MCB 317
Genetics and Genomics
MCB 317 Topic 10, part 1
A Story of Transcription
Eukaryotic Transcription
How We “Know” What we Know
Abbreviation for Transcription = Txn
Deletion and
Linker Scanner Analysis
In vitro
Txn Assay
Promoter not
sufficient in vivo
Identify and define TBP
and basal factors
Extract +
Prom.-Enh.
Basal Facts. +
Prom.-Enh.
Activated Txn
(Enhanced) &
Regulated Txn
Extract +
Prom.-Enh.
Identification of
Enhancers
Activators
Co-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 ElementsGeneral 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
HindIII
BamHI
PCR
XhoI
HindIII
Deletion
Analysis
HindIII
BamHI
PCR
XhoI
HindIII
HindIII
BamHI
PCR
XhoI
HindIII
BamHI
Deletion Series from the 3’
end
XhoI
HindIII
XhoI
BamHI
HindIII
XhoI
BamHI
HindIII
XhoI
BamHI
HindIII
Deletion Analysis Defines the Borders of Control
Regions
-100
-90
Txn
Yes
Yes
No
-80
Something between -80 and -90 nts required for txn
Deletion Analysis Defines the Borders of Control
Regions
-100
-90
Txn
Yes
Yes
No
-80
Something between -80 and -90 nts required for txn
+30
+20
+10
Something between +20
and +30 nts required for
txn
Control Region Between -90 and +30,
but how much reqiuired?
Construction of Linker-Scanner Mutant
HindIII
BamHI
XhoI
BamHI
XhoI
BamHI
XhoI
PCR
HindIII
HindIII
PCR
BamHI
HindIII
Construction of Linker-Scanner Mutant
BamHI
-100
XhoI
-19
HindIII
+300
-12
Linker-scanner mutations are substitution mutations
Length of mutant = same length as original clone
Wild-type except at the XhoI substitution site
ATGCGATGCTAGCTATTTAGATCGGATCGAATCGATCGATCGATAGGTC
ATGCGATCTCGAG
CTCGAGTATTTAGATCGGATCGAATCGATCGATCGATAGGTC
ATGCGATCTCGAGTATTTAGATCGGATCGAATCGATCGATCGATAGGTC
ATGCGATGCTAGCTATTTAGATCGGATCGAATCGATCGATCGATAGGTC
ATGCGATCTCGAGTATTTAGATCGGATCGAATCGATCGATCGATAGGTC
Site-directed Mutagenesis
Use of Oligos to Synthesize Mutant
Alleles
Txn
YES
YES
NO
YES
XhoI XhoI
“Gap”
Use of Oligos to Synthesize Mutant Alleles
HindIII
BamHI
BamHI
XhoI XhoI
HindIII
“Gap”
Wild-type
TAGCCGTGGCTCGA
ATCGGCACCGAGCT
Synthesized
Mutant allele
CTCGAGTAGCCGTAGCTCGACTCGAG
GAGCTCATCGGCATCGAGCTGAGCTC
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:
Promoters
Enhancers
Origins of Replication
Centromeres
Telomeres
ORFs
any DNA Sequence-dependent Process
“Run-off expt.”
Initial Result = Promoters are Sufficient for Txn
Linker-scanner Analysis -> Several Elements
Several small elements
None essential (in this case)
Watson 9-5
Eukaryotic Promoter Elements
-Promoter Elements Conserved Among Eukaryotes
-No Individual Element found at All Promoters
Deletion and
Linker 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?
Transfection
and
Electroporation
Transient
Transfection
Assay
Watson 12-7
Deletion and
Linker Scanner Analysis
In vitro
Txn Assay
Promoter
sufficient in vitro
Identify and define TBP
and basal factors
Extract +
Prom.-Enh.
Basal Facts. +
Prom.-Enh.
Activated Txn
(Enhanced) &
Regulated Txn
Extract +
Prom.-Enh.
In vivo Txn Assay
Promoter not Sufficient
Identification of
Enhancers
Activators
Co-activators + Enhancer &
TBP & TAFs
Promoter
“Activated” txn & Regulated txn
Biochemistry
1
Protein
Gene (Organism 2)
2
Ab
3
6
4
9
5
Gene
7
12
8
Expression
Pattern
10
Mutant Gene
Mutant Organism
11
Genetics
Molecular Genetics Summary
1.
2.
3.
4.
5.
6.
7.
8.
Column Chromatograpy (ion exch, gel filtr)
A. Make Polyclonal Ab; B. Make Monoclonal Ab
Western blot, in situ immuno-fluorescence (subcellular, tissue)
Screen expression library (with an Ab)
Screen library with degenerate probe, mass spec. & database
Protein expression (E. coli)
A. Differential hybridization
A. Northern blot, in situ hybridization, GFP fusion, RT-PCR and q-RT
PCR
9.
A. low stringency hybridization; B. computer search/clone by phone; C.
computer search PCR
10. Clone by complementation (yeast, E. coli)
11. A. Genetic screen; B. genetic selection
12. RNAi
Reverse Transcriptase PCR or RT-PCR
A Qualitative Test for Whether an mRNA is present
Quantitative PCR or qPCR or Real Time PCR
SYBR green
qPCR machine is a PCR machine that can measure
the fluorescence of the reaction after each cycle
CYCLE NUMBER
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
AMOUNT OF DNA
1
2
4
8
16
32
64
128
256
512
1,024
2,048
4,096
8,192
16,384
32,768
65,536
131,072
262,144
524,288
1,048,576
2,097,152
4,194,304
8,388,608
16,777,216
33,554,432
67,108,864
134,217,728
268,435,456
536,870,912
1,073,741,824
1,400,000,000
1,500,000,000
1,550,000,000
1,580,000,000
53
AMOUNT OF DNA
1600000000
AMOUNT OF DNA
1
2
4
8
16
32
64
128
256
512
1,024
2,048
4,096
8,192
16,384
32,768
65,536
131,072
262,144
524,288
1,048,576
2,097,152
4,194,304
8,388,608
16,777,216
33,554,432
67,108,864
134,217,728
268,435,456
536,870,912
1,073,741,824
1,400,000,000
1,500,000,000
1,550,000,000
1,580,000,000
1400000000
1200000000
1000000000
800000000
600000000
400000000
200000000
0
0
5
10
15
20
25
30
35
PCR CYCLE NUMBER
AMOUNT OF DNA
CYCLE NUMBER
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
10000000000
1000000000
100000000
10000000
1000000
100000
10000
1000
100
10
1
0
5
10
15
20
25
PCR CYCLE NUMBER
30
35
54
1400000000 1400000000
AMOUNT OF DNA
AMOUNT OF DNA
1600000000 1600000000
800000000
800000000
600000000
600000000
400000000
400000000
200000000
200000000
1200000000 1200000000
1000000000 1000000000
0
0
0
5
0 10
5 15
10 20
15 25
20 30
25 35
30
35
PCR CYCLE NUMBER
PCR CYCLE NUMBER
55
AMOUNT OF DNA
AMOUNT OF DNA
10000000000 10000000000
1000000000 1000000000
100000000 100000000
10000000
10000000
1000000
1000000
100000
100000
10000
10000
1000
1000
100
100
10
10
1
1
0
5
010
Linear range =
cycles 16-24
515
1020
1525
2030
2535
30
35
PCR CYCLE NUMBER
PCR CYCLE NUMBER
56
Linear range = cycles 16-24
57
SERIES OF 10-FOLD DILUTIONS
58
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
Enhancers= different combinations of elements found in other
enhancers
Some elements found in both promoters and enhancers
Several small elements
None essential (in this case)
Watson 9-5
Which element(s) are required for regulated txn?
Regulatory Elements v. Control Elements
Genes can have Multiple Enhancers Which
Regulate Different Responses
E1
E2
Pr
Coding Region
Transcription
Gluc Metal Neither
E1
E2
E1
E2
E1
E2
Pr
+
+
-
-
-
Pr
+
-
-
Pr
-
+
-