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

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

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  1. 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

  2. Topics

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

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

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

  6. 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?

  7. 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

  8. 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

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

  10. 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

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

  12. 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

  13. 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

  14. 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?

  15. 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?

  16. Regulatory sequences for eukaryotic genes are complex Histone H2B gene upstream region Thymidine kinase gene upstream region TATA box TATAAAA CAAT box GCCCAATCT GC box GGGCGG Octamer ATTGCAT What are these sites?

  17. 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.

  18. 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

  19. 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.

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

  21. 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.

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

  23. Eukaryotes vs BacteriaEukaryotes use signals at a distanceEffect on evolution Hair gene Toenail gene Hair gene

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