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Unformatted text preview: Lecture 21 Eukaryotic Genes and Genomes III Cis-acting sequences In the last lecture we considered a classic case of how genetic analysis could be used to dissect a regulatory mechanism. This analysis was contingent upon having clean phenotypes associated with the isolated mutants; e.g., mutations in the Gal80 gene produce a phenotype of constitutive Gal1 expression. However, it is sometimes very difficult to identify regulatory proteins by isolating mutants, because regulators that influence the expression of a wide variety of genes might be essential (i.e., mutations in these could be lethal), or their mutant phenotypes may be extremely complex and difficult to interpret. One solution to this has been to work backwards from the cis-acting promoter sequences for particular genes to identifying the proteins that bind to them. Lets take the Gal1 gene as an example. We have considered the fact that in the presence of galactose the Gal1 gene is transcriptionally upregulated (along with other Gal genes). What I havent told you is the fact that if glucose is present in addition to galactose, the induction of the Gal genes simply does not occur! This is known as glucose repression . This makes physiological sense because glucose is a more efficient energy source for yeast, and is therefore the preferred carbon source over galactose. Why bother metabolizing galactose as long as glucose is present? In fact, glucose represses a very large number of genes whose products metabolize a wide range of carbon sources (sucrose, maltose, galactose etc) that are less energy efficient than glucose, as well as repressing a whole host of other genes. GLUCOSE REPRESSION GLUCOSE REPRESSION It seems reasonable to expect that there is a transcriptional repressor that responds to glucose levels; this repressor would be ineffective when glucose is low or absent, and effective when glucose is present. It also seems reasonable that one could isolate trans- acting mutants that fail to repress galactose-induced Gal gene expression in the presence of glucose. However, it turns out that the very fact that glucose represses such a large number of different genes made it difficult to identify such mutants. + galactose and glucose + galactose and glucose + galactose and glucose + galactose and glucose Instead of looking for mutants that fail to execute glucose repression at the Gal1 gene, studies of the Gal1 promoter region itself provided the key to dissecting the mechanism of glucose repression. Specifically, the Gal1 promoter region was fused to the E. coli LacZ gene, on a plasmid that can replicate autonomously in S. cerevisiae . It was first important to establish that regulation of LacZ ( -galactosidase) from the plasmid mirrored the regulation of Gal1 (galactokinase) from its chromosomal locus; i.e., that galactosidase was induced by galactose in the absence of glucose, but not in its presence....
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- Fall '04