresses the recycling defect of rcy1[DELTA] cells. Conversely, overexpression of Gyp2p in wild-type cells interferes with recycling of GFP-Snc1p, and the cells accumulate membrane structures as evidenced by electron microscopy Gyp2p-GFP is distributed throughout the cytoplasm and accumulates in punctate structures, which concentrate in an actin-dependent manner at sites of polarized growth Taken together, our results suggest that the F-box protein Rcy1p may activate the Ypt6p GTPase module during recycling.

DNA polymerase [eta] (Pol[eta]) functions in the proficient bypass of a variety of DNA lesions. Relative to the replicative polymerases, Pol[eta] has a greater tolerance for distorted DNA geometries and possesses a low fidelity. X-ray crystal structures and studies with nucleotide analogs have implicated interactions with the DNA minor groove as being crucial for the high fidelity of replicative DNA polymerases. To determine whether Pol[eta] also makes such functionally important contacts with the DNA minor groove, here we examine the effects on Pol[eta]-catalyzed nucleotide incorporation when 3-deazaguanine, a base analog that lacks the ability to form minor-groove hydrogen bonds with the protein, is substituted for guanine at various positions in the DNA. From these studies, we conclude that Pol[eta] makes only a single functional contact with the DNA minor groove at the position of the incoming nucleotide; in this regard, Pol[eta] differs from high-fidelity DNA polymerases that are unable to replicate through DNA lesions. These results help explain the proficient ability of Pol[eta] for bypassing distorting DNA lesions.

Yme1p, an ATP-dependent protease localized in the mitochondrial inner membrane, is required for the growth of yeast lacking an intact mitochondrial genome. Specific dominant mutations in the genes encoding the [alpha]- and [gamma]-subunits of the mitochondrial [F.sub.1][F.sub.0]-ATPase suppress the slow-growth phenotype of yeast that simultaneously lack Yme1p and mitochondrial DNA. [F.sub.1][F.sub.0]-ATPase activity is reduced in yeast lacking Yme1p and is restored in yme1 strains bearing suppressing mutations in [F.sub.1]-ATPasc structural genes. Mitochondria isolated from yme1 yeast generated a membrane potential upon the addition of succinate, but unlike mitochondria isolated either from wild-type yeast or from yeast bearing yme1 and a suppressing mutation, were unable to generate a membrane potential upon the addition of ATP. Nuclear-encoded [F.sub.0] subunits accumulate in yme1 yeast lacking mitochondrial DNA; however, deletion of genes encoding those subunits did not suppress the requirement of yme1 yeast for intact mitochondrial DNA. In contrast, deletion of INH1, which encodes an inhibitor of the [F.sub.1][F.sub.0]-ATPase, partially suppressed the growth defect of yme1 yeast lacking mitochondrial DNA. We conclude that Yme1p is in part responsible for assuring sufficient [F.sub.1][F.sub.0]-ATPase activity to generate a membrane potential in mitochondria lacking mitochondrial DNA and propose that Yme1p accomplishes this by catalyzing the turnover of protein inhibitors of the [F.sub.1][F.sub.0]-ATPase.

The SIR2 (silent information regulator 2) gene family has diverse functions in yeast including gene silencing, DNA repair, cell-cycle progression, and chromosome fidelity in meiosis and aging. Human homologues, termed sirtuins, are highly conserved but are of unknown function. We previously identified a large imprinted gene domain on 11p15.5 and investigated the 11p15.5 sirtuin SIRT3. Although this gene was not imprinted, we found that it is localized to mitochondria, with a mitochondrial targeting signal within a unique N-terminal peptide sequence. The encoded protein was found also to possess NA[D.sup.+]-dependent histone deacetylase activity. These results suggest a previously unrecognized organelle for sirtuin function and that the role of SIRT3 in mitochondria involves protein deacetylation.

The Ume6 transcription factor in yeast is known to both repress and activate expression of diverse genes during growth and meiotic development. To obtain a more complete profile of the functions regulated by this protein, microarray analysis was used to examine transcription in wild-type and ume6[DELTA] diploids during vegetative growth in glucose and acetate. Two different genetic backgrounds (W303 and SK1.) were examined to identify a core set of strain-independent Ume6-regulated genes. Among genes whose expression is controlled by Ume6 in both backgrounds, 82 contain homologies to the Ume6-binding site (URS1) and are expected to be directly regulated by Ume6. The vast majority of those whose functions are known participate in carbon/nitrogen metabolism and/or meiosis. Approximately half of the Ume6 direct targets are induced during meiosis, with most falling into the early meiotic expression class (cluster 4), and a smaller subset in the middle and later classes (clusters 5-7). Based on these data, we propose that Ume6 serves a unique role in diploid cells, coupling metabolic responses to nutritional cues with the initiation and progression of meiosis. Finally, expression patterns in the two genetic backgrounds suggest that SK1 is better adapted to respiration and W303 to fermentation, which may in part account for the more efficient and synchronous sporulation of SK1.

B-boxes and TFIIIC are shown to limit the spread of heterochromatin at the silent mat region in the fission yeast genome. Studies reveal that the conservation of TFIIIC and B-boxes implies that the barrier function might span across species.

The structure of a new bioluminescent yeast based bioreporter (saccharomyces cerecisiave strain) for detecting androgens is presented. Results show that the strain is useful for high output testing of chemicals and environmental observation due to its speed and ability.

Key insights into the mechanism of Dot1 histone methylation are presented. It is found that Dot1 requires histone H4 for in vitro methytransferase activity and the histone H4 tail for Dot1-mediated methylation in yeast.

Homologous recombination is examined when breaks are reduced in yeast spo11 hypomorphs to understand the decision by which some breaks give crossovers while others follow a mostly noncrossover pathway(s). A previously unexpected expression of crossover control is defined whereby the crossover/noncrossover ratio can change to maintain crossovers and the theory that an obligate crossover is a genetically programmed event tied to crossover interference is supported by the results.

The tethering of a mammalian arginie-serine-rich (RS) domain to a yeast actin pre-mRNA rescues splicing of certain branchpoint or 5' splice site mutants in which U snRNA base-pairing was decreased. It is found that in the absence of other splicing factors an RS domain tethered to a pre-mRNA selectively contacts a double-stranded RNA region and enhances RNA-RNA base-pairing.

A meiosis-specific protein (Hed1) with a novel Rad51 regulatory function is described. It is proposed that Hed1 protein provides a mechanism to ensure the coordinated action of Rad51 and Dmc1 during meiosis, by down-regulating Rad51 activity when Dmc1 is unavailable.

A study employed 'gene-centered' high-throughput yeast one-hybrid (Y1H) assays to identify 283 interactions between 72 Caenorhabditis elegans digestive tract gene promoters and 117 proteins. The resulting protein-deoxyribonucleic acid interaction (PDI) network is highly connected and enriched for transcription factors (TFs) that are expressed in the digestive tract.

A topoisomerase II (Topo II)-dependent G2/M checkpoint is defined in a genetically amenable eukaryote, budding yeast, and it is demonstrated that this checkpoint enhances cell survival. Neither DNA damage-responsive pathways nor Pds1/securin are needed for this checkpoint, and compromised Topo II function activates a yeast checkpoint system that operates by a novel mechanism.

The experimental amenability of yeast as a unicellular model system has enabled the discovery of multiple sugar sensors and signaling pathways. Genetic analyses have revealed extensive interactions between sugar and plant hormone signaling, and a central role for hexokinase as a conserved glucose sensor.

A study is conducted to investigate whether in halophilic Hortaea werneckii and in halotolerant Aureobasidium pullulans osmotic adjustments of sodium and potassium also occur in response to osmotic stress. Small fluctuations of cation concentrations are observed after a hypersonic shock in nonadapted Aureobasidium pullulans similar to those in salt-adapted Hortaea werneckii, which additionally confirmed better regulation of ionic homeostasis in the latter.