mcb 317 genetics and genomics mcb 317 topic 10, part 1 a story of transcription
TRANSCRIPT
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MCB 317Genetics and Genomics
MCB 317 Topic 10, part 1A Story of Transcription
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Eukaryotic Transcription
How We “Know” What we Know
Abbreviation for Transcription = Txn
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Deletion andLinker Scanner Analysis
In vitro Txn Assay
Promoter not sufficient in vivo
Identification of Enhancers
Identify and define TBPand basal factors
Extract +Prom.-Enh.
Basal Facts. +Prom.-Enh.
Activated Txn(Enhanced) &Regulated Txn
Extract +Prom.-Enh.
ActivatorsCo-activators + Enhancer &TBP & TAFs Promoter
“Activated” txn & Regulated txn
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What is “True” Will Change as We Go Through the Story of Txn
Our “Knowledge” of Subjects Undergoing Active Research Evolves
“Knowledge” -> A Series of Models
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Discovery and Identification of Eukaryotic Promoters
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Identification of DNA Sequence Elements-General Strategy
1. Quick, rough look- 100’s bp to 10Kb-> example: Reporter Assay
2. Narrow to specific region- 100’s bp-> example: Gel mobility shift
3. High resolution analysis- Identify specific sequence element 10-20 bps
-> example: footprinting, site directed mutagenesis
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PCR-based construction of deletion mutants
Primer “tail” = BamHI site
Primer “tail” = HindIII site
PCR
Cut with BamHI and HindIII and clone
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Deletion Analysis
BamHI
XhoI
HindIII
PCRHindIII
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Deletion Analysis
BamHI
XhoI
HindIII
PCRHindIII
BamHI
XhoI
HindIII
PCRHindIII
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Deletion Series from the 3’ end
BamHI HindIIIXhoI
BamHI HindIIIXhoI
BamHI HindIIIXhoI
BamHI HindIII
XhoI
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Deletion Analysis Defines the Borders of Control Regions Txn
Yes
Yes
No
-100
-90
-80
Something between -80 and -90 nts required for txn
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Deletion Analysis Defines the Borders of Control Regions Txn
Yes
Yes
No
-100
-90
-80
Something between -80 and -90 nts required for txn
+30
+20
+10Something between +20 and +30 nts required for txn Control Region Between -90 and +30, but how much reqiuired?
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Construction of Linker-Scanner Mutant
BamHI
XhoI
HindIII
PCRHindIII
BamHIHindIIIPCR
BamHI XhoI
BamHI XhoI HindIII
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Construction of Linker-Scanner Mutant
BamHI XhoI HindIII
Linker-scanner mutations are substitution mutations
Length of mutant = same length as original clone
Wild-type except at the XhoI substitution site
-100 -19-12
+300
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ATGCGATGCTAGCTATTTAGATCGGATCGAATCGATCGATCGATAGGTC
ATGCGATCTCGAG
CTCGAGTATTTAGATCGGATCGAATCGATCGATCGATAGGTC
ATGCGATCTCGAGTATTTAGATCGGATCGAATCGATCGATCGATAGGTC
ATGCGATGCTAGCTATTTAGATCGGATCGAATCGATCGATCGATAGGTCATGCGATCTCGAGTATTTAGATCGGATCGAATCGATCGATCGATAGGTC
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Site-directed Mutagenesis
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Use of Oligos to Synthesize Mutant Alleles
XhoIXhoI
“Gap”
Txn
YES
YES
YES
NO
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Use of Oligos to Synthesize Mutant Alleles
XhoI HindIIIBamHI XhoI
BamHI HindIII
“Gap”
Wild-type
SynthesizedMutant allele
CTCGAGTAGCCGTAGCTCGACTCGAGGAGCTCATCGGCATCGAGCTGAGCTC
TAGCCGTGGCTCGA ATCGGCACCGAGCT
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Site directed mutagenesis, part 2
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Site directed mutagenesis, part 2
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Site directed mutagenesis, part 3
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Site directed mutagenesis, part 4
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Site directed mutagenesis, part 5
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Site directed mutagenesis,summary
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Mutational/Genetic Analysis of DNA
Can be used to Study:PromotersEnhancersOrigins of ReplicationCentromeresTelomeresORFsany DNA Sequence-dependent Process
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Initial Result = Promoters are Sufficient for Txn
“Run-off expt.”
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Watson 9-5Several small elementsNone essential (in this case)
Linker-scanner Analysis -> Several Elements
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Eukaryotic Promoter Elements
- Promoter Elements Conserved Among Eukaryotes- No Individual Element found at All Promoters
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Deletion andLinker Scanner Analysis
In vitro Txn Assay
Define Promoters Promoters sufficient for Txn
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Do Promoter Elements function in vivo similarly to the way the function in vitro?
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Watson 12-7
Transfectionand
Electroporation
Transient Transfection
Assay
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Deletion andLinker Scanner Analysis
In vitro Txn Assay
Promotersufficient in vitro
Identification of Enhancers
Identify and define TBPand basal factors
Extract +Prom.-Enh.
Basal Facts. +Prom.-Enh.
Activated Txn(Enhanced) &Regulated Txn
Extract +Prom.-Enh.
ActivatorsCo-activators + Enhancer &TBP & TAFs Promoter
“Activated” txn & Regulated txn
In vivo Txn AssayPromoter not Sufficient
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Ab
Protein
ExpressionPattern
Gene
Gene (Organism 2)
Mutant Gene
Biochemistry
Genetics
Mutant Organism
1
2
3
4
78
56 9
10
11
12
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Molecular Genetics Summary
1. Column Chromatograpy (ion exch, gel filtr)2. A. Make Polyclonal Ab; B. Make Monoclonal Ab3. Western blot, in situ immuno-fluorescence (subcellular, tissue)4. Screen expression library (with an Ab)5. Screen library with degenerate probe, mass spec. & database6. Protein expression (E. coli)7. A. Differential hybridization8. A. Northern blot, in situ hybridization, GFP fusion, RT-PCR and q-RT
PCR9. A. low stringency hybridization; B. computer search/clone by phone; C.
computer search PCR10. Clone by complementation (yeast, E. coli)11. A. Genetic screen; B. genetic selection12. RNAi
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Reverse Transcriptase PCR or RT-PCR
A Qualitative Test for Whether an mRNA is present
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Quantitative PCR or qPCR or Real Time PCR
qPCR machine is a PCR machine that can measure the fluorescence of the reaction after each cycle
SYBR green
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53
CYCLE NUMBER AMOUNT OF DNA0 11 22 43 84 165 326 647 1288 2569 512
10 1,02411 2,04812 4,09613 8,19214 16,38415 32,76816 65,53617 131,07218 262,14419 524,28820 1,048,57621 2,097,15222 4,194,30423 8,388,60824 16,777,21625 33,554,43226 67,108,86427 134,217,72828 268,435,45629 536,870,91230 1,073,741,82431 1,400,000,00032 1,500,000,00033 1,550,000,00034 1,580,000,000
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54
0
200000000
400000000
600000000
800000000
1000000000
1200000000
1400000000
1600000000
0 5 10 15 20 25 30 35
PCR CYCLE NUMBERA
MO
UN
T O
F D
NA
110100100010000100000100000010000000100000000100000000010000000000
0 5 10 15 20 25 30 35
PCR CYCLE NUMBER
AM
OU
NT
OF
DN
A
CYCLE NUMBER AMOUNT OF DNA0 11 22 43 84 165 326 647 1288 2569 512
10 1,02411 2,04812 4,09613 8,19214 16,38415 32,76816 65,53617 131,07218 262,14419 524,28820 1,048,57621 2,097,15222 4,194,30423 8,388,60824 16,777,21625 33,554,43226 67,108,86427 134,217,72828 268,435,45629 536,870,91230 1,073,741,82431 1,400,000,00032 1,500,000,00033 1,550,000,00034 1,580,000,000
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55
0
200000000
400000000
600000000
800000000
1000000000
1200000000
1400000000
1600000000
0 5 10 15 20 25 30 35
PCR CYCLE NUMBER
AM
OU
NT
OF
DN
A
0
200000000
400000000
600000000
800000000
1000000000
1200000000
1400000000
1600000000
0 5 10 15 20 25 30 35
PCR CYCLE NUMBER
AMOUNT OF DNA
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56
110100100010000100000100000010000000100000000100000000010000000000
0 5 10 15 20 25 30 35
PCR CYCLE NUMBERA
MO
UN
T O
F D
NA
110100100010000100000100000010000000100000000100000000010000000000
0 5 10 15 20 25 30 35
PCR CYCLE NUMBER
AMOUNT OF DNA
Linear range = cycles 16-24
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57
Linear range = cycles 16-24
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58SERIES OF 10-FOLD DILUTIONS
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qPCR
Can quantify the level of a given RNA in a sample by measuring the number of cycles it takes to produce a “threshold” level of PCR product.
The threshold level is the Ct value; which is a value in the linear range of amplification on a logarithmic plot.
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qRT-PCR
RT-PCR -> qPCR
Best method for quantitating levels of an mRNA in a sample
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RT-PCR
qPCR
qRT-PCR
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Properties of Enhancers
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Enhancers= short regions (typically ~ 200 bp)of densely packed consensus elements
Some elements found in both promoters and enhancers
Enhancers= different combinations of elements found in other enhancers
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Watson 9-5Several small elementsNone essential (in this case)
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Which element(s) are required for regulated txn?
Regulatory Elements v. Control Elements
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E1 E2 Pr Coding Region
E1 E2 Pr
E1 E2
E1 Pr
Gluc
Transcription
Metal Neither
E2 Pr
++ -
---
+
+
- -
--
Genes can have Multiple Enhancers Which Regulate Different Responses