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G e n e t i c s N e w s. Fly lab preliminary report due next week. Lab next week in Jepson?. James Sikorski (Searle Pharmaceuticals) Speaks on the process of drug discovery Friday, 10 November, 1:30 PM W-201 Informal discussion with students afterwards. Topics.

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G e n e t i c s n e w s

GeneticsNews

  • Fly lab preliminary report due next week

  • Lab next week in Jepson?

  • James Sikorski (Searle Pharmaceuticals)

  • Speaks on the process of drug discovery Friday, 10 November, 1:30 PM W-201 Informal discussion with students afterwards


Topics

Topics


Promoter recognition by sigma factors

Promoter recognition by sigma factors

Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.


Promoter recognition by sigma factors1

Promoter recognition by sigma factors

Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.


Promoter recognition by sigma factors2

Promoter recognition by sigma factors

Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.


Modes of regulation prokaryote

Modes of regulation (prokaryote)

Decreased transcription by negative regulators (repressors)

Promoter choice by alternative sigma factors

Increased transcription by positive regulators

  • SQ1. Which mutation affects expression of more genes: - in a gene encoding a repressor? - in a gene encoding a sigma factor?


Modes of regulation prokaryote1

Modes of regulation (prokaryote)

Decreased transcription by negative regulators (repressors)

Promoter choice by alternative sigma factors

Increased transcription by positive regulators

  • Heat shock

  • Starvation for N

  • Growth stops

  • All or none

  • Presence of glucose

  • Starvation for N

  • Broad, Graded

  • Presence of lactose

  • Presence of tryptophan

  • Specific, graded


Modes of regulation prokaryote2

Modes of regulation (prokaryote)

Decreased transcription by negative regulators (repressors)

Promoter choice by alternative sigma factors

Increased transcription by positive regulators

  • SQ2. Regulation by alternative sigma factor vs a repressor?

Phosphate starvation phosphate transporter


Regulation of transcription

Regulation of transcription

Prokaryotic

Eukaryotic

ß-globin

?

?

Goal

Find sequences necessary for transcription of ß-globin

Regulation


What regulates globin transcription experimental dissection of regulatory region mutagenesis

What regulates ß-globin transcription?Experimental dissection of regulatory regionMutagenesis

Step 1,2: Clone region

Step 3: Mutagenize

Step 4: Return mutant

region to ß-globin gene


What regulates globin transcription experimental dissection of regulatory region expression

What regulates ß-globin transcription?Experimental dissection of regulatory regionExpression

Step 5: Put construct in cells

Step 6: Allow expression

Step 7: Isolate RNA


G e n e t i c s n e w s

What regulates ß-globin transcription?Experimental dissection of regulatory regionQuantitation (part I)

Step 8: Make probe

Step 9: Mix probe + RNA

Step 10: Digest unbound

RNA and probe


G e n e t i c s n e w s

What regulates ß-globin transcription?Experimental dissection of regulatory regionQuantitation (part II)

Step 11: Apply samples to gel

Step 10: Blot gel, expose X-ray film

Step 11: Quantitate degree of exposure


What regulates globin transcription experimental dissection of regulatory region results

What regulates ß-globin transcription?Experimental dissection of regulatory regionResults

Relative Transcription Level

-100 -80 -60 -40 -20 CAP

SQ3. Are most mutations in upstream region detrimental?

SQ4. If this were lac, where would worst mutations be?


What regulates globin transcription experimental dissection of regulatory region results1

What regulates ß-globin transcription?Experimental dissection of regulatory regionResults

Relative Transcription Level

-100 -80 -60 -40 -20 CAP

AGAGCGACACCCTGGTAAGGGCCAATCTGC. . .AGAGCATATAAGGT

SQ5. Where were the most detrimental mutations?


Regulatory sequences for eukaryotic genes are complex

Regulatory sequences for eukaryotic genes are complex

Histone H2B gene upstream region

Thymidine kinase gene upstream region

TATA boxTATAAAA

CAAT boxGCCCAATCT

GC boxGGGCGG

OctamerATTGCAT

What are these sites?


Sites are binding sites for regulatory proteins

Sites are binding sites for regulatory proteins

SQ6. Why is TATA box position unimportant, CAAT box not?

SQ7. How might repression work in eukaryotes?

Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.


Rna processing

RNA Processing

DNA

Transcription

3’mG5’ppp

Capping

AUG

AAUAAA

Polyadenylation

E

I

E

I

E

AAAAAAAAAAAAAAAAAAA...

I

I

Splicing

Why???

AAAAAAAAAAAAAAAAAAA...

E

E

E


Special problems of eukaryotes

Special problems of Eukaryotes

eukaryote

bacterium

Cooperative binding

Linear search

O

Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.


Special problems of eukaryotes1

Special problems of Eukaryotes

Eukaryotes: Homogeneous in DNA, heterogeneous in form


Eukaryotes vs bacteria eukaryotes use signals at a distance

Eukaryotes vs BacteriaEukaryotes use signals at a distance

SQ10. Examples?

Transcriptional regulation

Bacteria

Eukaryotes

Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.


Eukaryotes vs bacteria eukaryotes use signals at a distance1

Eukaryotes vs BacteriaEukaryotes use signals at a distance

SQ10. Examples?

Translation regulation

Bacteria

AAGGAGGnnnnnAUG

Eukaryotes


Eukaryotes vs bacteria eukaryotes use signals at a distance effect on evolution

Eukaryotes vs BacteriaEukaryotes use signals at a distanceEffect on evolution

Hair gene

Toenail gene

Hair gene


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