Announcements

1. Announcements • 1. Exams are graded - available after class today - in lab. • - average = 70%; 105/150 • - high score = 97% (only one answer wrong) • - low score = • - see me (or email) if any questions and if you want to know your point total to date (I have 630 or 732 pts graded so far) 2. Reminder: no labs next week. Chemotaxis lab reports due 12/6. Start soon, so lab is fresh in your mind and you’ll have time to get answers to questions. Work together to do t-tests. Everyone will use same data set. 3. Genetics final exam is Monday, Dec. 9th. If you qualify to reschedule the exam, see me ASAP. Final is 200 pts of 1000 total pts; 1/2 of final is cumulative.

2. In the news….. Titanic accident - April 14, 1912 One unidentified infant - buried in “Titanic grave” Recently identified - how? Very small piece of tissue (tooth with piece of root) left in grave - most of body completely decomposed What technique used to make ID?

3. Review of lecture 34 Drosophila behavior and genetics Human behavior and genetics C. elegans behavior and genetics

4. Overview of lectures 35/36 • C. elegans chemotaxis behavior and genetics • Statistical analysis of chemotaxis data - t -tests • Genetics of cancer - Ch. 23 • cell cycle regulation • mutant genes confer predisposition to cancer • tumor suppressor genes • oncogenes • - translocations • genomic instability

5. I. Genetic approach to study chemotaxis Paper by Bargmann et al., 1993. Cell 74: 515-527. Known at start of study: in vertebrates, olfaction is used to detect presence of any volatile organic molecule and discriminate among different molecules Odorants bind to receptors in cilia of olfactory neurons and induce a signaling cascade in the cell Questions: 1. How specific is interaction of odorants and receptors? 2. How many receptors are expressed on a single olfactory neuron? 3. How is information about odorants trnsmitted to brain to generate appropriate behavior? Approach: determine whether C. elegans is attracted to volatile organic molecules; then screen for mutants that fail to chemotax to particular odorant; characterization of mutants will help address questions and allow for genes involved in process to be identified

6. Results of Bargmann study • tested 121 volatile organic chemicals: 50 strong attractants (12 alcohols), 11 variable, weak attractants; 60 not attractive (11 alcohols) • tested using attractance assay we’re using in lab this week • any generalizations/ rules about which alcohols are attractive to worms? Or is it random? • Specific size and shape are attractive: 4-6 carbons followed by hydroxyl group most attractive; very large numbers of carbons are repulsive

7. Results, continued from Bargmann study Is response to volatile chemicals non-specific OR is there a specific chemical recognition of particular odorants? How to distinguish between these two models? Use saturation assay - expose worms to uniform concentration of chemical 1; then add point source of chemical 2. If attraction to chemical 2 still occurs, then conclude a specific, saturable process is required for chemotaxis to each chemical. - 7 classes of volatile organic chemicals that are likely recognized by different receptor proteins

8. Characterization of odr mutants • Many different classes of mutants isolated: • some affect responses to all classes of volatile chemicals • what kind of protein affected? • some affect a subset of responses mediated by one • neuron type • - ex. odr-4 mutant does not respond to diacetyl but does respond to pyrazine (specificity in defect) • - what kind of protein affected? • results from a different study on chemotaxis identified the odr-10 mutant; it is also defective specifically to diacetyl • what kind of protein affected?

9. Do these “lab behaviors” relate to behavior in wild? Why does C. elegans chemotax to both water-soluble and organic, volatile chemicals? - short range to find nearby bacteria (food) - long range to find more distant food sources Why does C. elegans avoid certain chemicals? (ex. high osmolarity solutions) - they can cause paralysis and death

10. II. Statistical analysis of chemotaxis data Is there a significant difference between each index of each unknown and WT?

11. III. Genetics of Cancer Is cancer a single disease? In all cancers, mutations that alter gene expression are seen Most such mutations are somatic; 1% are germ-line - what is the difference? Cancer “runs in families” - known for over 200 years - but no clear-cut pattern of inheritance - usually one mutant allele of a cancer-causing gene is inherited - predisposes person to cancer - likelihood cancer develops depends on particular mutant allele, mutations in other genes, and environmental factors

12. Mutations play a central role in cancer • background rate of spontaneous mutation - due to ? • therefore, always baseline rate of cancer • above baseline, environmental agents that promote mutation also contribute to cancer = carcinogens • Which mutant genes are most likely to result in cancer? • How many mutations are needed to cause cancer? • How do mutations convert normal cells into malignant tumors? What are the differences between these cells? 1) uncontrolled growth 2) metastasis

13. The “cell cycle” Many cells alternate between dividing and “resting” or not dividing Gap 1; metabolic activity and cell growth G0(resting phase) Mitosis DNA synthesis 1 hour of 16 hour cell cycle Gap 2; metabolic activity and cell growth

14. Three main checkpoints in the cell cycle • 2001 Nobel Prize was awarded to 3 scientists who studied genes that regulate the cell cycle Is cell the correct size? Is DNA damaged? 2. Is DNA fully replicated? Is DNA damage repaired? 3. Have spindle fibers formed? Have they attached to chromosomes correctly? 1. 3. 2.

15. Why are cell cycle checkpoints important? What might result if DNA repair has not finished? Uncontrolled cell division could occur - cancerous cell Example: p53 protein normally targets cells with severe DNA damage to undergo programmed cell death. (this removes them from the population) If the p53 gene is mutated, damaged cells will not be removed and may continue dividing in an uncontrolled manner. Many different types of cancers involve mutations of p53.

16. Checkpoint Control of Cell Cycle Cdk-G1 cyclin Cdk-Mitotic cyclin (MPF)

17. Retinoblastoma • Diagnosis: “Cat’s eye” reflection (leukocoria) in affected eye. • Most common cancer of infants and children (1/20,000 U.S. live births). • Survival > 90% with early diagnosis and treatment. • Individuals at greater risk of developing other cancers.

18. Retinoblastoma Gene • A Tumor Suppressor, which normally suppresses unregulated cell growth. • Discovered as a regulator of growth of neuroblasts in developing retina of the eye. • Inactivation of both copies of the Rb Gene removes a “brake” on growth, leading to increased incidence of retinal cancer. • Since found to be active in all cells.

19. Retinoblastoma: Familial v. Sporadic “Loss of Heterozygosity” Common Rare

20. Rb Protein is Inactivated By CDK-Cyclin During G1  S

21. p53 Gene (tumor suppressor)Normal Functions • The “Last Gatekeeper” gene since malignant state not attained despite the presence of other cancer-causing mutations until p53 is inactivated by mutation. • Acts as a Transcription Factor to activate expression of p21, which inhibits CDK/G1 cyclin to halt the cell cycle. • Activates DNA repair. • Triggers apoptosis (programmed cell death) if damage can’t be repaired.

22. Role of p53 in Cell Cycle Control

23. p53 Mutations • Most commonly mutated gene in cancers (50% of total). • When p53 mutates, DNA-damaged cells are not arrested in G1 and DNA repair does not take place. This failure to arrest DNA-damaged cells will be repeated in subsequent cell cycles permitting other mutations to accumulate, culminating in neoplastic transformation... tumor formation and cancer.

24. Breast Cancer Tumor Suppressors • A small proportion of breast cancer is heritable. Two genes are associated with predisposition to breast cancer. • BRCA1 on chromosome 17 • BRCA2 on chromosome 13 • Normal function of both is in repair of ds DNA breaks.

25. Oncogenes • Arise from mutation in normal gene called a proto-oncogene. • Dominant mutation: one copy is sufficient to cause cancer. • First link between viruses and cancer proposed by Francis Peyton Rous in 1910 (Nobel Prize, 1966): cell-free extracts from chicken tumors injected into healthy chickens caused new tumors.

26. Rous Sarcoma Virus (RSV) • Discovered by Harold Varmus and Bishop, 1975-76 (Nobel Prize, 1989). • A transforming retrovirus: a cancer-causing single-stranded RNA virus that uses reverse transcriptase enzyme to make ssDNA, then ds DNA, which integrates into host DNA. • Note: not all retroviruses are TRV’s, not all oncogenes caused by viruses. • 100’s of oncogenes now known. • Human T-cell leukemia virus (HTLV) is only human TRV known; codes a TF.

27. Southern Blots Probed with viral src Gene Revealed Cellular Origin of Oncogenes Infected chicken #1 Infected chicken #2 Uninfected chicken (Negative Control) v-src c-src Proto-oncogene SURPRISE!

28. Origin of Transforming Retroviruses Capsid protein Reverse Transcriptase Envelope Protein Mutation creates oncogene

29. Ras Proto-oncogene • Mutated in 30% of all cancers. • A “molecular switch” in the signal transduction pathway leading from growth factors to gene expression controlling cell proliferation: GF  receptor   Ras    TF  target genes  growth. • A single amino acid change in Ras protein can cause constant stimulation of the pathway, even in the absence of growth factors.

30. Cancers Usually Result from a Series of Mutations in a Single Cell • Colon Cancer: oncogene Tumor suppressors oncogene

31. Tumor Progression: Evolution at the Cellular Level Benign tumor (polyp in epithelial cells) is confined by basal lamina; then additional mutation occurs. Malignant tumor (carcinoma in epithelial cells) grows very fast, becomes invasive, and metastasizes.

32. Cancer Cells Evade Two “Safety” Mechanisms Built into the Cell Cycle 1. Once p53 is inactivated, cells with DNA damage don’t arrest from G1 and don’t undergo apoptosis. 2. Telomerase enzyme is activated, avoiding the limit to cell divisions imposed by telomere shortening.