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Regulatory mutations and eukaryotic gene regulation
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Polling with TurningPoint XR devices(blue/beige clickers)
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Lac operon
Analyzing phenotypes in which gene regulation is disrupted
genotype -IPTG +IPTG interpretation
I+ Z+ - + normal
I- Z+ + + constitutive
I+ Z- - - uninducible
I- Z+ / F’ I+ Z+ - + I- is recessive
B-gal activity
Notation“F’ ABC“: Addition of separate strand of DNA that replicates within bacterial host (called a plasmid) that contains the ABC genes
Bacteria are haploid, but giving cells genes on the plasmid allows determination of the consequence of allele combinations
The “cis-versus-trans” test:I acts in trans
genotype -IPTG +IPTG interpretation
I+ Z+ - + normal
I- Z+ + + constitutive
I+ Z- - - uninducible
I- Z+ / F’ I+ Z+ - + I- is recessive
I+ Z-/ F’ I- Z+ - + I acts in trans
I- Z-/ F’ I+ Z+ - + I acts in trans
B-gal activity
The “cis-versus-trans” test:O acts in cis
genotype -IPTG +IPTG interpretation
O+ Z+ - + normal
O- Z+ + + constitutive
O+ Z- - - uninducible
O- Z+/ F’ O+
Z+
+ + O- is dominant
O- Z+/ F’ O+ Z- + +
O- Z-/ F’ O+
Z+
- +
B-gal activity
O acts in cis
Maltose operon
genotype -maltose +maltose
1 Q+ T+ - +
2 Q+ T- - -
3 Q- T+/ F’ Q+ T- - +
4 Q- T-/ F’ Q+ T+ - +
5 Q+ M+ - +
6 Q+ M- - -
7 Q- M+/ F’ Q+ M- - -
8 Q- M-/ F’ Q+ M+ - +
Interpretations?A. T acts in transB. T acts in cisC. M acts in transD. M acts in cis
What can be concluded?1. A and C2. B and C3. A and D4. B and D
Q activity
Maltose operon
genotype -maltose +maltose
1 Q+ T+ - +
2 Q+ T- - -
3 Q- T+/ F’ Q+ T- - +
4 Q- T-/ F’ Q+ T+ - +
5 Q+ M+ - +
6 Q+ M- - -
7 Q- M+/ F’ Q+ M- - -
8 Q- M-/ F’ Q+ M+ - +
What can be concluded?A. T is an activatorB. T is a repressorC. Not enough information
Q activity
I made a mistake in discussing this slide. The correct answer is A,because when T is missing there is no expression of Q. Therefore, T must be acting as an activator.
If T is a trans-acting activator, thenmaltose must induce T binding to DNA
M is a cis-acting sequence
Promoter QT
RNA polMaltose
Transcription of Q
M
Prediction: If T is missing no expression of Q becauseRNApol is not recruited to the poor promoter element
If T is a trans-acting Repressor, thenmaltose prevents T binding to DNA
M is a cis-acting sequence
Promoter QT
RNA pol
Maltose
Transcription of Q
M
Maltose
+ Prediction: If T is missingRNApol recruitment tothe promoter will not beblocked and Q expressionwill be constitutive.
Minimal requirements for specific gene regulation
1. An activity that can promote or inhibit gene expression upon a stimulus– Resides in a molecule that acts in trans
2. A way to target that activity to a particular gene – DNA/RNA sequences that act in cis
Essentially any step in gene expression can be regulated
Estrogen receptor
ERE
ERRNA pol
Estrogen
Nucleus
cytoplasm
ER
ER ER
Outside of cell
ER ER
MediatorCyclin D protein
Triggers S phase and cell proliferation
cyclinDTATA
TBP
Estrogen binding changes 3-D shape of ER protein, allowing it to dimerize
Estrogen receptor transactivation but not DNA binding is inhibited by tamoxifen
ERE
ER
Estrogen
Nucleus
cytoplasm
ER
ER ER
Outside of cell
ER ER
Tumor cell fails to proliferate
cyclinDTATA
tamoxifen
Sequence-specific RNA binding proteins can modulate post-transcriptional events such as splicing
sam68rasCancerous state
ex1 ex2 ex3 ex4 ex5 ex6 ex7 ex8 ex9
cap AAAAAAex1 ex2 ex3 ex4 ex5 ex6 ex7 ex8 ex9
cap AAAAAA1 2 3 4 5 6 7 8 9
CD44 gene
CD44 Exon5 inclusion: cancer cell migration (metastasis)
Translation can be controlled by sequence-specific RNA binding proteins
BRE
bicoid
4Ecap
AAAAAA
Ribosome can’t bind mRNA
AUG
STOP
caudal
caudalbicoid
Fly head
How to study regulation? Finding regulatory sequences
Luciferase
Luciferase
Luciferase
Luciferase
Luciferase
Luciferase
100%
Luciferase
100%
100%
1%
1%
100%
1%
Luciferase activity
Recombinant DNA: regulatory DNA region + reporter
Detecting mRNA: Northern blot• Use labeled antisense DNA or RNA, hybridizes
to mRNA
Detecting protein: Western blot• Use antibody, binds specific protein