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Control of Gene Expression: The lac operon Chapter 7: lac Operon; PCR Chapter 8: Recombinant DNA Much of what we know about how cells “decide” to express a particular gene is based on bacterial biology. One classic model of the regulation of gene expression is the lac operon in bacteria. Ch. 7 p. 182-188 Metabolism Some enzymes are always expressed; others are expressed only when needed. Dr. Amy Rogers Bio 139 Constitutive enzymes: always expressed e.g., glycolytic enzymes even if glucose is not present Inducible enzymes: expressed only when needed e.g., β-galactosidase (lacZ) only when lactose is present Lac operon Enzyme Induction To metabolize lactose (a disaccharide of glucose & galactose), bacteria need 3 special enzymes. If no lactose is present, it would be wasteful for the bacteria to express those enzymes. In an operon, several related genes are grouped together in the DNA 1. Structural genes: “classic” genes which carry information for the synthesis of a protein (enzyme) (expression of the enzymes is OFF) • In the presence of lactose, some bacteria WILL express the 3 enzymes. • (expression of the enzymes is ON) Default mode: genes OFF Lactose induces enzyme production Lac operon: 3 structural genes, for the 3 enzymes needed to use lactose Genes are named: – lacZ: codes for β-galactosidase (hydrolyzes lactose into monosaccharides glucose & galactose) – lacY – lacA 2. Regulatory sites / genes: • • DNA sequences that do NOT code for production of a protein They regulate / control expression of structural genes – Promoter: site in the DNA where RNA polymerase binds to start transcription – Operator: site in the DNA that determines whether transcription is ON or OFF (the switch) What controls “the switch” at the operator? Repressor protein (lacI) Lac repressor binds to the exact DNA sequence in the operator. Binding blocks movement of RNA polymerase. The operon is OFF (structural genes are NOT expressed). 1 Lac repressor protein • Lac “i” gene (for repressor protein) – Constitutively expressed (always ON) – lacI is NOT physically part of the operon, but is located somewhere else in the chromosome How to flip the switch? • Lactose, when present, will bind to the repressor protein • Shape of lac repressor is changed by lactose binding • Repressor bound to lactose CANNOT bind to the operator sequence In presence of lactose: Operon is “de-repressed”. Structural genes needed for lactose metabolism (lacZ, lacY, lacA) are expressed (turned on) What would happen if: Mutation in operator sequence? Repressor could not bind; structural genes expressed constitutively Mutation in lacZ gene? Expression still inducible by lactose, but β-galactosidase might not work Mutation in promoter sequence? RNA polymerase could not bind; structural genes never transcribed; bacteria could not use lactose Mutation in lacI gene? Depends on the mutation. 1. If repressor could no longer bind the operator, then constitutive expression of the structural genes (switch always ON) 2. If repressor could no longer bind lactose, then impossible to derepress, operator always bound by repressor (switch always OFF) Recombinant DNA ch. 8 p. 221-224 Transfer of DNA from one species to another • Genetic code is virtually the same in all life on earth • Gene for a (human) protein can be expressed in bacteria MANY applications; classic example: Using genetically engineered (recombinant) bacteria as factories for production of human protein: Insulin Insulin is a human hormone deficient in type I diabetics. Prior to 1982, these patients injected insulin purified from slaughtered pigs. Problems: Patients develop allergies to non-human (pig) insulin; contamination; cost 2 How to make recombinant bacteria 1. Cut human insulin gene from human chromosome 2. Cut open a vector Self-replicating gene carrier, often a plasmid 3. Paste human insulin gene into vector 4. Insert vector + insulin gene into bacteria 5. Select for recombinant bacteria by growing on antibiotic Restriction endonucleases How to cut DNA • Restriction endonucleases – also called restriction enzymes – Nuclease, enzyme that cuts DNA backbone – Endo-, cuts at internal sites, not just at the ends of a DNA molecule • Key features: – Sequence specific – Sticky ends Restriction endonucleases Sticky ends: Sequence specific: Each restriction enzyme will cleave DNA ONLY at a specific recognition sequence, usually 4 to 6 nucleotides long. EcoRI (famous restriction enzyme) Recognition sequence: GAATTC CTTAAG The enzyme cuts each DNA strand in a different place. Separation of the two strands then leaves single-strand overhangs, called sticky ends. “Sticky” because the single strand overhangs want to hydrogen bond/base pair with another complementary single stranded DNA Sticky ends EcoRI: G A A T T C CTTAAG G CTTAA AATTC G = cut site How to make recombinant bacteria (or, how to clone a gene) Steps 2 & 3. Cut open a vector; paste insulin gene into vector Step 1. Cut human insulin gene from human chromosome Insulin gene Restriction enzyme recognition sites Insertion of human insulin gene (with compatible sticky ends) Human Sticky ends Insulin gene insulin gene ligated (pasted) in 3 Step 4. Insert recombinant plasmid into bacteria How to make recombinant bacteria (transformation) 1. Cut human insulin gene from human chromosome Use a restriction enzyme that cuts on both sides of the gene insulin gene 2. Cut open a vector Use the same restriction enzyme to make compatible sticky ends 3. Paste human insulin gene into vector Ligase closes the breaks in the DNA backbone 4. Insert recombinant plasmid into bacteria Use transformation to get the plasmid inside 5. Select for recombinant bacteria by growing on antibiotic Only bacteria carrying the recombinant plasmid are resistant; wildtype bacteria die That’s just the beginning… Once you have a pure culture of the recombinant bacteria, they will express the foreign gene and manufacture human insulin! Most insulin-dependent diabetics in the U.S. use human insulin manufactured by recombinant bacteria (“humulin”) Polymerase Chain Reaction (PCR): Large-scale amplification of tiny quantity of DNA A few uses: • Medical diagnostics • Test for infectious disease, genetic abnormalities • Forensics • • • • • • Human growth hormone Erythropoietin (epo) Hepatitis A & B vaccines Bt toxin (insecticide) in agriculture Vitamin production in food crops Genetic engineering of mammals to produce desired proteins for isolation from milk Principle of PCR • Start with one DNA molecule; replicate it • Repeat, repeat, repeat = exponential increase in the # of DNA copies • Rape, murder cases • Study of DNAs from long ago • Replication occurs in cycles • Jurassic Park, evolutionary studies 4 PCR: Some details •Each cycle starts with denaturation of the DNA to separate the strands PCR: Some details • Thermal cycling is key to PCR: •Denaturation is done by heating almost to boiling •DNA polymerase does the job of actually copying the DNA •A thermostable DNA polymerase is used that continues working despite repeated exposure to boiling temperatures • HIGH temperature to denature DNA • LOWER temperatures to anneal primers and synthesize new DNA • After each cycle of high to low, start over with high again (e.g., Taq polymerase from Thermus aquaticus) PCR: Primers • Primers define the region to be amplified • Short single-stranded DNAs • In PCR, always used in pairs • You choose their sequences • Primers will hybridize to complementary sequences in the target DNA to be amplified • Taq polymerase synthesizes new DNA onto the 3’ end of each primer • Whatever DNA lies between the “upstream” and “downstream” primers is what gets amplified 5