7.03, 2005, LECTURE 21 EUKARYOTIC GENES AND GENOMES II In the last lecture we considered the structure of genes in eukaryotic organisms and went on to figure out a way to identify S. cerevisiae genes that are transcriptionally regulated in response to a change in environment. The ability to regulate gene expression in response to environmental cues is a fundamental requirement for all living cells, both prokaryote and eukaryote. We considered how many genes each organism has, about 4,000 for E. coli, 6,000 for yeast and a little over 20,000 for mouse and humans. But only a subset of these genes is actually expressed at any one time in any particular cell. For multicellular organisms this becomes even more apparent…it is obvious that skin cells must be expressing a different set of genes than liver cells, although of course there must be a common set of genes that are expressed in both cell types; these are often called housekeeping genes. There are a number of ways that gene regulation in eukaryotes differs from gene regulation in prokaryotes. • Eukaryotic genes are not organized into operons. • Eukaryotic regulatory genes are not usually linked to the genes they regulate. • Some of the regulatory proteins must ultimately be compartmentalized to the nucleus, even when signaling begins at the cell membrane or in the cytoplasm. • Eukaryotic DNA is wrapped around nucleosomes Today we will consider how one can use genetics to begin to dissect the mechanisms by which gene transcription can be regulated. For this we will take the example of the yeast GAL genes in S. cerevisiae. GALACTOSE METABOLISM IN YEAST ReactionD-galactoseD-galactose-1-phosphateUDP-D-galactoseUDP-D-glucoseD-glucose-1-phosphateD-glucose-6-phosphateGLYCOLYSISEnzymeGalactokinaseGalactose transferaseGalactose epimeraseUDP-glucosePhosphorylasePhosphoglucomutaseGeneGAL1GAL7GAL10ReactionD-galactoseD-galactose-1-phosphateUDP-D-galactoseUDP-D-glucoseD-glucose-1-phosphateD-glucose-6-phosphateGLYCOLYSISEnzymeGeneGAL1GAL7GAL10ReactionD-galactoseD-galactose-1-phosphateUDP-D-galactoseUDP-D-glucoseD-glucose-1-phosphateD-glucose-6-phosphateGLYCOLYSISGalactokinaseGalactose transferaseGalactose epimeraseUDP-glucosePhosphorylasePhosphoglucomutaseEnzymeGeneGAL1GAL7GAL10GalactokinaseGalactose transferaseGalactose epimeraseUDP-glucosePhosphorylasePhosphoglucomutaseGAL1 encodedGAL1, GAL7, GAL10 transcription all induced in thepresence of glucose. How is this achieved. GAL1 encodedGAL1, GAL7, GAL10 transcription all induced in thepresence of glucose. How is this achieved.Once a gene has been identified as being inducible under certain inducing conditions, in this case in the presence of galactose, we can begin to dissect the regulatory mechanism by isolating mutants; i.e., mutants that constitutively express the GAL genes even in the absence of galactose, and mutants that have lost the ability to induce the GAL genes in the presence of galactose. If we were studying galactose regulation today we would probably use a lacZ reporter system as we discussed in the last lecture. However, when the Gal regulatory system was fist genetically dissected, it was done by actually measuring the induction of Gal1 encoded galactokiase activity, so this is how we will discuss the genetic dissection of the system. Another approach is to simply measure galactokinaseactivity in the presence or absence of GalactoseAnother approach is to simply measure galactokinaseactivity in the presence or absence of GalactoseMutagenized What we know is that Gal4 mutants are uninducible and that Gal80 and Gal81 mutants constitutively express the Gal1 galactokinase gene, along with the other Gal genes. Let’s analyze each mutant in turn: Gal4 mutant: It was first established that, like Gal1-, the Gal4- mutant phenotype is recessive, because heterozygous diploids generated by mating Gal4- to wild type have normal regulation. It was then established that the mutation in the Gal4- strain lies in a new gene, and not simply in the GAL1 galactokinase gene; Gal1- mutants don’t express galactokinase activity in the presence of galactose, just as was seen for the Gal4- mutant. That Gal1- and Gal4- mutants have mutations in different genes was shown by complementation analysis, (diploids from mating Matα Gal4- with Mata Gal1- behave like wild type) and the fact that the GAL4 and GAL1::Tn7lacZ fusion strain grown on: GLYCEROLGLYCEROL + X-GalGALACTOSE+ X-GalConstitutiveUninducibleMutagenized GAL1::Tn7lacZ fusion strain grown on: GLYCEROLGLYCEROL + X-GalGALACTOSE+ X-GalMutagenized GAL1::Tn7lacZ fusion strain grown on: GLYCEROLGLYCEROL + X-GalGALACTOSE+ X-GalConstitutiveUninducibleGAL1 genes are unlinked was established by tetrad analysis. You should think about what the tetrads from the aforementioned diploids would look like. Put together the simplest model is that Gal4 is a positive regulator of Gal1 (and the other Gal genes). The + sign indicates that Gal4 increases Gal expression, but does not indicate whether this is direct or indirect. Gal80 mutant: The next useful regulatory mutant isolated was Gal80-, in which the Gal1 encoded galactokinase is expressed even in the absence of galactose and is not further induced in its presence. Again, heterozygous diploids (Gal80-/wt) showed that Gal80- is recessive, Tetrad analysis showed that Gal80 is not linked to Gal1, Gal4 or any of the Gal genes. If a mutant Gal80 results in constitutive Gal1 expression, the simplest model is that Gal80 negatively regulates the Gal genes. Since Gal4 positively regulates, and Gal80 negatively regulated Gal1 expression, we have to figure out how these two gene products work together to achieve such regulation. Assuming that Gal4 and Gal80 act in series there are two formal possibilities: Model 1 is that Gal4 positively regulates Gal1, and that Gal80 negatively regulates Gal4; the presence of galactose somehow inhibits Gal80 function thus releasing Gal4 to positively activate Gal1 expression. Model 2 is that Gal80 negatively regulates Gal1, and Gal4 negatively regulates Gal80; here the presence of galactose positively activates Gal4 which in turn negatively regulates Gal80, thus relieving inhibition of Gal1 expression. Model 1Model 2Model 1Model 2We can distinguish between these two models by doing what’s called an epistasis test to establish the epistatic relationship between Gal4- and Gal80-. This involves making a double Gal4- /
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