Ch 7 Mutation. A heritable change in DNA Random Source of genetic variation in a species may be advantageous, deleterious, neutral

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1 Ch 7 Mutation A heritable change in DNA Random Source of genetic variation in a species may be advantageous, deleterious, neutral Mutation (+ sexual reproduction) + natural selection = evolution

2 Types of mutations germ-line mutation in sperm or egg (meiosis), will be passed to offspring somatic mutation body cells, not passed on to offspring spontaneous mutation natural aspect of DNA replication induced UV light, chemicals, X-ray etc.

3 Germ line or somatic? cat Drosophila Euphorbia basal cell tumor Spontaneous? Induced?

4 Mutation and natural selection

5 Point mutation single base substitution, deletion, or addition

6 Point mutations 1. Base pair substitutions transition = G to A (purines) or T to C (pyrmidines) transversion =?

7 2. Missense mutation Wrong amino acid encoded May lead to a neutral or non-conservative amino acid change posit Sickle cell Hb Subsititution, addition or deletion? TS or TV? Which codon position?

8 Non-conservative mutation

9 3. Nonsense mutation Premature stop codon Codon AAA UAA lys stop

10 5' - ATG ACT CAC CGA GCG CGA AGC TGA - 3 3' - TAC TGA GTG GCT CGC GCT TCG ACT 5' mrna: 5' - AUG ACU CAC CGA GCG CGA AGC UGA - 3 Protein: Met Thr His Arg Ala Arg Ser Stop 5' - ATG ACT CAC TGA GCG CGA AGC TGA - 3 3' - TAC TGA GTG ACT CGC GCT TCG ACT - 5' mrna: 5' - AUG ACU CAC UGA GCG CGU AGC UGA - 3' Protein: Met Thr His Stop

11 4. Neutral mutation - mutation does not change amino acid folding - codon AAA AGA lys arg (both basic aa)

12 5. Silent mutation No amino acid change, usually 3 rd position TS codon GCA GCG arg arg

13 6. Frameshift mutation Shifts reading frame --> scrambled message May lead to a premature stop codon The sun was hot but the man did not get his hat.

14 Other mutations Larger insertions, trinucleotide repeats, deletions, duplications, translocation, inversions

15 pg. 475 Fragile X Syndrome FMR-1 gene X-linked dominant 2 nd leading cause of mental retardation

16 (Reprinted from Medical Genetics, 2nd ed., Jorde LB, et al, 2000, with permission from Elsevier Science.)

17 CGG repeat in promoter region Normal = 6-54 repeats Normal carriers = repeats Fragile X syndrome = repeats

18 Mutation rate Mutation rates per gene differ. May be hot spots

19 Causes of mutations 1. Spontaneous mistakes by DNA polymerase = replication errors Most repaired by DNA proofreading and repair systems

20 2. Induced mutations Chemicals Mustard gas, industrial waste, PCBs, DDT Ethidium bromide Base analogues Example: Bromouracil substitutes for thymine Radiation sun (UV), radon, X-ray Breaks, dimers High doses kill cells

21 The Ames test Indirect assay for carcinogens Plate 10 8 auxotrophic bacteria (his-) Add filter disc with chemical Look for revertants to his+ = mutations

22 Spontaneous reversion rate to His+ colonies 1000X higher rate with mutagen

23 Repair of mutations 1. Direct reversal Example: Mismatch repair 3 5 proofreading by DNA polymerase

24 2. Excision repair Ex. base excision repair - single strand incision at both sides of error; - Excise mistake - DNA synthesis to replace the gap - Ligation

25 Defective excision repair: Xeroderma pigmentosum (1/250,000) Autosomal recessive Extreme sun sensitivity High risk of skin cancer (1000X) average by age 8 Metastatic melanoma Corneal damage

26 Knockout mice for excision repair (XPA gene) XPA gene knocked out UV sensitive and develop tumors Study cancer, drugs

27 Cancer Ch 20 Terms Tumor (neoplasm) = mass of cells Benign = cells in a single, contained, mass Malignant = invades surrounding tissue cells may break off and move = metastasis Transformed cell has lost normal growth controls Loses contact inhibition Immortal Oncogenesis = initiation of cancer

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29 Benign vs. malignant

30 metastasis

31 The cell cycle Time from one cell division to the next G1 (Gap 1) Prepare S G2 Synthesize DNA Gap 2 Prepare to divide

32 Arrest cell cycle repair damage or send cell to apoptosis G 1 -S checkpoint Checkpoints Should cell continue to S? G 2 -M M Is the DNA replicated Is the cell large enough? Are chromosomes attached to the spindle?

33 cell cycle regulation Growth Factors stimulate cell division in target cells Cyclins cyclic concentration with cell cycle Cyclin-dependent kinases (CDK) phosphorylate other proteins Signal transducers Transcription factors bind promoters, enhancers in DNA to regulator transcription

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35 Cyclin/CDK 1. cyclin binds a CDK 2. CDK phosphorylates another protein 3. cell continues in cell cycle to next checkpoint and cyclin degraded Detailed overview Sinauer Detailed overview WH Freeman

36 Figure 20.2

37 Cancer is genetic pg. 581 Hereditary cancers Predisposition genes Ex. some forms of colon cancer ~ 10 % of cancers Sporadic cancers ~90% of cancers Descendents of cancerous cells all cancerous Mutagens increase cancer risk Certain mutations cause certain cancers

38 Cyclins levels:cell cycle

39 Genes and cancer 1. Proto-oncogenes (Harold Varmus and Michael Bishop Nobel Prize 1989) Genes involved in cell cycle If mutated oncogene Tumor cells exhibit oncogenes Dominant mutation Cancer is a multi-hit disease Mutations in more than one cell cycle gene required

40 Protooncogenes (100 identified) Table Growth factors stimulate cells to divide 2. Growth factor receptors bind growth factor at cell membrane signal transduction cell division

41 3. Signal transducers Ex. H-ras signal cascade EGR binds to EGFR ras binds GTP cell division Growth factor receptor complex cascade

42 Why is mutation in a protooncogene relevant? EXAMPLES: Expression of a PDGF in cell that does not normally express it Mutant EGFR or signal transducer gene may be always on

43 Fig 20.3 Signal transduction Mutation in gene encoding signal transduction protein Loss of control of cell cycle neoplastic cell

44 How do proto-oncogenes become oncogenes? Spontaneous or induced mutation in gene coding region promoter region Point mutation, deletion, gene amplification, translocation HPV in humans (viral genes speed cell cycle) KNOW THE FLOW

45 2. Tumor suppressor genes Table 20.3 Protein products of suppress uncontrolled cell proliferation Recessive why? Both copies must be inactivated for loss of function = 2 mutations in one cell required 2 hit model (Knudson 1971)

46 Retinoblastoma Eye cancer develops in childhood Hereditary can be passed on Sporadic one eye only (13q14.1-q14.2.) 11 cases per million children aged 1 4 in US/yr treat with laser therapy

47 Sporadic develop 2 mutations in 1 cell after birth Child born RB/RB Hereditary inherited 1 mutation in all body cells, need 1 more in any cell = Loss of heterozygosity (LOS) Child born RB/rb

48 Fig. 20.9

49 Function of prb tumor suppressor protein prb is a G1 S checkpoint protein 1. E2F protein binds prb E2F is a transcription factor E2F is not active if bound to prb Cell cycle arrested = cell does not move to S

50 2. Cyclin/CDK phosphorylates prb 3. E2F released to travel into nucleus 4. E2F stimulates transcription of genes cell moves to S phase (DNA replication) 5. Cyclin degraded (no more phosphorylation of prb), EF2 bound If RB gene is mutant then

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52 Telomerase in cancer cells Telomeres at ends of chromosomes TTAGGG repeated many times Chromosome shortens with each cell division Ex. human fibroblasts in culture senesce as telomeres shorten No telomerase in normal cells Cancer cells make telomerase immortalized

53 Cancer is a multi-step disease Accumulation of mutations in a number of genes in single cell Can build up over decades Vogelstein model FAP colorectal cancer

54 Colorectal cancer

55 FAP APC tumor suppressor gene mutation is inherited (adenoma class I is benign tumor) Ras Oncogene Tumor suppressor gene DCC (Adenoma class III) Tumor suppressor gene TP53 metastasis

56 angiogenesis Tumor obtains its own blood supply HHMI animation

57 metastasis Tumor cells move to new location metastasis Pancreatic cancer liver

58 p53 tumor suppressor Involved in ~50% of cancers

59 Example of hereditary cancer predisposition Li-Fraumeni syndrome Inherit one mutant copy of TP53 gene One more mutation (single cell).. Develop a number of cancers Bone, Blood cell, Brain, Breast, Colon, Bladder cancer >90% lifetime risk of cancer (Very rare, 17p13.1)

60 Role of p53 tumor suppressor Monitors signals that indicate DNA damage/mutation Damage cell increase p53 protein

61 Damage DNA p53 DNA repair, cell cycle arrest or apoptosis genome integrity

62 When normal cells are damaged beyond repair, they are eliminated by apoptosis (A). Cancer cells avoid apoptosis and continue to multiply in an unregulated manner (B).

63 p53 can activate apoptosis pathway Apoptosis Programmed cell death HeLa cell apoptosis Garland science

64 p53/p53 knockout mice Develop normally, within 10 months 100% of mice have cancer

65 Evidence that p53 is a tumor suppressor Moshe Oren Weizmann Institute/Israel

66 Types of cancer Carcinomas; 90% of cancers epithelial cells Sarcomas; rare tumors of connective tissues and muscle Leukemias and lymphomas; 8% of tumors. Basal cell carcinoma Kaposi s sarcoma of blood vessels leukemia Hodkins lymphoma in lymph node