1. Lab 7: Cell Division: Mitosis and Meiosis

2. Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles AIM: Describe how the cell cycle is regulated. Are there molecular signals in the cytoplasm that regulate the cell cycle?

3. Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles AIM: Describe how the cell cycle is regulated. Are there molecular signals in the cytoplasm that regulate the cell cycle? Conclusion: The results suggest that molecules are present in the cytoplasm of cells in the S or M phase controlling the progression of phases. What are these molecules you ask?

4. Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles AIM: Describe how the cell cycle is regulated. The cell cycle clock: Cyclins and Cyclin-Dependent Kinases What is a cyclin-dependent kinase (cdk)? It’s a group of related kinases (enzymes that phosphorylates molecules – usually proteins) that are activated by a group of proteins called cyclins. What is a cyclin? A group of proteins that are made in cycles (hence the name) and are allosteric activators of the cdk’s (activate them upon binding). Ex. “Cyclin B”

5. Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles AIM: Describe how the cell cycle is regulated. Expression of human cyclins throughout the cell cycle

6. Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles AIM: Describe how the cell cycle is regulated. The cell cycle clock: Cyclins and Cyclin-Dependent Kinases Give an example: Ex. “Cyclin B” (the cyclin in the figure to the right) begins to be made at the end of S phase and slowly increases in concentration throughout G2. When the concentration gets high enough, Cyclin B will bind to Cdk-1 (the Cdk shown in the diagram to the right), activating it. “Cyclin B” + Cdk-1 = MPF MPF stand for maturation-promoting factor –(aka mitosis-promoting factor). ****All molecular binding (binding of two molecules) is concentration dependent.

7. Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles AIM: Describe how the cell cycle is regulated. The cell cycle clock: Cyclins and Cyclin-Dependent Kinases Give an example: Ex. MPF will activate mitosis at the G2 checkpoint by going around and phosphorylating a number of proteins including: 1. Condensins Proteins involved in condensing the chromosomes. 3. Proteins involved in microtubule spindle fiber formation 4. Lamins The proteins that hold the nuclear envelope together (nuclear lamina) 2. Histones – to condense DNA

8. Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles AIM: Describe how the cell cycle is regulated. The cell cycle clock: Cyclins and Cyclin-Dependent Kinases Give an example: How will the MPF complex be turned off? High levels of MPF result in the phosphoylation of proteins that will degrade (break down) Cyclin-B - negative feedback

9. Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles AIM: Describe how the cell cycle is regulated. All of the various cyclins and Cdk’s are part of the… Cell Cycle Control System

10. Lab 7: Cell Division: Mitosis and Meiosis Recall that mitotic cell division is regulated by Cyclin and Cdk’s (cyclin dependent kinases).

11. Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles AIM: Describe how the cell cycle is regulated. Checkpoints of the Cell Cycle Control System There are three major checkpoints: A. G1 checkpoint - Passing G1 checkpoint = point of no return - Cell can halt at this checkpoint and remain there for their lifetime -external and internal signals required to pass this checkpoint -Growth Factor (external signal) like PDGF (platelet derived growth factor), which stimulates division near a wound. - Enough nutrients/enzymes (internal)

12. Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles AIM: Describe how the cell cycle is regulated. B. The G2 checkpoint Ex) Is all the DNA replicated? Ex) Is the DNA too damaged from replication (too many mistakes made) to continue? If yes, apoptosis; if no, continue to mitosis. -Internal signals only Checkpoints of the Cell Cycle Control System There are three major checkpoints:

13. Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles AIM: Describe how the cell cycle is regulated. C. The M (metaphase) checkpoint Ex) Are all the sister chromatids present and attached to microtubules and ready to be pulled apart? -internal signals only Checkpoints of the Cell Cycle Control System There are three major checkpoints:

14. Chapter 12/13 - Cell Cycle, Meiosis, and Sexual cycles AIM: Describe how the cell cycle is regulated. Checkpoints Review

15. Lab 7: Cell Division: Mitosis and Meiosis

16. Lectin - Highly specific carbohydrate-binding proteins - Present throughout nature Ex) Concanavalin A Jack Bean ConA – pdb code: 3CNAJack Bean - Promotes cell division in plant cell by binding to cell surface receptors that have been glycosylated with very specific polysaccharide chains. Lab 7: Cell Division: Mitosis and Meiosis

17. What’s a Lectin? Ex) Concanavalin A Jack Bean ConA – pdb code: 3CNAJack Bean Lab 7: Cell Division: Mitosis and Meiosis

20. After the procedure you should see this for both the lectin-treated (experimental) and untreated (control) groups: Lab 7: Cell Division: Mitosis and Meiosis

21. Root TipInterphaseMitoticTotal 17821100 29235127 3641579 Total23471306 Root TipInterphaseMitoticTotal 18432116 210244146 39139130 Total277115392 Untreated (Control) Lectin-Treated (Expt) Data from one group: Lab 7: Cell Division: Mitosis and Meiosis

23. Root TipInterphaseMitoticTotal 17821100 29235127 3641579 Total23471306 Root TipInterphaseMitoticTotal 18432116 210244146 39139130 Total277115392 Untreated (Control) Lectin-Treated (Expt) Percentage of cell in interphase = 234/306*100=76.5 % We now know to expect that in a standard onion plant, 76.5% of the cells will be in interphase…. Therefore, if we have 392 total cells, we expect, if standard, 392*.765 to be in interphase = 300 cells – expected value Lab 7: Cell Division: Mitosis and Meiosis

24. Root TipInterphaseMitoticTotal 18432116 210244146 39139130 Total277115392 Lectin-Treated (Expt)300 cells in interphase – expected value Therefore 92 cells in mitotic phase – expected value InterphaseMitotic Phase Observed277115 Expected30092 Now calculate the chi2 and determine a p-value… Lab 7: Cell Division: Mitosis and Meiosis

25. InterphaseMitotic Phase Observed277115 Expected30092 Now calculate the chi2 and determine a p-value… = (277-300) 2 /300 + (115-92) 2 /92 = 529/300 + 529/92 = 1.76 + 5.75 = 7.51 DOF = # groups – 1 = 2 -1 = 1 P <.01 There is less than 1% probability that there is no difference (null hypothesis). Therefore, there is >99% that there is a difference and therefore the data supports the alternative hypothesis. Lab 7: Cell Division: Mitosis and Meiosis

29. Hela cell karyotypeNormal cell karyotype

30. Lab 7: Cell Division: Mitosis and Meiosis Hela cell karyotype

31. Lab 7: Cell Division: Mitosis and Meiosis HPV – Human Papillomavirus -most common STD - Infects genital areas as well as mouth and throat - Nearly everyone gets it at some point in their lives - 90% of cases fought off by immune systom and no symptoms arise. - The other 10%: - Genital Warts - Cervical Cancer *Nearly all cases of cervical cancer are caused by HPV!!!

32. Lab 7: Cell Division: Mitosis and Meiosis How does HPV cause cancer? - HPV is a small DNA virus - Ligand/receptor interactions allow virus access to cell. - Genome of virus enters nucleus by unknown mechanism - Between 10 and 200 copies are inserted into persons nuclear DNA. - Protein E6 and E7 are the major oncogenic (cancer causing) proteins - “E” stands for early because the gene is transcribed early in the viral life cycle. “L” for late. HPV genome - ~8000bp

33. Lab 7: Cell Division: Mitosis and Meiosis Why are E6 and E7 oncogenic? Both p53 (guardian angel) and pRb are tumor suppressor proteins. E6 functions by ubiquitinating p53 Recall that adding the small protein ubiquitin to a protein targets it to be degraded by the proteosome!

34. Lab 7: Cell Division: Mitosis and Meiosis p53 p53 (“the guardian of the genome”) bound to an enhancer region of DNA - Transcription factor “53” because its apparent mass is 53kDa or 53,000Da or 53,000amu. - Turns on genes that code for proteins having to do with DNA repair, cell cycle arrest and apooptosis. - Goes to work when the cell is in bad shape (DNA damage, etc…) - Like batman

35. Lab 7: Cell Division: Mitosis and Meiosis pRb pRb (Retinoblastoma Protein) - Inhibits cell cycle progression at G1 checkpoints - Binds and inhibits transcription factors that want to turn on genes and push the cell into S phase. - Named such because if you both copies are mutated early in life you will get retinoblastoma (cancer of the eye’s retina) pRb is inactivated when it is phosphorylated by CDK/cyclin!!

36. Lab 7: Cell Division: Mitosis and Meiosis

37. http://www.youtube.com/watch?v=pOROHmEmqmU Focus on meiosis part