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Announcements

Announcements. 1. Today – in class problem solving: including handout/worksheet on recombinant DNA technology, putting it all together AND restriction analysis problems

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Announcements

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  1. Announcements • 1. Today – in class problem solving: including handout/worksheet on recombinant DNA technology, putting it all together AND restriction analysis problems • 2. Based on your input, I’ve compiled slides from lectures that are good overviews of different topics covered. As time allows, I will go over “big picture” and main points of topics requested – in order of “popularity”. • 3. Wednesday is review day – when I take your specific questions from problem sets, end of chapter problems, etc… • 4. Exam 3 is Thursday, Friday, and Sunday at testing center. You will need a bluebook, pencil, calculator, ID – as usual. • 5. GRE workshop Nov. 13 - Bovee UC 7pm- detail by main office

  2. Learning Check Linear DNA fragment What is the restriction map of this cloned DNA fragment, showing the locations of the restriction sites and relative distance between sites? Marker EcoRI BamHI EcoRI/BamHI 15 10 9 8 8 7 6 6 5 4 2 5

  3. Transcription factors TBP-TATA binding protein TAFs- TATA assoc. factors

  4. Problems of Multicellularity • All of our genes are present in every cell, but only certain proteins are needed. • Expression of a gene at the wrong time, in the wrong type of cell, or in abnormal amounts can lead to deleterious phenotypes or death - even when the gene itself is normal. Pancreatic cell Neuron + insulin - insulin - neurotransmitter +neurotransmitter

  5. Promoters: Eukaryotic vs. Prokaryotic RNA pol II RNA pol Promoters: sequences adjacent to genes, where RNA pol binds to initiate transcription Euk. - Chromatin and TFs Prok. - Naked DNA and no TFs needed

  6. Chromatin remodeling

  7. TFs can recruit HATs or HDs

  8. Bacterial Strategy • If glucose is present, • then use glucose as a carbon source. • If glucose is not present, and if lactose is present, • then use lactose (indirectly) as carbon source. Levels of enzymes needed to use lactose as carbon source increase dramatically when lactose is present; enzymes are inducible and lactose is the inducer.

  9. The Operon Model - components R pol L Repressor protein has 2 key binding sites

  10. pol R R pol pol R R L L

  11. pol pol R pol pol R

  12. Learning Check pol Will transcription and translation of Z, Y,and A enzymes occur? What would happen if a wild-type copy of I was added? R L

  13. Catabolite Repression of lac Operon -/+ Glucose

  14. Goal: efficiency, don’t waste energy converting lactose, when glucose available

  15. Tryptophan Operon - Repressor Binds when tryptophan is present

  16. Other Theoretical Possibilities

  17. Separation of Nucleic Acids by CeCl Gradient Centrifugation

  18. Meselson-Stahl Experiment DNA Labeling with 15N Subsequent Generations Labeled with 14N Cesium Chloride Gradient Banding

  19. Expected Results From Conservative or Dispersive Reproduction If Conservative: Two bands, heavy and light, in 1st and 2nd generations If Dispersive, one smeary band in 1st and 2nd generations

  20. Expected Results if Semiconservative These results were obtained. A related experiment was performed in plants (Fig. 12.5)

  21. Steps of DNA Synthesis • Denaturation and Unwinding • Priming and Initiation • Continuous and Discontinuous Synthesis • Including Proofreading and Error Correction • Removal of Primer • Ligation of nicks in backbone

  22. Continuous and Discontinuous Synthesis • Continuous • on Leading Strand. • Discontinuous • on Lagging Strand • creates Okazaki • fragments. • DNA ligase joins • nicks in backbone.

  23. IV. The Eukaryotic Problem of Telomere Replication RNA primer near end of the chromosome on lagging strand can’t be replaced with DNA since DNA polymerase must add to a primer sequence. Do chromosomes get shorter with each replication???

  24. Solution to Problem: Telomerase • Telomerase enzyme adds TTGGGG repeats to end of lagging strand template. • Forms hairpin turn primer with free 3’-OH end on lagging strand that polymerase can extend from; it is later removed. • Age-dependent decline in telomere length in somatic cells, not in stem cells (germ cells), cancer cells.

  25. Aminoacyl tRNA synthetase Charging the tRNA

  26. Special Anticodon-Codon Base-Pairing Rules

  27. Overview of Prokaryotic Translation 1 2 E P A 3

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