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Ethanol can be used as a complete fuel or as an octane enhancer, and has the advantages of being renewable and environmentally friendly. Ethanol produced by a fermentation process, generally referred to as bioethanol, is considered to be a partial solution to the worldwide energy crisis. Traditionally, industrial bioethanol fermentation involves two major steps: starch hydrolysis and fermentation. Since the key microorganism, Saccharomyces cerevisiae, lacks amylolytic activity and is unable to directly utilize starch for proliferation and fermentation, it requires intensive amount of energy and pure starch hydrolyzing enzymes to gelatinize, liquefy and dextrinize the raw starch before fermentation. It has been suggested that genetically engineered yeast which expresses amylolytic enzymes could potentially perform simultaneous starch hydrolysis and fermentation. This improvement could greatly reduce the capital and energy costs in current bioethanol producing plants and make bioethanol production more economical. In this project, a novel yeast strain of Saccharomyces cerevisiae was genetically engineered in such a way that barley alpha-amylase was constitutively expressed and immobilized on the yeast cell surface. This particular alpha-amylase was selected based on its superior kinetic properties and its pH optimum which is compatible with the pH of yeast culture media. The cDNA encoding barley Ñ-amylase, with a secretion signal sequence, was fused to the cDNA encoding the C-terminal half of a cell wall anchoring protein, alpha-agglutinin. The fusion gene was cloned downstream of a constitutive promoter ADH1 in a yeast episomal plasmid pAMY. The pAMY harbouring yeast showed detectable amylolytic activity in a starch plate assay. In addition, alpha-amylase activity was detected only in the cell pellet fraction and not in the culture supernatant. In batch fermentation studies using soluble wheat starch as sole carbon source, even though pAMY harbouring yeast was able to hydrolyse soluble starch under fermentation conditions, no ethanol was produced. This was probably due to insufficient alpha-amylase activity which resulted from the enzyme being anchored on the cell wall by alpha-agglutinin. Further research using alternative cell surface anchoring system might be able to produce yeast with industrial applications.

Ethanol can be used as a complete fuel or as an octane enhancer, and has the advantages of being renewable and environmentally friendly. Ethanol produced by a fermentation process, generally referred to as bioethanol, is considered to be a partial solution to the worldwide energy crisis. Traditionally, industrial bioethanol fermentation involves two major steps: starch hydrolysis and fermentation. Since the key microorganism, Saccharomyces cerevisiae, lacks amylolytic activity and is unable to directly utilize starch for proliferation and fermentation, it requires intensive amount of energy and pure starch hydrolyzing enzymes to gelatinize, liquefy and dextrinize the raw starch before fermentation. It has been suggested that genetically engineered yeast which expresses amylolytic enzymes could potentially perform simultaneous starch hydrolysis and fermentation. This improvement could greatly reduce the capital and energy costs in current bioethanol producing plants and make bioethanol production more economical. In this project, a novel yeast strain of Saccharomyces cerevisiae was genetically engineered in such a way that barley alpha-amylase was constitutively expressed and immobilized on the yeast cell surface. This particular alpha-amylase was selected based on its superior kinetic properties and its pH optimum which is compatible with the pH of yeast culture media. The cDNA encoding barley Ñ-amylase, with a secretion signal sequence, was fused to the cDNA encoding the C-terminal half of a cell wall anchoring protein, alpha-agglutinin. The fusion gene was cloned downstream of a constitutive promoter ADH1 in a yeast episomal plasmid pAMY. The pAMY harbouring yeast showed detectable amylolytic activity in a starch plate assay. In addition, alpha-amylase activity was detected only in the cell pellet fraction and not in the culture supernatant. In batch fermentation studies using soluble wheat starch as sole carbon source, even though pAMY harbouring yeast was able to hydrolyse soluble starch under fermentation conditions, no ethanol was produced. This was probably due to insufficient alpha-amylase activity which resulted from the enzyme being anchored on the cell wall by alpha-agglutinin. Further research using alternative cell surface anchoring system might be able to produce yeast with industrial applications.

The foundation of Luecke’s course M: 396 Geometry and Topology is that collaboration amongst mathematicians and biologists caused tremendous gains in DNA research. The field of topology has led to significant strides in understanding of the topological properties of the genetic molecule DNA. Through the integration of biological phenomena and knowledge of topology and Euclidean geometry, biologists can describe and quantize enzyme mechanisms and therefore determine enzyme mechanisms causing the changes. Understanding mathematical applications in contexts outside of mathematics on any level helps to explain why mathematics is a core content area in primary and secondary education. Requiring secondary educators to take such a course could result in mathematics taught with real world application on the secondary level as well as on the graduate level, as shown in Luecke’s course. / text

Benzene is a ubiquitous environmental pollutant and a known human leukemogen. Early-life exposure to environmental carcinogens, including benzene, may lead to genomic instability in the fetus, ultimately leading to an increased risk for the development of childhood cancers including leukemia. It is possible that exposure to benzene results in DNA damage that may either be left unrepaired or be repaired erroneously causing genotoxicity. The first objective of this study was to determine if exposure of fetal hematopoietic cells to p-benzoquinone, a known toxic metabolite of benzene, increased DNA recombination in the pKZ1 model of mutagenesis. A significant increase in recombination was observed following exposure to 25 μM and 50 μM p-benzoquinone for 2, 4, 8, and 24 hours. A significant increase in recombination was also observed following exposure to 25 μM p-benzoquinone for 30 min, 45 min, and 1 hour, but not 15 min as compared to vehicle alone. Secondly, this study determined if exposure of fetal hematopoietic cells to p-benzoquinone resulted in DNA damage using γ-H2A.X as a marker for DNA double strand breaks and 8-hydroxy-2’-deoxyguanosine as a marker of oxidative DNA damage. A significant increase in γ-H2A.X foci formation was observed following exposure to 25 μM p-benzoquinone for 30 min, 45 min and one hour. Exposure of fetal hematopoietic cells to 25 μM p-benzoquinone did not significantly increase oxidative DNA damage at any of the examined time points. The third objective of this study was to determine whether or not reactive oxygen species were involved in the observed increase in DNA damage and recombination. Exposure to 25 μM p-benzoquinone for 15 min and 30 min, but not 45 min or one hour, led to an increase in reactive oxygen species production as measured by 5-(and-6)-chloromethyl-2-7-dichlorodihydrofluorescein diacetate fluorescence. Additionally, pretreatment with 400 U/mL PEG-catalase, an antioxidative enzyme, attenuated the increases in both DNA recombination and DNA double strand breaks as compared to treatment with p-benzoquinone alone. These studies indicate that p-benzoquinone is able to induce DNA damage and recombination in fetal hematopoieitic cells and that reactive oxygen species and oxidative stress may be important in the mechanism of toxicity. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2010-07-23 15:44:05.381

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Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 121-134).

Motivated by genome rearrangements that take place in some species of ciliates we introduce a combinatorial model for these processes based on spatial graphs. This model builds up on two earlier models for pointer-guided DNA recombination (intramolecular model introduced in [22, 23] and intermolecular model introduced in [35, 36]) and is influenced by a molecular model for RNA guided DNA recombination (introduced in [2]). Despite their differences, the intermolecular and intramolecular model formalize the recombination events through rewriting operations applied on formal words. Both models predict the same set of molecules as a result of correct rearrangement. Here, we give an algorithm that for an input of scrambled gene structure outputs a set of strings which represents the expected set of molecules after complete assembly. Moreover, we prove that both the set of all realistic words (words that model a possible gene structure) and the set of all nonrealistic words are closed under the rewriting operations in the intramolecular model. We investigate spatial graphs that consist of 4-valent rigid vertices, called assembly graphs. An assembly graph can be seen as a representation of DNA molecule during certain recombination processes, in which 4-valent vertices represent molecular alignment of the recombination sites. We introduce a notion of polygonal path in assembly graph as a model for a single gene. Polygonal paths are defined as paths that make “90◦ -turn” at each vertex of the assembly graph and define smoothing of the vertices visited by the paths. Such vertex smoothing models a homologous DNA recombination. We investigate the minimal number of polygonal paths that visit all vertices of a given graph exactly once, called assembly number. We prove that for every positive integer n there is assembly graph with assembly number n. We also study the relationship between the number of vertices in assembly graph and its assembly number. One of the results is that every assembly graph with assembly number n has at least 3n − 2 vertices. In addition, we show that there is an embedding in three dimensional space of each assembly graph with a given set of polygonal paths, such that smoothing of vertices with respect to the polygonal paths results in unlinked circles. We study the recombination strategies by subsets of vertices. Such a subset is called a successful set if smoothing of all vertices from the set with respect to a polygonal path results in a graph that contains the polygonal path in a single component. We characterize the successful sets in a given assembly graph by a notion of complementary polygonal path. Furthermore, we define a smoothing strategy in assembly graph relative to a polygonal path as a sequence of successful sets which model a successive DNA recombinations for correct gene assembly. Recent experimental results suggest that there might be different pathways for unscrambling a gene. These results lead to a mathematical model for gene recombination that builds upon the intermolecular model. We introduce assembly words as a formalization of a set of linear and circular DNA molecules. Assembly words are partially ordered, so that any linearly ordered subset models a pathway for gene rearrangement. We suggest two different pathways for unscrambling of the actin I gene in O.Trifallax and we prove that they are the only theoretically possible pathways.

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This diploma thesis is focused on topic of genetically modified organisms and their use in the practical sectors of human life. Theoretical part of the thesis defines general terms GMO, plasmid, genetic engineering, biotechnology. It also records historical milestones relating to the problematic, deals with individual techniques of genetic engineering and briefly states legislative procedures in context of dealing with GMO. It gives examples of transgenic organisms and summarizes advantages and disadvantages of their use.Practical part of the thesis contains educational program called "Genetically modified organisms", which was conceived by the author and includes a draft of a lesson inclusive of teaching materials - powerpoint presentations, worksheets, interactive worksheets, auxiliary text for teacher and written preparation. Research part deals with high school students change of view about using GMOs after completing the educational program. Due to analysis was proven that most of the attitudes and knowledge about GMO was changed after completing the educational program (for example in issues of willingness to consume GM food and animal products, perception of advantages and disadvantages etc.) Data was still unchanged in questions which cannot be affected by the program (control of food packaging or...