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