Download Handout #11 - MSU Billings

VII. Structure of a Eukaryotic Gene
Regulation of Prokaryotic Gene Expression
Enzyme induction: enzymes needed to break down a
molecule are only synthesized when molecule is present
1
Regulation of Prokaryotic Gene Expression
Enzyme repression: enzymes needed to synthesize a molecule
are not made if molecule is present
Negative Control
Repressors: proteins that negatively regulate transcription
bind to DNA sequences (promoters) and inhibit RNA polymerase
polymerase
repressor
GENE OFF
GENE ON
mRNA
2
Negative Control
Repressors: proteins that negatively regulate transcription
bind to DNA sequences (promoters) and inhibit RNA polymerase
inducers interrupt repressor binding to the promoter
polymerase
repressor
GENE OFF
inducer
GENE OFF
GENE ON
mRNA
Negative Control
Repressors: proteins that negatively regulate transcription
bind to DNA sequences (promoters) and inhibit RNA polymerase.
co-repressors induce repressor binding to the promoter
polymerase
Repressor +
co-repressor
GENE OFF
GENE OFF
GENE ON
mRNA
3
Positive Control
Activators: proteins that induce transcription; usually interact with RNA
polymerase and stabilize transcription initiation complex
polymerase
GENE OFF
activator
GENE ON
mRNA
Positive Control
Activators: proteins that induce transcription; usually interact with RNA
polymerase and stabilize transcription initiation complex
inducer induces binding to the promoter
polymerase
+
inducer
GENE OFF
+
activator
GENE ON
mRNA
4
Positive Control
Activators: proteins that induce transcription; usually interact with RNA
polymerase and stabilize transcription initiation complex
inhibitors prevent binding to the promoter
polymerase
GENE OFF
GENE ON
inhibitors
mRNA
Regulation of the Lac operon
A repressor and an activator work in concert with two different
inducers. (Francois Jacob and Jacques Monod 1950s)
Lac repressor
GENES OFF
allolactose (inducer)
CAP (activator)
cAMP (inducer)
5
Regulation of the Lac operon
If lactose is not present, the repressor binds the promoter and transcription is
repressed.
Lac repressor
GENES OFF
allolactose (inducer)
CAP (activator)
cAMP (inducer)
Regulation of the Lac operon
If lactose is present, the repressor binds the inducer and transcription is
derepressed.
Lac repressor
GENES OFF
allolactose (inducer)
CAP (activator)
cAMP (inducer)
6
Regulation of the Lac operon
If lactose is present, the repressor binds the inducer and transcription is
derepressed.
Lac repressor
GENES OFF
allolactose (inducer)
CAP (activator)
cAMP (inducer)
Regulation of the Lac operon
The polymerase still cannot initiate transcription without the
activator.
Lac repressor
GENES OFF
allolactose (inducer)
CAP (activator)
polymerase
GENES OFF
cAMP (inducer)
7
Regulation of the Lac operon
If lactose levels are high and glucose levels are low, cAMP levels
rise and cAMP binds the activator.
Lac repressor
GENES OFF
allolactose (inducer)
CAP (activator)
polymerase
GENES OFF
cAMP (inducer)
Regulation of the Lac operon
cAMP-activator bind the promoter and the transcription of the operon is
activated.
Lac repressor
GENES OFF
allolactose (inducer)
CAP (activator)
polymerase
GENES OFF
cAMP (inducer)
GENES ON
mRNA
8
Regulation of the Lac operon
What happens if glucose levels are low and lactose levels are also
low?
Lac repressor
allolactose (inducer)
CAP (activator)
GENES?
cAMP (inducer)
Regulation of the Lac operon
What happens if glucose levels are low and lactose levels are also
low?
Lac repressor
allolactose (inducer)
CAP (activator)
GENES OFF
cAMP (inducer)
9
Regulation of the Lac operon
It is all about the balance (ratio) of activator or repressor and their
respective inducers.
Lac repressor
allolactose (inducer)
CAP (activator)
GENES OFF
cAMP (inducer)
Regulation of the L-ara operon
Two activators work in concert with different inducers.
GENES OFF
arabinose activator
Arabinose (inducer)
CAP (activator)
cAMP (inducer)
10
Regulation of the L-ara operon
If arabinose is present, the activator binds the arabinose (inducer).
GENES OFF
arabinose activator
GENES OFF
Arabinose (inducer)
CAP (activator)
cAMP (inducer)
Regulation of the L-ara operon
If both arabinose and cAMP are present, the activators binds the co-activators
and the operon is turned on.
GENES OFF
arabinose activator
GENES OFF
Arabinose (inducer)
CAP (activator)
polymerase
GENES ON
cAMP (inducer)
mRNA
11
Regulation of the L-ara operon
The arabinose activator regulates its own promoter.
GENES OFF
arabinose activator
Arabinose (inducer)
CAP (activator)
polymerase
GENES ON
cAMP (inducer)
mRNA
polymerase
GENE ON
arabinose repressor
Arabinose (inducer)
Regulation of the L-ara operon
The arabinose activator regulates its own promoter as a
repressor.
GENES OFF
arabinose activator
Arabinose (inducer)
CAP (activator)
polymerase
GENES ON
cAMP (inducer)
mRNA
polymerase
GENE ON
arabinose repressor
Arabinose (inducer)
12
Regulation of the L-ara operon
The arabinose activator regulates its own promoter as a
repressor.
GENES OFF
arabinose activator
Arabinose (inducer)
CAP (activator)
polymerase
GENES ON
cAMP (inducer)
mRNA
polymerase
Feedback loop
GENE ON
arabinose repressor
Arabinose (inducer)
Regulation of the L-ara operon
The arabinose activator regulates its own promoter as a
repressor.
GENES OFF
arabinose activator
Arabinose (inducer)
CAP (activator)
polymerase
GENES ON
cAMP (inducer)
mRNA
polymerase
GENE OFF
Feedback loop
arabinose repressor
Arabinose (inducer)
13
Regulation of the L-ara operon
The arabinose activator regulates its own promoter as a
repressor.
GENES OFF
arabinose activator
Arabinose (inducer)
CAP (activator)
polymerase
GENES ON
cAMP (inducer)
mRNA
polymerase
GENE ON
Feedback loop
arabinose repressor
Arabinose (inducer)
Transcription regulates the expression of eukaryotic genes
during development to determine a cells FATE.
1
GENE OFF
2
GENE OFF
3
GENE OFF
polymerase
GENE ON
1
2
polymerase
GENE ON
polymerase
3
GENE ON
14
Transcription regulates the expression of eukaryotic genes
during development to determine a cells FATE.
Transcription regulates the expression of eukaryotic genes
during development to determine a cells FATE.
1
GENE OFF
2
GENE OFF
3
GENE OFF
polymerase
GENE ON
1
2
polymerase
GENE ON
polymerase
3
GENE ON
15
Gene Technology
I.
II.
III.
IV.
V.
VI.
Vocabulary
Genetic Engineering Techniques
PCR
Gel Electrophoresis
DNA Fingerprinting
Biotechnology Applications
I. Some of the Vocabulary of Molecular Biologists
! Enzymes
- Restriction Endonuclease: cuts DNA at
Restriction Sites
- DNA Ligase: joins DNA strands
! Recombinant DNA: DNA joined from different genomes
! Vector: the agent used to carry new genes into cells
! Plasmids: extrachromosomal circular DNA
! Phages: viruses that infect bacterial cells
! Cloning Genes: the production of many copies of genes
of interest
16
II. Genetic Engineering Techniques
Restriction Endonuclease (or just Restriction Enzymes)
II. Genetic Engineering Techniques
Restriction Endonuclease (or just Restriction Enzymes)
17
II. Genetic Engineering Techniques
Enzymes Cleave & Splice DNA
GAATTC
CTTAAG
GAATTC
CTTAAG
Restriction endonuclease
G
A
CTTA
AATTC
G
DNA of interest
Cuts DNA
G
CTTAA
G
CTTAA
AATTC
G
AATTC
G
Vector DNA
Vector DNA
G AATTC
CTTAA G
G AATTC
CTTAA G
DNA ligase
joins DNA
Recombinant DNA molecule
II. Genetic Engineering Techniques
Recombinant DNA techniques use
biological vectors like plasmids and
viruses to carry foreign genes into cells
A. Plasmids
– Small, circular bacterial
chromosomes
– Independent of main circular
chromosome
– Has its own:
• origin of replication
• cloning site
• set of genes
18
Plasmid Selection and Screening
lacZ encodes !-galactosidase, an
enzyme that cleaves the disaccharide
lactose into glucose and galactose.
Some bacteria do not receive
plasmids—no antibiotic resistance
Culture plates with both antibiotic
and X-gal (a sugar). When X-gal is
metabolized it turns Blue.
A. Plasmids (cont.)
– Transferred by conjugation
– Several types:
• Metabolic factors (e.g. oil-eating)
• F factors (fertility – allows
conjugation)
• R factors (resistance to
antibiotics)
– Originate as parts of transposons
• Pieces of DNA that are readily
moved around
Pilus / Pili
Pilus
19
A. Plasmids (cont.)
Cloning a gene in a bacterial plasmid
Bacterial Cell 1
2
Human cell
1. Isolate DNA from two sources
2. Cut both DNAs
with restriction enzyme
Plasmid
Sticky ends
3
3. Mix the DNAs: join by base-pairing
4 4. Add DNA ligase to bond DNA
Recombinant DNA plasmid
Human Gene
5 5. Reinsert plasmid into bacterium
Recombinant bacterium
6 6. Grow bacteria (cloning)
7. Produce copies of the human gene;
or use bacterial cell machinery to
produce protein products coded in gene
B. Phages
Viruses are infectious particles that
contain genetic material to which a
new gene can be added. The virus can
carry the new gene into a recipient cell
in the process of infecting that cell.
20
Recombinant DNA Technology
Produce new genetic varieties of plants and animals,
Genetically Modified (GM) organisms
Agrobacterium
tumefaciens
DNA containing
gene for desired trait
Ti
plasmid
1
Insert plant gene
into plasmid using
restriction enzyme
and DNA ligase
Recombinant
Ti plasmid
T DNA
Restriction
site
Plant cell
2
Plant with
new trait
3
Regeneration
of plant
Introduction
Into
plant cells
T DNA carrying new
gene within plant chromosome
Luciferase gene expressed
in a tobacco plant
Examples of genetically modified (GM) crop plants
Applications of
Recombinant Technologies
European Corn Borer
Genetically Engineered Corn=Bt corn
= A corn plant that has been developed
though biotechnology so that the plant
tissues express a protein derived from
a bacterium, Bacillus thuringiensis,
which is toxic to some insects but nontoxic to humans and other mammals.
Bacillus thuringiensis
1. bacterium
2. produces Bt toxin
3. kills larvae of Corn Borer
Bt Corn
Bt Corn
Non-Bt Corn
21
Examples of genetically modified (GM) crop plants
Glyphosate Resistance
= ROUNDUP (non-selective herbicide)
- Cotton
- Corn
- Soybeans
- Canola
- Wheat
Bt Crops
- Cotton
- Corn
Other engineered crops
- Papaya
virus resistance
- Carnation
longevity
- Flax
herbicide resistance
- Lentil
herbicide resistance
- Potato
insect resistance
- Squash
virus resistance
- Sugar beet
herbicide resistance
- Cucumber
virus resistance
- Watermelon
virus resistance
Enhancement of Nutritional
Value/Longevity
1. Rice
2. “Flavr Savr” Tomato
22
Other Gene Transfer Techniques
While recombinant DNA techniques use biological vectors to
carry foreign genes into cells, here are some alternatives:
! Microinjection: inject genetic material containing the new gene
into the recipient cell. Where the cell is large enough the injection
can be done with a fine-tipped glass needle. The injected genes find
the host cell genes and incorporate themselves among them.
! Electro- and Chemical Poration: create pores or holes in
the cell membrane to allow entry of the new genes. Cells are bathed
in solutions of special poration chemicals or subjected to weak
electric current (electroporation).
! BioBallistics (cool): Uses a shotgun to deliver geneticallycoated slivers directly into the cell
Target sequence
P P
P
Heat P
P
Primers
1 Denaturation
Cool
PCR
– Kary Mullis (1983)
– 3 Repeated Steps:
• Denaturation (High
temperature)
• Primer Annealing
• Polymerization or
Extension
– Millions of Copies
Produced
– Effective with genomic
ranges of < 2500 bp
P
III. Polymerase Chain Reaction (PCR)
Cycle
1
P 2 Annealing of primers
DNA polymerase
Free nucleotides
P3
Primer extension
P
2
copies P
Heat
Cycle
4 copies
2
Cool
P
P
P
P
Heat
Cool
Cycle 8 copies
3
P
P
P
P
P
P
P
P
23
IV. Gel Electrophoresis
Separation of proteins or nucleic acids based on mass and charge
DNA has a net
negative charge
IV. Gel Electrophoresis
Load PCR product alone or incubate with restriction
enzymes before loading into gel
DNA and
restriction
endonuclease
– Cathode
Longer fragments
Power
source
Gel
Mixture of DNA fragments
at top of gel
+ Anode
Electric current applied
Completed gel
Shorter fragments
24
IV. Gel Electrophoresis
Stain gel and view under UV light.
One Option: Excise bands of interest for further study
or DNA sequencing.
V. DNA Fingerprinting
! Restriction fragment length polymorphisms (RFLPs)
! Restriction Fragments reflect differences in DNA sequences!
25
V. DNA Fingerprinting
Larger
fragments
Restriction endonuclease
cutting sites
Smaller
fragments
–
+
–
+
–
+
Single base-pair
change
Sequence duplication
Cut DNA
Three different
DNA duplexes
Gel electrophoresis of
restriction fragments
V. DNA Fingerprinting
1. Amplify blood or other fluid at the scene of the crime using PCR
2. Digest with restriction enzymes
3. Run fragments on a gel and visualize
Crime scene
Suspect
CG
CG
GC
GC
z
AT
CG
GC
GC
y
CG
Cut C
G
GC
GC
w
Cut
–
Longer
fragments
x
x
Cut
y
CG
CG
GC
GC
z
Shorter
fragments
w
y
y
+
DNA from chromosomes
26
V. DNA Fingerprinting
Guilty or Not Guilty?
Defendant’s
blood
Blood from
defendant’s clothes
Victim’s
blood
VI. Biotechnology: Gene Therapy
ADA adenosine deaminase deficiency
27
28