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.

No description available.

No description available.

No description available.

The budding yeast Saccharomyces cerevisiae is the most well characterized model organism for systematic analysis of fundamental eukaryotic processes. Approximately 19% of S. cerevisiae genes are considered essential. Essential genes tend to be more highly conserved from yeast to humans when compared to nonessential genes. The set of essential yeast genes spans diverse biological processes and while the primary role of most essential yeast genes has been characterized, the full breadth of function associated with essential genes has not been examined, due, at least in part, to the lack of adequate genetic reagents for their conditional and systematic perturbations. To systematically study yeast essential gene functions using synthetic genetic array analysis and to complement the current yeast deletion collection, I constructed a collection of temperature-sensitive yeast mutants consisting of 795 ts strains, covering 501 (~45%) of the 1,101 essential yeast genes, with ~30% of the genes represented by multiple alleles. This is the largest collection of isogenic ts yeast mutants constructed to date. I confirmed the correct integration of over 99% of the ts alleles using PCR-based strategy and the identity of the ts allele by complementation of the ts phenotype with its cognate plasmid. The ts mutant collection was characterized by high-resolution profiling of the temperature sensitivity of each ts strain, distribution analysis of gene ontology molecular function and biological process, and comparison of ts allele strains to the strains carrying Tet-repressible alleles of essential genes. The results demonstrated that the ts collection is a powerful reagent for the systematic study of yeast essential gene functions and provides a valuable resource to complement the current yeast deletion collection. I validated and demonstrated the usefulness of the ts collection in a number of different ways. First, I carried out detailed temperature profiling of each mutant strain using liquid growth assays and found that ts mutants that define particular biological pathways often show highly similar profiles. Second, I showed that the ts mutant array can be used to screen compounds for suppression of growth defects and thus is useful for exploration of chemical-genetic interactions. Third, I demonstrated that the ts collection represents a key reagent set for genetic interaction analysis because essential genes tend to be highly connected hubs on the global genetic network. Fourth, I further validated the ts array as a key resource for quantitative phenotypic analysis by using a high-content screening protocol to score six different fluorescent markers, diagnostic for different subcellular compartments or structures, in hundreds of different mutants. Quantification of the marker behaviour at the single-cell level enabled integration of this data set to generate a morphological profile for each ts mutant to reveal both known and previously unappreciated functions for essential genes, including roles for cohesion and condensin genes in spindle disassembly.

In a previous study, three determinants of fitness were identified as mutant alleles (each designated "e") that arose in yeast populations propagated in divergent environments. In a low-glucose environment, MDS3e and MKT1e interacted positively to confer a fitness advantage. PMA1e from a high-salt environment interacted negatively with MKT1e in low glucose, indicating a mechanism of reproductive isolation. In this thesis, I demonstrated that the negative interaction between PMA1e and MKT1e is mediated by alteration in intracellular pH and likely by a delay of the cell division cycle, while the positive interaction between MDS3e and MKT1e is mediated by changes in gene expression affecting glucose transporter genes. I also confirmed the evolutionary significance of the positive interaction by showing that an MDS3e genetic background is required for the recapitulation of the MKT1e mutation. Collectively, these results illustrate how epistasis can play a central role in both adaptation and speciation.

The budding yeast Saccharomyces cerevisiae is the most well characterized model organism for systematic analysis of fundamental eukaryotic processes. Approximately 19% of S. cerevisiae genes are considered essential. Essential genes tend to be more highly conserved from yeast to humans when compared to nonessential genes. The set of essential yeast genes spans diverse biological processes and while the primary role of most essential yeast genes has been characterized, the full breadth of function associated with essential genes has not been examined, due, at least in part, to the lack of adequate genetic reagents for their conditional and systematic perturbations. To systematically study yeast essential gene functions using synthetic genetic array analysis and to complement the current yeast deletion collection, I constructed a collection of temperature-sensitive yeast mutants consisting of 795 ts strains, covering 501 (~45%) of the 1,101 essential yeast genes, with ~30% of the genes represented by multiple alleles. This is the largest collection of isogenic ts yeast mutants constructed to date. I confirmed the correct integration of over 99% of the ts alleles using PCR-based strategy and the identity of the ts allele by complementation of the ts phenotype with its cognate plasmid. The ts mutant collection was characterized by high-resolution profiling of the temperature sensitivity of each ts strain, distribution analysis of gene ontology molecular function and biological process, and comparison of ts allele strains to the strains carrying Tet-repressible alleles of essential genes. The results demonstrated that the ts collection is a powerful reagent for the systematic study of yeast essential gene functions and provides a valuable resource to complement the current yeast deletion collection. I validated and demonstrated the usefulness of the ts collection in a number of different ways. First, I carried out detailed temperature profiling of each mutant strain using liquid growth assays and found that ts mutants that define particular biological pathways often show highly similar profiles. Second, I showed that the ts mutant array can be used to screen compounds for suppression of growth defects and thus is useful for exploration of chemical-genetic interactions. Third, I demonstrated that the ts collection represents a key reagent set for genetic interaction analysis because essential genes tend to be highly connected hubs on the global genetic network. Fourth, I further validated the ts array as a key resource for quantitative phenotypic analysis by using a high-content screening protocol to score six different fluorescent markers, diagnostic for different subcellular compartments or structures, in hundreds of different mutants. Quantification of the marker behaviour at the single-cell level enabled integration of this data set to generate a morphological profile for each ts mutant to reveal both known and previously unappreciated functions for essential genes, including roles for cohesion and condensin genes in spindle disassembly.

In a previous study, three determinants of fitness were identified as mutant alleles (each designated "e") that arose in yeast populations propagated in divergent environments. In a low-glucose environment, MDS3e and MKT1e interacted positively to confer a fitness advantage. PMA1e from a high-salt environment interacted negatively with MKT1e in low glucose, indicating a mechanism of reproductive isolation. In this thesis, I demonstrated that the negative interaction between PMA1e and MKT1e is mediated by alteration in intracellular pH and likely by a delay of the cell division cycle, while the positive interaction between MDS3e and MKT1e is mediated by changes in gene expression affecting glucose transporter genes. I also confirmed the evolutionary significance of the positive interaction by showing that an MDS3e genetic background is required for the recapitulation of the MKT1e mutation. Collectively, these results illustrate how epistasis can play a central role in both adaptation and speciation.