A Comparison of the 5' Flanking Regulatory Regions of the Yeast Triose Phosphate Isomerase and Pyruvate Kinase Genes
Dadabay, Carolyn Y.
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One means by which eukaryotes regulate the expression of genes is by controlling the rate at which transcription is initiated. Sequences which lie in the regions 5' to gene coding sequences have been implicated in the efficiency with which RNA polymerase II binds to the DNA and initiates transcription. These sequences madiate the levels of gene expression in response to environmental stimuli. Highly expressed genes, such as those coding for yeast glycolytic enzymes, carry 5' flanking regions which direct the initiation of transcription at high levels. In yeast, these regions have been termed upstream activation sequences (UAS), and are induced by high levels of fermentable carbon sources. In yeast, the study of these regions is accomplished by in vitro fusions of 5' regulatory sequences to the lac Z gene of Escherichia coli, using hybrid plasmid vectors. The level of B-galactosidase expression then falls under the control of yeast regulatory elements, and can easily be detected using photometric assays. In this project, the construction of such plasmid vectors was attempted, but could not be completed due to technical difficulties in isolating DNA sequences. Two previously constructed vectors, containing 5' fusions of the pyruvate kinase (PYK) and triose phosphate isomerase (TPI) genes fused to lac Z were introduced into the yeast Saccharomyces cerevisiae, and the resulting B-galactosidase activity compared. Initial results showed that the TPI fusion directed higher levels of expression; however, this greater level of activity cannot be attributed solely to more efficient initiation of transcription until the actual maintenance of the plasmid vectors in yeast has been studied and compared.