Genetics NewsGenetics News Fly lab preliminary report due next week James Sikorski (Searle Pharmaceuticals) Speaks on the process of drug discovery Friday, 10 November, 1:30 PM W-201 Informal discussion with students afterwards Lab next week in Jepson?

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

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) Promoter choice by alternative sigma factors Increased transcription by positive regulators Decreased transcription by negative regulators (repressors) SQ1. Which mutation affects expression of more genes: - in a gene encoding a repressor? - in a gene encoding a sigma factor?

 Modes of regulation (prokaryote) Promoter choice by alternative sigma factors Increased transcription by positive regulators Decreased transcription by negative regulators (repressors) 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 (prokaryote) Promoter choice by alternative sigma factors Increased transcription by positive regulators Decreased transcription by negative regulators (repressors) SQ2. Regulation by alternative sigma factor vs a repressor? Phosphate starvation phosphate transporter

Prokaryotic Regulation of transcription ß-globin Eukaryotic Regulation ?? Goal Find sequences necessary for transcription of ß-globin

What regulates ß-globin transcription? Experimental dissection of regulatory region Mutagenesis Step 1,2: Clone region Step 3: Mutagenize Step 4: Return mutant region to ß-globin gene

What regulates ß-globin transcription? Experimental dissection of regulatory region Expression Step 5: Put construct in cells Step 6: Allow expression Step 7: Isolate RNA

What regulates ß-globin transcription? Experimental dissection of regulatory region Quantitation (part I) Step 8: Make probe Step 9: Mix probe + RNA Step 10: Digest unbound RNA and probe

What regulates ß-globin transcription? Experimental dissection of regulatory region Quantitation (part II) Step 11: Apply samples to gel Step 10: Blot gel, expose X-ray film Step 11: Quantitate degree of exposure

What regulates ß-globin transcription? Experimental dissection of regulatory region Results Relative Transcription Level CAP SQ3. Are most mutations in upstream region detrimental?SQ4. If this were lac, where would worst mutations be?

What regulates ß-globin transcription? Experimental dissection of regulatory region Results Relative Transcription Level CAP SQ5. Where were the most detrimental mutations? AGA GCGACACCC TGGTAAG GGCCAATC TGC... AGAGC ATATAA GGT

Regulatory sequences for eukaryotic genes are complex TATA boxTATAAAA CAAT boxGCCCAATCT GC boxGGGCGG OctamerATTGCAT Histone H2B gene upstream region Thymidine kinase gene upstream region What are these sites?

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 DNA AUG 3’mG5’ppp AAAAAAAAAAAAAAAAAAA... Capping Polyadenylation AAAAAAAAAAAAAAAAAAA... Splicing EEEII EEE I I Transcription AAUAAA Why???

Special problems of Eukaryotes O Linear search Cooperative binding bacterium eukaryote Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.

Special problems of Eukaryotes Eukaryotes: Homogeneous in DNA, heterogeneous in form

Eukaryotes vs Bacteria Eukaryotes use signals at a distance SQ10. Examples?  Bacteria Eukaryotes Transcriptional regulation Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.

Eukaryotes vs Bacteria Eukaryotes use signals at a distance SQ10. Examples? Translation regulation Bacteria Eukaryotes AAGGAGGnnnnnAUG

Eukaryotes vs Bacteria Eukaryotes use signals at a distance Effect on evolution Hair gene Toenail gene Hair gene