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MCB 317Genetics and Genomics
MCB 317 Topic 10, part 1A Story of Transcription
Eukaryotic Transcription
How We “Know” What we Know
Abbreviation for Transcription = Txn
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
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
Discovery and Identification of Eukaryotic Promoters
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
PCR-based construction of deletion mutants
Primer “tail” = BamHI site
Primer “tail” = HindIII site
PCR
Cut with BamHI and HindIII and clone
Deletion Analysis
BamHI
XhoI
HindIII
PCRHindIII
Deletion Analysis
BamHI
XhoI
HindIII
PCRHindIII
BamHI
XhoI
HindIII
PCRHindIII
Deletion Series from the 3’ end
BamHI HindIIIXhoI
BamHI HindIIIXhoI
BamHI HindIIIXhoI
BamHI HindIII
XhoI
Deletion Analysis Defines the Borders of Control Regions Txn
Yes
Yes
No
-100
-90
-80
Something between -80 and -90 nts required for txn
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?
Construction of Linker-Scanner Mutant
BamHI
XhoI
HindIII
PCRHindIII
BamHIHindIIIPCR
BamHI XhoI
BamHI XhoI HindIII
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
ATGCGATGCTAGCTATTTAGATCGGATCGAATCGATCGATCGATAGGTC
ATGCGATCTCGAG
CTCGAGTATTTAGATCGGATCGAATCGATCGATCGATAGGTC
ATGCGATCTCGAGTATTTAGATCGGATCGAATCGATCGATCGATAGGTC
ATGCGATGCTAGCTATTTAGATCGGATCGAATCGATCGATCGATAGGTCATGCGATCTCGAGTATTTAGATCGGATCGAATCGATCGATCGATAGGTC
Site-directed Mutagenesis
Use of Oligos to Synthesize Mutant Alleles
XhoIXhoI
“Gap”
Txn
YES
YES
YES
NO
Use of Oligos to Synthesize Mutant Alleles
XhoI HindIIIBamHI XhoI
BamHI HindIII
“Gap”
Wild-type
SynthesizedMutant allele
CTCGAGTAGCCGTAGCTCGACTCGAGGAGCTCATCGGCATCGAGCTGAGCTC
TAGCCGTGGCTCGA ATCGGCACCGAGCT
Site directed mutagenesis, part 2
Site directed mutagenesis, part 2
Site directed mutagenesis, part 3
Site directed mutagenesis, part 4
Site directed mutagenesis, part 5
Site directed mutagenesis,summary
Mutational/Genetic Analysis of DNA
Can be used to Study:PromotersEnhancersOrigins of ReplicationCentromeresTelomeresORFsany DNA Sequence-dependent Process
Initial Result = Promoters are Sufficient for Txn
“Run-off expt.”
Watson 9-5Several small elementsNone essential (in this case)
Linker-scanner Analysis -> Several Elements
Eukaryotic Promoter Elements
- Promoter Elements Conserved Among Eukaryotes- No Individual Element found at All Promoters
Deletion andLinker Scanner Analysis
In vitro Txn Assay
Define Promoters Promoters sufficient for Txn
Do Promoter Elements function in vivo similarly to the way the function in vitro?
Watson 12-7
Transfectionand
Electroporation
Transient Transfection
Assay
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
Ab
Protein
ExpressionPattern
Gene
Gene (Organism 2)
Mutant Gene
Biochemistry
Genetics
Mutant Organism
1
2
3
4
78
56 9
10
11
12
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
Reverse Transcriptase PCR or RT-PCR
A Qualitative Test for Whether an mRNA is present
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
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
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
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
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
57
Linear range = cycles 16-24
58SERIES OF 10-FOLD DILUTIONS
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.
qRT-PCR
RT-PCR -> qPCR
Best method for quantitating levels of an mRNA in a sample
RT-PCR
qPCR
qRT-PCR
Properties of Enhancers
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
Watson 9-5Several small elementsNone essential (in this case)
Which element(s) are required for regulated txn?
Regulatory Elements v. Control Elements
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