The yeast Saccharomyces cerevisiae is a prevalent system for studying gene regulation because of the ease of experimental methods and the simplicity of its gene structure. Here, I describe my work that aims to identify the sequences and factors responsible for demarcating genes within the genome sequence. With comparative genomics and RNA-Seq, we are quite adept at identifying gene structure. However, the cell does not have access to this kind of information. Instead, it uses the specificities of DNA- and RNA-binding proteins to read and interpret the sequence of the genome; it is this process that I have studied in my thesis. In the first chapter, I describe my work collecting yeast transcription factor specificities. I evaluated these specificities using available confirmatory data to determine which one best represents the transcription factor; this gave me a high-confidence description of what DNA sequences yeast transcription factors recognize. Next, I look for over- and under-represented DNA words within and surrounding gene structures and attempt to explain these in terms of the specificities of known factors or other known biological phenomena. I found that the sequences in the 5' and 3' gene ends are very similar and can often be explained by similar phenomena. I also provide evidence that several factors may be involved in regulating transcription in non-canonical ways. In the final chapter, I describe my efforts to build a model that uses my collection of transcription factor specificities as well as DNA structural features to identify gene structure as we think the cell would. This model is comprised of two classifiers that identify mRNA initiation and termination sites, and these are used to provide evidence to a hidden Markov model that predicts gene structure. I test that the predicted determinants of promoter structure are sufficient to initiate transcription, and that the transcription arising from randomly-generated DNA is correctly predicted. Overall, my work demonstrates that the sequence elements demarcating yeast genes are relatively simple in nature, which has implications for how transcription is regulated and how genes evolve.

In the last few years few reports on the incidence of yeasts from natural sources have been published, and only a few of these investigations concerned more than one source of isolation. Most investigations on microflora of various habitats were directed towards the number rather than the types of bacteria, yeasts and fungi. Studies on wild yeasts were directed toward their fermentative capabilities and the identification of these yeasts was neglected. [...]

Deletion mutant collections allow for the systematic study of gene function by linking a genotype to a phenotype. Furthermore, these collections permit the parallel and quantitative study of phenotypes, which is the foundation of functional genomics. I begin by summarizing the methods used and data derived from the field of functional genomics using the Baker’s yeast Saccharomyces cerevisiae, and provide important background information on the origins of the filamentous growth-competent S.cerevisiae strain Σ1278b, and the developmental process of fungal dimorphism. I describe my efforts in creating a complete deletion mutant collection in the filamentous growth-competent S.cerevisiae strain Σ1278b, and the subsequent phenotypic analysis of that deletion mutant collection. By quantitatively measuring mutant phenotypes of cells undergoing haploid invasive growth, biofilm mat formation and diploid pseudohyphal growth, I studied the clinically relevant developmental process of fungal dimorphism. I present the first genome-wide and quantitative phenotypic analysis of fungal dimorphism and identify a novel transcription factor encoded by the open reading frame YDL233W, which I named FMR1for Filamentation Master Regulator 1. By performing genetic, cell biological, biochemical, and expression analysis, I demonstrate that Ydl233w acts by forming a protein complex with the DNA-binding transcription factors Flo8 and Mss11 and this complex binds to a specific element within the promoter of the surface adherence mediating gene FLO11. I directly compare the essential gene sets between the Σ1278b deletion collection and the reference deletion collection made in the S288c genetic background completed by the Yeast Deletion Consortium in 2002. I find that most essential genes are shared between these two strains but a number of genes are essential for viability in only one genetic background, a phenomenon termed conditional essentiality. I describe the genetic basis of conditional essentiality as a consequence of the complex inheritance of background-specific alleles. Lastly, I summarize the scientific advancements of my research using the Σ1278b deletion collection, and highlight some potential applications for both the data derived from my research and the deletion mutant collection itself. The Σ1278b deletion collection provides a valuable resource for yeast geneticists, evolutionary biologists, researchers of fungal disease, and researchers interested in modeling the genetics that underlie complex traits and diseases.

Peroxisomes are virtually found in all eukaryotic cells, but unlike mitochondria and chloroplasts, they do not contain DNA or a protein secretory apparatus. Therefore, all of their proteins must be imported by a process called peroxisomal biogenesis. This requires a group of protein factors referred to as peroxins which are encoded by the pex genes. Currently, there are approximately thirty-two known peroxisomal proteins. Among all the peroxisomal proteins, two enzymes namely GPD1, LYS1 and a peroxin, PEX7 were selected for the research. GPD1 is a NAD+ -dependent glycerol 3-phosphate dehydrogenase1 that catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate which is crucial for growth under osmotic stress. Its purification was achieved using ion exchange chromatography and the pure protein was crystallized for structure determination. Diffraction data sets were obtained to a resolution of 2.2 Å which were used to solve the C-terminal portion of the structure. Unfortunately, the N-terminal portion remained disordered. LYS1 is the terminal enzyme of α-aminoadipate pathway and catalyzes the reversible NAD-dependent oxidative cleavage of saccharopine to yield L-lysine and α-ketoglutarate. The purification of LYS1 was carried out using affinity chromatography. Another protein, PEX7 is responsible for peroxisome biogenesis by importing newly synthesized proteins bearing PTS2 (peroxisome targeting signal sequence2) into peroxisomes. PEX7 presented the greatest challenge among the three proteins at both the expression stage and the purification stage. Its soluble fraction was purified using ion exchange and affinity chromatographies, although the final yield was too low for crystallization trials. A much large proportion of the protein was found in the insoluble cell debris and attempts were made to purify this fraction after denaturation. An alternative, protocol involving the formation of a GPD1-PEX7 complex proved to be effective route to co-purification of the two proteins and crystallization trials are proceeding. Having known the structures of peroxisomal proteins, it would be helpful for studying the development and maintenance of the organelle related to its metabolic diseases in the eukaryotic cells.

No description available.

No description available.

The translation of a series of <I>cat </I>mRNAs containing either the <I>HSP26 </I>5'- leader or various artificial 5'-leaders (Vega Laso <I>et al., </I>1993) were analysed during growth. From this study, the relative translational efficiencies of these mRNAs were shown to vary from 2% to 100% during mid-exponential phase as observed previously (Vega Laso <I>et al.,</I>1993). However, upon analysing the translation of the various <I>cat </I>constructs during growth, their relative translational efficiencies did not change significantly as yeast cells approached stationary phase. A new set of <I>lacZ </I>mRNAs carrying different natural 5'-leaders (<I>PGK1, PYK1, RpL3, Rp29, GDH1, HSP26, HSP12 </I>and <I>TH14</I>) were constructed. These <I>lacZ </I>mRNAs were placed under the control of the promoters taken from genes expressed during different phases of growth (<I>PGK1 </I>and <I>HSP26</I>). Even though the various <I>PGK1-lacZ </I>and <I>HSP26-lacZ </I>mRNAs were translated differentially, the ability of these mRNAs to compete for the translational apparatus did not appear to change as cells entered stationary phase. The translation of a variety of natural mRNAs encoding a wide range of functions was then analysed by determining their polysomal distribution at various points during growth. Irrespective of the growth phase, a large proportion of each mRNA was detected in the polysomal fractions, suggesting that they continued to be translated in stationary phase. Overall, the data strongly suggest that, under the conditions tested, an excess translational capacity exists in stationary phase yeast cells. Hence gene expression may be largely regulated by transcription upon entry to stationary phase.

The aim of this work was to establish the relative contribution of genetic regulation of the <I>PYK1</I>, <I>PFK1</I> and <I>PFK2</I> genes to the control of glycolysis. A series of isogenic mutant strains were constructed where the promoters and 5' untranslated sequences of the <I>PYK1, PFK1</I> and <I>PFK2</I> genes were replaced with those from <I>PGK1</I>. In addition , a second series of mutant strains were constructed where synthesis of Pyk1p and Pflkp was driven by the <I>PGK1_Δuas</I> promoter. These latter series of mutants were designed to contain weak expression of Pyklp and Pflkp. Analysis of UKC1 (<I>PGK::PYK1)</I> in shake flask cultures revealed similar growth rates on glucose and on lactate and similar rates of ethanol production and glucose consumption to those of the wild-type strain. This suggested that the native genetic regulation did not appear to play a significant role in the control of glycolysis. Nonetheless, analysis of this strain in the fermentor revealed that genetic regulation of <I>PYK1</I> may be important in co-ordinating Pyk1p synthesis, under the conditions studied. Analysis of YKC11 (<I>PGK_Δuas::PYK1)</I> in both shake flask and fermentor experiments showed that genetic control was important in maintaining Pyk1p levels in order to sustain glycolytic flux. Shake flask analysis of the single and double <I>PFK</I> mutants under the control of the <I>PGK1</I> promoter revealed that the genetic regulation of the <I>PFK1</I> and <I>PFK2</I> genes did not appear to be important in the control of glycolysis. Weak expression of the <I>PFK1</I> and <I>PFK2</I> genes, under the control of the <I>PGK_Δuas</I> promoter showed the importance of genetic regulation in maintaining Pflkp levels to support glycolytic flux, under the conditions studied.

The four cyanogen bromide fragments of yeast 3-phosphoglycerate kinase (PGK) have been isolated and characterised. After digestion with proteolytic enzymes and specific cleavage reagents, the resuting peptides were purified by various methods and sequenced using the manual dansyl-Edman technique and the Beckman 890C liquid phase sequencer. The entire sequence of yeast PGK (415 residues) has been determined using a combination of amino acid sequence data and nucleotide sequence data. Nucleotide sequence data were supplied by Dr. A. Kingsman, University of Oxford. The yeast PGK sequence data have been fitted tothe 2.S electron density map and the nucleotide binding site has been fully characterised. The fitting of sequence data to the electron density map permitted identification of additional electron density which is probably attributable to the triose phosphate substrate. This binding site has also been characterised. The construction of the 1g:1cm model of yeast PGK permitted interpretation of chemical modification, NMR, hydrodynamic and kinetic data from a structural point of veiw, thereby allowing a catalytic mechanism to be proposed. This mechanism involves a major conformational change, triggered by the breaking of a salt-bridge between glutamate 190 and histidine 388 concommittant with the formation of the ternary enzyme-substrates complex. The conformational change brings the two substrates into close proximity, thereby permitting the in-line, direct, associative,phosphoryl transfer reaction to take place. The hydrodynamic properties of yeast PGK were examined in order to determine conditions under which PGK adopted its closed , catalytically active conformation. The solubility of yeast PGK in organic solvents commonly used as crystallising media was examined and experiments performed which were designed to crystallise a) the closed conformation of yeast PGK, and b) the substrate-free form of yeast PGK. No crystals have yet been observed in these experiments.

The yeast Saccharomyces cerevisiae is a prevalent system for studying gene regulation because of the ease of experimental methods and the simplicity of its gene structure. Here, I describe my work that aims to identify the sequences and factors responsible for demarcating genes within the genome sequence. With comparative genomics and RNA-Seq, we are quite adept at identifying gene structure. However, the cell does not have access to this kind of information. Instead, it uses the specificities of DNA- and RNA-binding proteins to read and interpret the sequence of the genome; it is this process that I have studied in my thesis. In the first chapter, I describe my work collecting yeast transcription factor specificities. I evaluated these specificities using available confirmatory data to determine which one best represents the transcription factor; this gave me a high-confidence description of what DNA sequences yeast transcription factors recognize. Next, I look for over- and under-represented DNA words within and surrounding gene structures and attempt to explain these in terms of the specificities of known factors or other known biological phenomena. I found that the sequences in the 5' and 3' gene ends are very similar and can often be explained by similar phenomena. I also provide evidence that several factors may be involved in regulating transcription in non-canonical ways. In the final chapter, I describe my efforts to build a model that uses my collection of transcription factor specificities as well as DNA structural features to identify gene structure as we think the cell would. This model is comprised of two classifiers that identify mRNA initiation and termination sites, and these are used to provide evidence to a hidden Markov model that predicts gene structure. I test that the predicted determinants of promoter structure are sufficient to initiate transcription, and that the transcription arising from randomly-generated DNA is correctly predicted. Overall, my work demonstrates that the sequence elements demarcating yeast genes are relatively simple in nature, which has implications for how transcription is regulated and how genes evolve.