In the near future, biomass-derived energy is predicted to substantially complement that generated from petroleum. However, certain types of biomass employed as substrates in the microorganism-mediated production of renewable fuelethanol contain significant amounts of the recalcitrant hexose sugar galactose. The consumption of galactose in hexose sugar-fermenting yeasts is often delayed with respect to other sugars, such as glucose and mannose, because of an intrinsic preference for carbon sources requiring less energy in the preparatory reactions preceding glycolysis. This work comprised the search for, and characterization of anethanologenic yeast capable of efficiently assimilating galactose. Screening experiments conducted with wild-type Saccharomyces cerevisiae strains identified one isolate (Y-1528) exhibiting exceptionally fast galactose fermentation. The absence of conventional glucose repression, including a preference for galactose as carbon source and notable delays in the utilization of glucose and mannose, was demonstrated in mixed sugar fermentations. Endogenous extracellular glucose was observed during double sugar fermentations of galactose and mannose. This glucose was traced to supplied galactose by radioisotope labeling, suggesting involvement of UDP-galactose 4-epimerase in the responsible reaction mechanism(s).Sub-cellular fractionation was employed in an attempt to ascertain enzyme localization in Y-1528. Fermentations of lignocellulosic substrate mixtures by Y-1528 illustrated better performance than that accomplished by a reference yeast strain, and again showed a preference for galactose. Mixed cultures of Y-1528 and the same reference strain demonstrated accelerated hexose sugar consumption, and no detrimental effects from competition, during synthetic and lignocellulosic substrate fermentations. Glucose repression was absent in mixed culture fermentations. Fermentations of synthetic sugar mixtures augmented with lignocellulosic inhibitory compounds showed Y-1528 to have better performance than a reference yeast strain, despite a global detrimental effect relative to inhibitor-free fermentations. Cell recycle batch fermentations of spent sulfite liquor illustrated the toxic effect of the hardwood variant, as well as a net loss of performance from all strains tested. Y-1528 was taxonomically confirmed as S. cerevisiae. UDP-galactose 4-epimerase chromatographic purification was unsuccessful, but a partial sequence of the enzyme, showing complete identity with type sequence, was obtained by electrophoretic separation, liquid chromatography, and mass spectrometry. A significantly mutated UDP-galactose 4-epimerase gene was successfully sequenced.

DNA post-replication repair (PRR) is a cellular tolerance mechanism by which eukaryotic cells survive lethal lesions during or after DNA synthesis. In the yeast Saccharomyces cerevisiae, modification of proliferating cell nuclear antigen (PCNA) by ubiquitin and by small ubiquitin-like modifier (SUMO) plays an important role in PRR. PCNA ubiquitination is dependent on Rad6, a ubiquitin-conjugating enzyme (E2) and Rad18, a ubiquitin ligase (E3). Rad6 and Rad18 form a stable complex. PCNA sumoylation is dependent on Ubc9, an E2 specific to SUMO modification. <p>PRR in mammalian cells is less well understood. However, human Rad18 (hRad18) has been found to interact with human Rad6 (HHR6A/B). In this study, we detected physical interaction between hRad18 and human Ubc9 (hUbc9) through yeast two-hybrid assays. In order to define the domain(s) of hRad18 involved in the formation of a complex with HHR6B or hUbc9, a series of yeast two-hybrid constructs containing various hRAD18 gene deletions and mutations were made. A C-terminal region of hRad18, containing the putative HHR6A/B binding domain (amino acids 340 to 395), interacts with HHR6A/B while the N-terminus (amino acids 1-93) does not. Yeast Rad18 has a homologous fragment of the HHR6A/B binding domain and this fragment is sufficient to interact with yeast Rad6 in yeast two-hybrid assays, so we infer that hRad18 interacts with HHR6B through the same domain. Surprisingly, both the N-terminal and C-terminal fragments of hRad18 can interact with hUbc9, suggesting the existence of two separate domains in hRad18 interacting with hUbc9. The N-terminal fragment of hRad18 contains only a RING finger domain (amino acids 25-64), which is probably responsible for binding to hUbc9. The C-terminal fragment of hRad18 with HHR6A/B binding domain deletion can still interact with hUbc9, suggesting that the HHR6A/B binding domain is not involved in hUbc9 interaction. A key cysteine mutation (C28F) in the RING finger domain abolished the interactions of hRad18 with both HHR6A/B and hUbc9. This amino acid substitution is likely to alter the three-dimensional structure of the protein, thus making the protein unstable. Taken together, results obtained from this study suggest that hRad18 may regulate the modification status of PCNA by interacting with two different E2s, HHR6A/B and hUbc9, through distinct domains.

The Arabidopsis TGA family of basic leucine zipper transcription factors regulate the expression of pathogenesis-related genes and are required for resistance to disease. Members of the family possess diverse properties in respect to their ability to transactivate and interact with NPR1, the central regulator of systemic acquired resistance in Arabidopsis. Two TGA factors, TGA1 and TGA2, have 83 % amino acid similarity but possess differing properties. TGA1 does not interact with NPR1 but is able to transactivate, while TGA2 interacts with NPR1 but is unable to transactivate. This study uses these two TGA factors to identify amino acids that are responsible for their function. <p>Four cysteines residues within TGA1 were targeted for study by site-directed mutagenesis and the resulting mutants were tested for interaction with NPR1 in yeast. The construct containing a mutation of cysteine 260 (Cys-260) interacted well with NPR1, while those with mutations at Cys-172 or Cys-266 interacted poorly. The Cys-260 mutant also displayed the greatest decrease in transactivation potential in yeast, while mutation of Cys-172 or Cys-266 resulted in smaller decreases. Mutation of Cys-287 had no effect on NPR1 interaction or transactivation. Combining various point mutations in a single protein did not increase NPR1 interaction or transactivation levels, indicating that Cys-260 is crucial for regulating TGA1 properties. Cysteines possess the unique ability of forming reversible disulfide bonds which have been shown to regulate several mammalian cellular processes. The observation that mutation of a single TGA1 cysteine (Cys-260) greatly alters the proteins properties provides a convincing argument that oxidoreduction of this residue is important for its regulation, possibly through the formation of a disulfide bond with either Cys-172 or Cys-266. <p>To test whether other members of the TGA family could be regulated by oxidoreduction, several TGA2 constructs were created that introduced Cys at positions corresponding to those found in TGA1. When tested in yeast none were able to transactivate but continued to interact with NPR1.

Much of the work on how the cell cycle is regulated has focused on Cyclin-Dependent Kinase (CDK)-mediated regulation of factors that control the coordinate expression of genes required for entry into the cell cycle. In Saccharomyces cerevisiae, SBF and MBF are related transcription factors that co-ordinately activate a large group of genes at the G1/S transition, and their activation depends on the Cln3-Cdk1 form of the cyclin-dependent kinase. However, cells are viable in the absence of Cln3, or SBF and MBF, indicating that other regulatory pathways must exist that activate the budding yeast cell cycle. The known CDK-independent pathways are made up of various phosphatases and plasma membrane transporters that control ion homeostasis in early G1 phase, a time when cells assess environmental growth conditions in order to commit to cell cycle entry. The enigmatic Bck2 protein is thought to act within these CDK-independent pathways, but the means by which it activates G1/S-regulated genes is not known. Bck2 contains little sequence homology to any known protein. In order to understand how CDK-independent pathways operate, I have studied the Bck2 protein using multiple approaches. In one approach, I have screened for novel SBF/MBF-binding proteins in order to determine if other non-CDK proteins, such as Bck2, might activate SBF and MBF. I have also investigated which region of Bck2 is required for its activity in order to determine if Bck2’s transcriptional activation region is essential. Using one of the iii truncation derivatives from this analysis, I have screened for proteins that interact with Bck2. One of these novel proteins is Mcm1, a global transcriptional activator of genes involved in cell cycle progression, mating gene transcription and metabolism. My studies suggest that Bck2 regulates the activity of Mcm1 in early G1 phase to activate the expression of SWI4, CLN3, and others. My evidence suggests that Bck2 competes for binding to a specific pocket on Mcm1 that is also bound by an Mcm1 repressor called Yox1. My findings suggest that CDK-independent pathways function through Bck2, in order to induce the initial suite of genes required for entry into the cell cycle.

Global quantitative analysis of genetic interactions provides a powerful approach for deciphering the roles of genes and mapping functional relationships amongst path-ways. Using colony size as a proxy for fitness, I developed a method for measuring ge-netic interactions from high-density arrays of yeast double mutants generated by synthet-ic genetic array (SGA) technology. I identified several experimental sources of systematic variation and developed normalization strategies to obtain accurate fitness measurements. I used this scoring method to map quantitative genetic interactions among 5.4 million yeast double mutants and generated the first functionally unbiased genetic interaction map of a eukaryotic cell. My map produced an unprecedented view of the cell in which genes of similar biological processes cluster together in coherent subsets and functionally interconnected bioprocesses map next to each other. We discovered several physiological and evolutionary gene features that are characteristic of genetic interaction hubs, and explored the relationship between genetic and protein-protein interaction networks. In particular, by comparing quantitative single and double mutant phenotypes, we identified specific cases of positive genetic interactions, termed genetic suppression, and constructed a global network of suppression interactions among protein complexes. I also demonstrated that an extensive and unbiased mapping of genetic interactions provides a key for interpreting chemical-genetic interactions and identifying drug targets. In addition, I used genome-wide SGA data to map profiles of genetic linkage along all sixteen yeast chromosomes. These linkage profiles recapitulated previously identified recombination patterns and uncovered an unexpected correlation between chromosome length and the extent of centromere-related recombination repression. These findings suggest a chromosome size-dependent mechanism for ensuring proper chromosome segregation and highlight the SGA methodology as a unique approach for systematic analysis of yeast meiotic recombination.

Eukaryotic organisms utilize post-translational modifications of highly conserved histone proteins to control gene expression programs. Methylation of lysine 4 on histone H3 (H3K4me) in particular, is thought to be associated with actively transcribed DNA. Paradoxically, recent evidence has suggested that H3K4me has a repressive function as well. Pho23, a member of the highly conserved ING family of tumour suppressor proteins, binds H3K4me and is a component of the gene repressive complex, Rpd3L. My genetic analysis suggests that Pho23 controls transcriptional repression via H3K4me and that Pho23 is itself regulated by the sequence-specific DNA-binding protein Ume6. Moreover, this Ume6-regulated function appears to be governed by Ume6 phosphorylation by Mck1, an evolutionarily conserved kinase. Finally, while Ume6/Pho23 are known to function together with the histone deacteylase Rpd3, my findings suggest the existence of an Rpd3-independent function for Pho23.

Eukaryotic organisms utilize post-translational modifications of highly conserved histone proteins to control gene expression programs. Methylation of lysine 4 on histone H3 (H3K4me) in particular, is thought to be associated with actively transcribed DNA. Paradoxically, recent evidence has suggested that H3K4me has a repressive function as well. Pho23, a member of the highly conserved ING family of tumour suppressor proteins, binds H3K4me and is a component of the gene repressive complex, Rpd3L. My genetic analysis suggests that Pho23 controls transcriptional repression via H3K4me and that Pho23 is itself regulated by the sequence-specific DNA-binding protein Ume6. Moreover, this Ume6-regulated function appears to be governed by Ume6 phosphorylation by Mck1, an evolutionarily conserved kinase. Finally, while Ume6/Pho23 are known to function together with the histone deacteylase Rpd3, my findings suggest the existence of an Rpd3-independent function for Pho23.

The goal of this research is to understand how three different parameters affect single chain variable fragment (scFv) binding capacity. The parameters that were varied include the number of variable complementarity determining regions (CDRs), the number amino acids used to diversify CDRs, and configuration of the structure. How the parameters affect the binding capacity will be tested using the yeast two hybrid assay against five different protein domains. Eight scFv libraries were generated; the genes expressing the scFvs were constructed and the CDRs were randomized using PCR amplification. Genes expressing scFvs were cloned, using the homologous gap repair mechanism in <i>Saccharomyces cerevisiae</I>. Representative members of scFv libraries were sequenced to confirm correct construction.<p> Library diversity was calculated from the library transformation efficiency. Transformation efficiency refers to the number of cells that grew at the time of transformation of the scFv gene into yeast cells. There were significant differences in the diversity of the scFv libraries, which created difficulty in comparing the library binding capacities. Sequencing the scFv libraries revealed that on average 50% of each library contained correct scFv sequences. The percent of correct sequences within each library was then used to calculate the functional diversity.<p> The yeast two-hybrid assay was used to screen the scFv libraries for interactions and to test binding capacity. The binding capacity of the scFv libraries was tested and compared in five different yeast two-hybrid assays using five protein domains as the targets for each screen. The screening results showed that in all cases cyclic scFv libraries had a statistically significant higher binding capacity than linear scFv libraries despite a diversity bias against the cyclic libraries. There was no clear trend in binding capacity with the other two parameters; however, the four amino acid three CDR libraries dominated over the other libraries in almost every screen.<p> Some of the scFvs isolated from the screens were expressed in <i>E. coli</i> and <i>S. cerevisiae</i>to analyze for proper expression and correct size. All the scFvs that were isolated and analyzed were the correct size and could be purified using a poly histidine tag.<p> Due to its bioaffinity and specificity, scFvs were constructed to profile disease patterns, and to identify potential drug targets. In addition to its original application to health-related studies, scFvs could also be extended to locate potential metabolic bottlenecks, to alter metabolic flux to enhance productivity, and regulate metabolic bionetworks. Industrial microorganisms are generally carrying more than two sets of chromosomes, making it difficult to be genetically engineered when conventional approaches are employed. With the availability of scFvs as reported in this thesis, we are able to design specific scFvs that selectively bind to target proteins, resulting in re-routing of metabolic flux within the microorganism, toward a high productivity of desired product. ScFvs can be applied to industrial microorganisms directly, leading to the development of new fermentation processes.

Much of the work on how the cell cycle is regulated has focused on Cyclin-Dependent Kinase (CDK)-mediated regulation of factors that control the coordinate expression of genes required for entry into the cell cycle. In Saccharomyces cerevisiae, SBF and MBF are related transcription factors that co-ordinately activate a large group of genes at the G1/S transition, and their activation depends on the Cln3-Cdk1 form of the cyclin-dependent kinase. However, cells are viable in the absence of Cln3, or SBF and MBF, indicating that other regulatory pathways must exist that activate the budding yeast cell cycle. The known CDK-independent pathways are made up of various phosphatases and plasma membrane transporters that control ion homeostasis in early G1 phase, a time when cells assess environmental growth conditions in order to commit to cell cycle entry. The enigmatic Bck2 protein is thought to act within these CDK-independent pathways, but the means by which it activates G1/S-regulated genes is not known. Bck2 contains little sequence homology to any known protein. In order to understand how CDK-independent pathways operate, I have studied the Bck2 protein using multiple approaches. In one approach, I have screened for novel SBF/MBF-binding proteins in order to determine if other non-CDK proteins, such as Bck2, might activate SBF and MBF. I have also investigated which region of Bck2 is required for its activity in order to determine if Bck2’s transcriptional activation region is essential. Using one of the iii truncation derivatives from this analysis, I have screened for proteins that interact with Bck2. One of these novel proteins is Mcm1, a global transcriptional activator of genes involved in cell cycle progression, mating gene transcription and metabolism. My studies suggest that Bck2 regulates the activity of Mcm1 in early G1 phase to activate the expression of SWI4, CLN3, and others. My evidence suggests that Bck2 competes for binding to a specific pocket on Mcm1 that is also bound by an Mcm1 repressor called Yox1. My findings suggest that CDK-independent pathways function through Bck2, in order to induce the initial suite of genes required for entry into the cell cycle.

La respuesta de adaptación de la levadura Saccharomyces cerevisiae a la alcalinización ambiental provoca una remodelación de su expresión génica. Una diana clave es el gen ENA1 que codifica una ATPasa de sodio, y cuya inducción por pH alcalino está mediada por las vías de calcineurina y el Rim101.En un estudio previo se identificaron dos regiones del promotor de ENA1 responsables de su respuesta al álcali, la ARR1 (Alkaline Responsive Region 1) que es calcineurina-dependiente y ARR2 que es calcineurina-independiente. En este trabajo restringimos la región responsable de la respuesta alcalina de ARR2 a un pequeño fragmento de 42 nucleótidos que denominamos MCIR (por Minimum Calcineurin Independent Response). MCIR contiene un elemento MIG, capaz de unir a los represores Mig1 y Mig2. Observamos que la respuesta a pH alcalino de la MCIR se anula en células que carecen de Snf1, la quinasa que regula la actividad represora de Mig1 en función de la disponibilidad de glucosa. En cambio, su respuesta se ve moderadamente reducida en cepas rim101, mientras que el doble mutante mig1 mig2 presenta altos niveles de expresión a pH alcalino. Además, la deleción de NRG1 resulta en una expresión elevada y la inducción de MCIR es marginal en el cuádruple mutante nrg1,2 mig1,2. También demostramos que Nrg1 se une al extremo 5' de la ARR2 in vitro e in vivo. Por lo tanto, la respuesta de ENA1 que es calcineurina independiente esta regulada por Rim101 (a través de Nrg1) y por Snf1 (a través de Nrg1 y Mig2). De esta manera, la inducción del promotor de ENA1 por pH alcalino en un mutante rim101snf1 en presencia del inhibidor químico de la calcineurina FK506 se anula totalmente. Por lo tanto, la respuesta transcripcional de ENA1 a estrés alcalino, integra tres vías de señalización, cuya importancia relativa es Snf1 > calcineurina > Rim101.La CK2 es una quinasa que está conservada en eucariotas y participa en diversos procesos celulares. En S. cerevisiae cepas que carecen de las subunidades reguladoras Ckb1 y/o Ckb2 de la CK2 son muy sensibles a cationes de litio y de sodio. En este estudio confirmamos observaciones anteriores que describían que la respuesta de ENA1 a estrés salino y alcalino está disminuida en células que carecen de Ckb1 y/o Ckb2. Además demostramos que los mutantes ckb son sensibles a pH alcalino. Las tres vías de señalización (Rim101, calcineurina, Snf1) responsables de la regulación de ENA1 en condiciones de estrés alcalino se examinaron para posibles interacciones con la CK2. Nuestros resultados sugieren que CK2 y calcineurina regulan la expresión de ENA1 de manera independiente. Además, mostramos que la deleción de RIM101 resulta en inducción de la expresión de ENA1, disminuida en condiciones de estrés salino, y que la deleción simultanea de CKB agrava solo ligeramente el defecto de las cepas rim101 en la expresión salina y alcalina de ENA1. Deleción del factor de transcripción Nrg1 en un fondo genético ckb resulta en niveles de expresión de ENA1 relativamente altos en condiciones de estrés salino y alcalino. Estos resultados, junto con datos anteriores que muestran que Nrg1 se fosforila por CK2 en estas condiciones de estrés, son compatibles con una supuesta interacción entre CK2 y la vía Rim101. Cabe destacar que la deleción de CKB repara el defecto que presentan las células snf1 en la expresión de ENA1 bajo estrés salino y alcalino y que los mutantes ckb1,2 snf1 presentan un crecimiento en litio mayor que la cepa salvaje, sugiriendo la existencia de una interacción compleja entre CK2 y Snf1. / The adaptive response of the yeast Saccharomyces cerevisiae to environmental alkalinization results in remodeling of gene expression. A key target is the gene ENA1, encoding a sodium ATPase, whose induction by alkaline pH was shown to integrate at least two different signals, mediated by the calcineurin and the Rim101 pathways.Early work in our laboratory identified two regions in the ENA1 promoter required for full response to alkalinization, the ARR1 (from Alkaline Responsive Region 1), whose response is calcineurin-dependent and ARR2, whose response is calcineurin-independent. In this work we have restricted the alkaline response of ARR2 to a smaller fragment of 42 nucleotides that we denominated MCIR (from Minimum Calcineurin Independent Response). MCIR contains a MIG element, able to bind Mig1 and Mig2 repressors. We observe that high pH-induced response driven from MCIR is largely abolished in cells lacking Snf1, the protein kinase that regulates repressor activity of Mig1 with respect to glucose availability, and results moderately reduced in a rim101 strain, whereas the double mig1 mig2 mutant presents high levels of expression upon alkaline stress. In addition, deletion of NRG1 results in increased expression and induction from the MCIR region is marginal in a quadruple nrg1,2 mig1,2 mutant. We also demonstrate that Nrg1 binds to the 5´-end of the ARR2 region in vitro and in vivo. Therefore, the calcineurin-independent response of the ENA1 gene is under the regulation of Rim101 (through Nrg1) and Snf1 (through Nrg1 and Mig2). Accordingly, induction by alkaline stress of the entire ENA1 promoter in a snf1 rim101 mutant in the presence of the calcineurin inhibitor FK506 is completely abolished. Thus, the transcriptional response to alkaline stress of the ENA1 gene integrates three different signaling pathways, whose relative potency is Snf1 > calcineurin > Rim101. CK2 is a well conserved kinase among eukaryotes that participates in many different cellular processes. In Saccharomyces cerevisiae, strains lacking the regulatory subunits Ckb1 and/or Ckb2 of this kinase are hypersensitive to sodium and lithium cations. However, the mechanism by which CK2 affects yeast salt tolerance is not known. In this study we confirm previous observations that the alkaline and saline response of ENA1 is decreased in strains lacking Ckb1 and/or Ckb2. Furthermore, we show that ckb mutants are sensible at alkaline pH. The three pathways (Rim101, calcineurin and Snf1) responsible for ENA1 regulation under alkaline stress conditions were examined for any possible interaction with CK2. Our results suggest that CK2 and calcineurin regulate ENA1 expression under alkaline and lithium stress in an independent fashion. Moreover, we show that deletion of RIM101 results in decreased ENA1 induction under lithium stress conditions and that simultaneous mutation of CKB only slightly aggravates the defect that presents the rim101 strain in ENA1 alkaline and saline expression. Mutation of the Nrg1 transcription factor in a ckb background leads to relatively high levels of ENA1 expression under alkaline and lithium stress conditions. These results, together with previous data showing that Nrg1 is phosphorylated by CK2 under these stress conditions, support a possible interaction between the CK2 and the Rim101 pathways. Remarkably, deletion of CKB partially counteracts the defect that snf1 mutants present in ENA1 saline and alkaline expression, and ckb1,2 snf1 mutants are as tolerant as wild type cells to lithium ions, revealing a complex interaction between CK2 and Snf1.