Download Enzyme Induction

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).
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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
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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
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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
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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
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