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Lecture Notes 22 - Lecture 22 Biosc 0350 Genetics Lecture...

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Lecture 22 4/3/2008 1 Biosc 0350 Genetics Lecture 22 - Eukaryotic gene regulation 1 Prokaryotes ( Lecture 21 ) The lactose operon - Negative regulation by a repressor (lacI) - positive regulation by an activator (CRP) Eukaryotes cis - and trans -acting elements Mechanisms of activation and repression Two examples - tissue specific regulation in Drosophila - Myc/Max system in humans 4/3/08 Martens. Biosc 0350, University of Pittsburgh 2 β -galactosidase activity Noninduced Induced wild type No (actually a tiny bit) Yes lacO C Yes Yes ( constitutive ) lacI - Yes Yes ( constitutive ) Both types of mutations prevent repression lacO C defined cis -acting DNA element lacI - defined a trans -acting factor Genetics determined that these two mutations mapped to different chromosomal locations How did they distinguish the mutations in order to come up with the operon model?
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Lecture 22 4/3/2008 2 4/3/08 Martens. Biosc 0350, University of Pittsburgh 3 Demonstrate cis and trans effects using partial diploids (meroploids) I - Z + Y + I - Z + Y + / I + Z + Y + I + Z + Y + I + Z + Y + I - Z + Y + Construct strains with different lac mutations on either the chromosome or F' lac chromosome F factor F' lac lac lac lac Conjugation: E. coli F (fertility) factor F' is an F factor that contains a few chromosomal genes F' lac plasmid has the lac operon Wild type cells containing F' lac are partial diploids 4/3/08 Martens. Biosc 0350, University of Pittsburgh 4 Function of lacO in partial diploids O c Z + Y - O + Z - Y + -IPTG Conclusions: lacO c on F' has no effect on the lac operon on the chromosome - lacO c is not complemented by lacO + Therefore, lacO (operator) is a cis -acting element (acts on same molecule)
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Lecture 22 4/3/2008 3 4/3/08 Martens. Biosc 0350, University of Pittsburgh 5 Function of lacI in partial diploids Conclusions: lacI + is dominant to lacI - (or lacI + complements lacI -) Product of lacI, no matter where it is encoded, acts on the lac operon on the chromosome as well as F’ Therefore, product of lacI is a trans -acting factor that can diffuse within the cytoplasm (Lac repressor) I - Z - I + Z + -IPTG I + Z - I - Z + -IPTG 4/3/08 Martens. Biosc 0350, University of Pittsburgh 6 Another type of lacI mutation lacI s superrepressor can bind DNA, cannot bind inducer non-inducible β - galactosidase activity Noninduced Induced lacI + No Yes lacI S No No
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Lecture 22 4/3/2008 4 4/3/08 Martens. Biosc 0350, University of Pittsburgh
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