Systematic genomic studies in the budding yeast S. cerevisiae have greatly increased our capacity to conduct functional profiling of the eukaryotic genome. I describe a new method that makes use of yeast “Barcoder” strains to uniquely tag strains in a yeast overexpression collection (FLEX) and to systematically examine the effects of gene overexpression on cell fitness. This novel system is compatible with the so-called Synthetic Genetic Array (SGA) method, which automates yeast genetics and allows introduction of marked query alleles of interest into arrayed collections of yeast mutants. I identified SDL interactions for two key regulatory kinases, Dun1 and Mck1, using my system. I also used my array and approach to identify SDL interactions for Dun1 that are only manifest in the presence of DNA damage. These studies demonstrate the utility of the pooled SGA-SDL method for systematic discovery of condition-specific genetic interactions in conserved biological pathways.

In the presence of a non-fermentable carbon source, nitrogen-starved diploid cells of the yeast Saccharomyces cerevisiae undergo a meiotic program called sporulation to form gametes called spores. While four spores are produced under standard laboratory sporulation conditions, spore number is known to be regulated by carbon availability: under carbon-depleted conditions, yeast cells package a portion of the four haploid meiotic nuclei into spores. Our lab has demonstrated that these unpackaged meiotic products undergo programmed nuclear destruction (PND) that is associated with apoptotic-like DNA fragmentation. Nevertheless, the mechanism that mediates PND remained to be elucidated. Here, I describe the execution of PND through an unusual form of autophagy that has not been documented previously in yeast. This form of autophagy is most similar to megaautophagy in plants and lysosomal membrane permeabilization in mammals. My results demonstrate further diversity in cell death programs in unicellular microbes that is potentially conserved across eukaryotes.

The Membrane Yeast Two-Hybrid (MYTH) technology was used in this study to find protein-protein interactors of Saccharomyces cerevisiae ATP binding cassette (ABC) transporters Nft1p, Pdr10p, Pdr18p and Vmr1p. There were 23 interactors for Nft1p, 22 interactors for Pdr10p, 4 interactors for Pdr18p and 1 interactor for Vmr1p. The 43 unique interactors belong to a wide variety of functional categories. There were 11 interactors involved in metabolism, 9 interactors involved in transport, 8 interactors with unknown function, 4 interactors involved in trafficking and secretion, 3 interactors involved in protein folding, 2 interactors involved in stress response, and 1 interactor in each of the following categories: cell wall assembly, cytoskeleton maintenance, nuclear function, protein degradation, protein modification and protein synthesis. Follow up experiments also showed that Pdr15p and Pdr18p play an important role in zinc homeostasis because deletion of these ABC transporters results in sensitivity to zinc shock.

Heavy metals, such as copper and cadmium have been linked to a number of cellular dysfunctions in single and multicellular organisms that are associated with apoptosis. The yeast, Saccharomyces cerevisiae, provides a valuable model for elucidating apoptosis mechanisms, and this study extends that capability to Cu and Cd-induced apoptosis. We demonstrate that S. cerevisiae undergoes a glucose-dependent, programmed cell death in response to low cadmium concentrations, which is initiated within the first hour of Cd exposure. The response was associated with induction of the yeast caspase, Yca1p, and was abolished in YCA1∆ mutant. Other apoptotic markers, including sub-G1 DNA fragmentation and hyper-polarization of mitochondrial membranes, were also evident among Cd-exposed cells. We also show that low levels of copper can induce a similar apoptotic response in yeast within the first hour of exposure. Such cellular responses were verified by analyzing mitochondrial perturbation, generation of superoxide ions, activation of the yeast caspase1, and the eventual fragmentation of nuclear DNA (through TUNEL). In analyzing the response of yeast to the different metals, we also demonstrated that the metal-induced PCD is instigated through the sequential activity of at least two caspase-like proteins (i.e., Yca1 and Atg4), both of which appear to be in involved in the process of inducing mitochondrial stress. The additional caspase-like activity is shown to be derived from an enzyme involved in the latter stages of autophagy (Atg4), and provides an intriguing association of apoptosis with autophagy. Here we also demonstrate that metals such as copper and cadmium causes oxidative damage to mitochondrial proteins. Such oxidative attack is targeted and we show that oxidation of certain crucial proteins is required for apoptosis upon metal exposure. By showing that such targeted protein oxidation is dependent on YCA1 and ATG, we also confirm the finding that in yeast that have been exposed to a heavy metal, YCA1 and ATG are essential for damaging mitochondria and to initiate apoptosis. These novel findings highlight several new perspectives about the mechanism of metal-dependent apoptosis, while opening up future analyses to the power of the yeast model system.

Saccharomyces cerevisiae exhibits an apoptotic response upon exposure to toxic metals such as cadmium (Cd) and copper (Cu). Preliminary findings indicate that this response is dependent –to some extent- on the presence of a fermentable carbon source, glucose. To investigate this dependency we monitored the apoptotic response to both metals in the presence and absence of glucose and have shown that glucose is absolutely necessary in order to induce apoptosis in yeast at least during the exposure to metal. We have also looked at the biochemical changes that are taking place in yeast when treated with Cd using Fourier Transform Infra-Red (FTIR) Spectroscopy. Our results suggest that there are definitive changes in cellular activities that are discernable at 1660-1640cm-1 (amide I), 1540-1510cm-1 (amide II) and 1140-1080cm-1(DNA absorption bands).

<p>RPL23a is one of the ~80 ribosomal proteins (r-proteins) of the cytoplasmic ribosome in the model plant <i>Arabidopsis thaliana</i>. The objectives of this research were to establish Arabidopsis RPL23a as a functional r-protein, characterize expression patterns for the two genes (RPL23aA and B) encoding RPL23a using reverse transcription PCR (RT-PCR), and identify regulatory elements controlling the expression of RPL23aA and B. Complementation of a yeast l25 mutant with AtRPL23aA demonstrated that AtRPL23aA can fulfill all the essential functions of L25 in vivo. A survey of various Arabidopsis tissue types showed that, while RPL23aA and B expression patterns both showed increased transcript abundance in mitotically active tissues, RPL23aB transcript levels were generally lower than those of RPL23aA and responded differently to abiotic stresses. In order to determine cis regulatory elements controlling RPL23aA and B expression, the 5 regulatory region (RR) of each gene was characterized via plants carrying a series of 5 RR deletion fragments upstream of a reporter. Transcript start sites and 5 untranslated regions (UTRs) for both RPL23aA and B were also characterized using primer extension, and transcripts from 5 deletion transgenics were amplified using RT-PCR. No correlation was observed between putative cis-acting elements and the expression patterns from the RPL23aA and B deletion transgenics, although a 102 bp sequence in the RPL23aB 5 RR was found to contain pollen-specific elements. 5 leader introns were found in each RPL23a gene, and amplification of transgene transcripts from deletion series plants indicated the importance of post-transcriptional and translational regulation in RPL23aA and B expression. This thesis work is the first demonstration of a plant RPL23a protein as a functional member of the L23/L25 (L23p) conserved r-protein family, and is one of the few in-depth studies of the regulation of r-protein genes in plants. While the majority of previous research on plant r-protein gene expression has focused solely on transcript levels, I show herein that post-transcriptional mechanisms have a critical role in regulating these genes, and thus plant r-protein genes more strongly resemble their mammalian counterparts than those of yeast in terms of structure and regulation.

he influence of two levels of yeast cell wall on phase one laying hen performance was investigated in this study. A total of 75 Lohmann W-36 replacement pullets, 17-weeks-old, were distributed among 75 laying hen cages (1 bird per pen). A total of 3 treatments were sequentially assigned to pens and each treatment had 25 replicates. Feeds were prepared according to the management guide for those birds and a single basal diet was divided into three treatments. First was the control basal diet only with no feed additives. The second was the basal diet supplemented with 250 ppm of yeast cell wall (YCW 250) and the third treatment was the basal diet supplemented with 500 ppm of yeast cell wall (YCW 500). Individual birds per cage served as the experimental unit for this study. Feed and water were offered ad libitum. Data were collected when birds were 21 weeks old and hen day egg production was > 90%. Treatment YCW 250 resulted in significantly higher egg weight than the control and YCW 500 treatment in the first and second production period and was higher than YCW 500 in the third and fourth production period. Feed consumed per dozen eggs was significantly lower in treatment YCW 500 versus treatment YCW 250, but not significantly lower than the control for the first production period and all treatments were not different from each other for the rest of the study. Average feed consumed per bird per day, and monthly cumulative egg production was not different between treatments. Period feed conversion ratios were lower for the second and fourth period (P = 0.15 and 0.18 respectively). There was no treatment effect on interior egg quality except for yolk color for the YCW 500 treatment in the fourth period which had significantly higher Roche color scores than the YCW 250 treatment. Specific gravity, egg shell thickness, egg shell weight, and percent shell weight were significantly higher in hens fed YCW 500 versus YCW 250. Egg shell breaking force was significantly higher in hens fed YCW 250 versus the control group. Overall, feeding a diet supplemented with yeast cell wall improved laying hen performance and 250 ppm YCW had the most significant influences, particularly with respect to increasing egg weight in early production laying hens.

The Saccharomyces cerevisiae Mms4 and Mus81 proteins are required for repairing DNA alkylation damage, but not damage caused by ionizing radiations. Previous studies have demonstrated that Mms4 and Mus81 form a specific complex in vivo, which functions as an endonuclease specific for branched DNA molecules. <p> In an effort to further understand the role of the Mus81-Mms4 complex in vivo, the structural and functional characteristics of this complex were investigated in this study. The epistatic analysis revealed that RAD52 was epistatic to MMS4 with respect to killing by methyl methanesulfonate (MMS), suggesting that MMS4 is involved in the RAD52 dependent homologous recombinational repair pathway. However, the mms4 rad51, mms4 rad54 and mms4 rad50 double mutants showed more sensitivity to MMS than either corresponding single gene disruptant. Since Rad51 and Rad54 are required to form the Holliday junction during recombinational repair pathway, it is unlikely that the Mus81-Mms4 complex functions as a Holliday junction resolvase in vivo. <p> The role of MMS4 in DNA damage induced mutagenesis has been investigated. Deletion of MMS4 had no obvious effects on damage-induced basepair mutations, but increased frame-shift mutations by 3 fold when the yeast cells were treated with MMS. This suggests that the Mus81-Mms4 complex plays a role in limiting the damage-induced frame-shift mutagenesis. <p> Through a yeast two-hybrid assay, Mus81 and Mms4 have been demonstrated to form a stable and specific complex in vivo. This result is consistent with previous studies. To localize the domains of the Mms4 and Mus81 proteins involved in herterodimer formation, a series of deletion mutants were constructed for the yeast two-hybrid assay. The Mus81-binding domain of Mms4 was mapped to the extreme C-terminal region between amino acids 598-691. The Mms4-binding domain of Mus81 was mapped to a domain between amino acids 527-632. The results from co-immunoprecipitation experiment were consistent with those from the yeast two-hybrid assay. The Mms4-1 (Gly173Arg) protein was found to lose its interaction with Mus81, and this kind of amino acid substitution is very likely to alter the three-dimension structure of the protein. Thus we hypothesize that the three-dimensional structure is also important for Mms4 to interact with Mus81. <p> By studies on green fluorescent protein (GFP) fusion proteins and their subcellular localization, we demonstrated that Mms4 and Mus81 are nuclear proteins. When the putative nuclear localization sequence 1 (residues 244-263) in Mms4 was deleted, the truncated protein lost the ability to enter the nucleus. On the contrary, deletion of the putative nuclear localization sequence 2 (residues 539-555) had no effect on the localization of the protein. Furthermore, the nuclear localization of Mus81 was proven to be independent of its interaction with Mms4, and the N-terminal half of Mus81 is necessary and sufficient for its localization to the nucleus.

The genome-wide HaploInsufficieny Profiling (HIPHOP) technique has been validated as a method to quantify the relative abundance of uniquely tagged yeast deletion strains using a microarray readout. The massive throughput of next generation sequencing presents a new technology for assessing HIPHOP profiles. I developed a new method called Barcode analysis by Sequencing (Bar-seq) that applies deep sequencing to genome-scale fitness. I show that Bar-seq outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex genome-scale fitness assay, Bar-seq quantitatively identifies drug-targets, exceeding the performance of the microarray assay. I also established that Bar-seq is well suited to a multiplex format and provides a dramatic increase in throughput. I used the genome-wide HIPHOP assay and other functional genomics tools to explore the mechanisms underlying drug-drug synergies. Drug combination therapy, and synergistic combinations in particular, have several advantages over monotherapies. Synergistic drug combinations allow the dose of each agent to be reduced, often with the benefit of diminishing side effects while maintaining efficacy and decreasing the chances of drug resistance. I used my yeast model to identify synergistic drug combinations and found that inhibitors of ergosterol biosynthesis are highly synergistic with several agents, including those targeting other points within the same pathway. I also devised a method that enriches for synergistic interactions during screening of compound combinations. This new synergy prediction method can aid in the rapid identification of anti-proliferative combinations and can be readily applied to other organisms for further characterization and/or confirmation. Finally, I examined synergistic combination HIPHOP profiles and identified Gene Ontology enrichments that are combination-specific.

The Saccharomyces cerevisiae Mms4 and Mus81 proteins are required for repairing DNA alkylation damage, but not damage caused by ionizing radiations. Previous studies have demonstrated that Mms4 and Mus81 form a specific complex in vivo, which functions as an endonuclease specific for branched DNA molecules. <p> In an effort to further understand the role of the Mus81-Mms4 complex in vivo, the structural and functional characteristics of this complex were investigated in this study. The epistatic analysis revealed that RAD52 was epistatic to MMS4 with respect to killing by methyl methanesulfonate (MMS), suggesting that MMS4 is involved in the RAD52 dependent homologous recombinational repair pathway. However, the mms4 rad51, mms4 rad54 and mms4 rad50 double mutants showed more sensitivity to MMS than either corresponding single gene disruptant. Since Rad51 and Rad54 are required to form the Holliday junction during recombinational repair pathway, it is unlikely that the Mus81-Mms4 complex functions as a Holliday junction resolvase in vivo. <p> The role of MMS4 in DNA damage induced mutagenesis has been investigated. Deletion of MMS4 had no obvious effects on damage-induced basepair mutations, but increased frame-shift mutations by 3 fold when the yeast cells were treated with MMS. This suggests that the Mus81-Mms4 complex plays a role in limiting the damage-induced frame-shift mutagenesis. <p> Through a yeast two-hybrid assay, Mus81 and Mms4 have been demonstrated to form a stable and specific complex in vivo. This result is consistent with previous studies. To localize the domains of the Mms4 and Mus81 proteins involved in herterodimer formation, a series of deletion mutants were constructed for the yeast two-hybrid assay. The Mus81-binding domain of Mms4 was mapped to the extreme C-terminal region between amino acids 598-691. The Mms4-binding domain of Mus81 was mapped to a domain between amino acids 527-632. The results from co-immunoprecipitation experiment were consistent with those from the yeast two-hybrid assay. The Mms4-1 (Gly173Arg) protein was found to lose its interaction with Mus81, and this kind of amino acid substitution is very likely to alter the three-dimension structure of the protein. Thus we hypothesize that the three-dimensional structure is also important for Mms4 to interact with Mus81. <p> By studies on green fluorescent protein (GFP) fusion proteins and their subcellular localization, we demonstrated that Mms4 and Mus81 are nuclear proteins. When the putative nuclear localization sequence 1 (residues 244-263) in Mms4 was deleted, the truncated protein lost the ability to enter the nucleus. On the contrary, deletion of the putative nuclear localization sequence 2 (residues 539-555) had no effect on the localization of the protein. Furthermore, the nuclear localization of Mus81 was proven to be independent of its interaction with Mms4, and the N-terminal half of Mus81 is necessary and sufficient for its localization to the nucleus.