1. SC435 Genetics Seminar Welcome to our Unit 8 Seminar
We will continue our discussion of mutation and cancer
The seminar will begin at 9:00PM ET

2. Unit 8 Discussion board Unit 8 Quiz Unit 8 Exam Looking ahead
Final Project

3. Unit Readings
Chapter 12
Chapter 13

4. Mutations A mutation is any heritable change in the genetic material
Mutations are classified in a variety of ways
Most mutations are spontaneous: they are random, unpredictable events
Each gene has a characteristic rate of spontaneous mutation, measured as the probability of a change in DNA sequence in the time span of a single generation

5. Table 12.1

6. Mutations
Rates of mutation can be increased by treatment with a chemical mutagen or radiation, in which case the mutations are said to be induced
Mutations in cells that form gametes are germ-line mutations; all others are somatic mutations
Germ-line mutations are inherited; somatic mutations are not
A somatic mutation yields an organism that is genotypically a mixture (mosaic) of normal and mutant tissue

7. Mutations
Among the mutations that are most useful for genetic analysis are those whose effects can be turned on or off by the researcher
These are conditional mutations: they produce phenotypic changes under specific (permissive conditions) conditions but not others (restrictive conditions)
Temperature-sensitive mutations: conditional mutation whose expression depends on temperature

8. Mutations
Mutations can also be classified according to their effects on gene function:
A loss-of-function mutation (a knockout or null) results in complete gene inactivation or in a completely nonfunctional gene product
A hypomorphic mutation reduces the level of expression of a gene or activity of a product
A hypermorphic mutation produces a greater-than-normal level of gene expression because it changes the regulation of the gene so that the gene product is overproduced
A gain-of-function mutation qualitatively alters the action of a gene. For example, a gain-of-function mutation may cause a gene to become active in a type of cell or tissue in which the gene is not normally active.

9. Mutations Mutations result from changes in DNA
A base substitution replaces one nucleotide pair with another
Transition mutations replace one pyrimidine base with the other or one purine base with the other. There are four possible transition mutations

10. Mutations
Transversion mutations replace a pyrimidine with a purine or the other way around. There are eight possible transversion mutations
Spontaneous base substitutions are biased in favor of transitions:
Among spontaneous base substitutions, the ratio of transitions to transversions is approximately 2:1

11. Fig

12. Mutations
Mutations in protein-coding regions can change an amino acid, truncate the protein, or shift the reading frame:
Missense or nonsynonymous substitutions result in one amino acid being replaced with another
Synonymous or silent substitutions in DNA do not change the amino acid sequence
Silent mutations are possible because the genetic code is redundant

13. Mutations A nonsense mutation creates a new stop codon
Frameshift mutations shift the reading frame of the codons in the mRNA
Any addition or deletion that is not a multiple of three nucleotides will produce a frameshift

14. Sickle-cell anemia
The molecular basis of sickle-cell anemia is a mutant gene for b-globin
The sickle-cell mutation changes the sixth codon in the coding sequence from the normal GAG, which codes for glutamic acid, into the codon GUG, which codes for valine
Sickle-cell anemia is a severe genetic disease that often results in premature death
The disease is very common in regions where malaria is widespread because it confers resistance to malaria

15. Spontaneous Mutations
Mutations are statistically random events—there is no way of predicting when, or in which cell, a mutation will take place
The mutational process is also random in the sense that whether a particular mutation happens is unrelated to any adaptive advantage it may confer on the organism in its environment
A potentially favorable mutation does not arise because the organism has a need for it

16. Spontaneous Mutations
Several types of experiments showed that adaptive mutations take place spontaneously and were present at low frequency in the population even before it was exposed to the selective agent
One experiment utilized a technique developed by Joshua and Esther Lederberg called replica plating
Selective techniques merely select mutants that preexist in a population

17. Fig

18. Mutation Hot Spots
Mutations are nonrandom with respect to position in a gene or genome
Certain DNA sequences are called mutational hotspots because they are more likely to undergo mutation than others
For instance, sites of cytosine methylation are usually highly mutable

19. Mutagenes
Almost any kind of mutation that can be induced by a mutagen can also occur spontaneously, but mutagens bias the types of mutations that occur according to the type of damage to the DNA that they produce

20. DNA Repair Mechanisms Many types of DNA damage can be repaired
Mismatch repair fixes incorrectly matched base pairs
The AP endonuclease system repairs nucleotide sites at which the base has been lost
Special enzymes repair damage caused to DNA by ultraviolet light
Excision repair works on a wide variety of damaged DNA
Postreplication repair skips over damaged bases

21. Mismatch Repair
Mismatch repair fixes incorrectly matched base pairs: a segment of DNA that contains a base mismatch excised and repair synthesis followed
The mismatch-repair system recognizes the degree of methylation of a strand and preferentially excises nucleotides from the undermethylated strand
This helps ensure that incorrect nucleotides incorporated into the daughter strand in replication will be removed and repaired.

22. Mismatch Repair
The daughter strand is always the undermethylated strand because its methylation lags somewhat behind the moving replication fork
Fig

23. Mismatch Repair
The most important role of mismatch repair is as a “last chance” error-correcting mechanism in replication
Fig

24. AP Repair
Deamination of cytosine creates uracil which is removed by DNA uracil glycosylase from deoxyribose sugar. The result is a site in the DNA that lacks a pyrimidine base (an apyrimidinic site)
Purines in DNA are somewhat prone to hydrolysis, which leave a site that is lacking a purine base (an apurinic site)
Both apyrimidinic and apurinic sites are repaired by a system that depends on an enzyme called AP endonuclease

25. Fig

26. Excision Repair
Excision repair is a ubiquitous, multistep enzymatic process by which a stretch of a damaged DNA strand is removed from a duplex molecule and replaced by resynthesis using the undamaged strand as a template
Fig

27. Postreplication repair
Sometimes DNA damage persists rather than being reversed or removed, but its harmful effects may be minimized. This often requires replication across damaged areas, so the process is called postreplication repair
Fig

28. Ames test
In view of the increased number of chemicals used and present as environmental contaminants, tests for the mutagenicity of these substances has become important
Furthermore, most agents that cause cancer (carcinogens) are also mutagens, and so mutagenicity provides an initial screening for potential hazardous agents
A genetic test for mutations in bacteria that is widely used for the detection of chemical mutagens is the Ames test

29. The Cell Cycle There are two major parts in the cell cycle:
Interphase: G1 = gap1 S = DNA synthesis G2 = gap2
Mitosis: M
There are two essential functions of the cell cycle:
To ensure that each chromosomal DNA molecule is replicated only once per cycle
To ensure that the identical replicas of each chromosome are distributed equally to the two daughter cells 29

30. 30
Fig. 13.1

31. The Cell Cycle The cell cycle is under genetic control
A fundamental feature of the cell cycle is that it is a true cycle: it is not reversible
Many genes are transcribed during the cell cycle just before their products are needed
Mutations affecting the cell cycle have helped to identified the key regulatory pathways 31

32. The Cell Cycle
Progression from one phase to the next is propelled by characteristic protein complexes, which are composed of Cyclins and cyclin-dependent protein kinases (CDK)
Expression of mitotic cyclins E, A, and B are periodic, whereas cyclin D is expressed throughout the cell cycle in response to mitosis stimulating drugs (mitogens)
The cyclin-CDK complexes phosphorylate targeted proteins, changing dramatically their activity 32

33. 33
Fig. 13.8

34. The Retinoblastoma Protein
The retinoblastoma (RB) protein controls the initiation of DNA synthesis.
RB maintains cells at a point in G1 called the G1 restriction point or start by binding to the transcription factor E2F, until the cell has attained proper size
If the cycling cell is growing properly and becomes committed to DNA synthesis, several cyclin-CDK complexes inactivate RB by phosphorylation
After cell enters S phase E2F becomes phosphorylated as well and loses its ability to bind DNA 34

35. 35
Fig

36. The Cell Cycle
The progression from G2 to M is controlled by a cyclin B-CDK2 complex know as maturation-promoting complex
Protein degradation (proteolysis) also helps regulate the cell cycle. The anaphase-promoting complex (APC/C), which is a ubiquitin–protein ligase responsible for adding the 76-amino-acid protein ubiquitin to its target proteins and marking them for destruction in the proteasome
Proteolysis eliminates proteins used in the preceding phase as well as proteins that would inhibit progression into the next 36

37. Checkpoints
Cells monitor their external environments and internal state and functions
Checkpoints in the cell cycle serve to maintain the correct order of steps as the cycle progresses; they do this by causing the cell cycle to pause while defects are corrected or repaired
Checkpoints in the cell cycle allow damaged cells to repair themselves or to self-destruct 37

38. Three Main Checkpoints
A DNA damage checkpoint
A centrosome duplication checkpoint
A spindle checkpoint 38

39. 39
Fig

40. A DNA Damage Checkpoint
A DNA damage checkpoint arrests the cell cycle when DNA is damaged or replication is not completed.
In animal cells, a DNA damage checkpoint acts at three stages in the cell cycle: at the G1/S transition, in the S period and at the G2/M boundary
The p53 transcription factor is a key player in the DNA damage checkpoint. 40

41. A DNA Damage Checkpoint
In normal cells, level of activated p53 is very low
Protein Mdm2 keeps p53 inactivated by preventing phosphorylation and acetylation of p53 and by exporting p53 from the nucleus
Damaged DNA leads to activation of p53 and its release from Mdm2 41

42. A DNA Damage Checkpoint
Activated p53 triggers transcription of a number of genes - p21, s, Bax, Apaf1, Maspin,GADD45
DNA damage detected in G1 blocks cell G1/S transition
DNA damage in S phase reduces processivity of DNA polymerase and gives the cell time for repair
Processivity: number of consecutive nucleotides that replicate before polymerase detaches from template
DNA damage detected in S or G1 arrests cells at G2/M transition 42

43. 43
Fig

44. A DNA Damage Checkpoint
DNA damage also triggers activation of a pathway for apoptosis = programmed cell death
When the apoptotic pathway is activated, a cascade of proteolysis is initiated that culminates in cell suicide
The proteases involved are called caspases 44

45. Centrosome Duplication Checkpoint
Monitors spindle formation
Functions to maintain the normal complement of chromosomes
Sometimes coordinates with the spindle checkpoint and the exit from mitosis 45

46. The Spindle Checkpoint
Monitors assembly of the spindle and its attachment to kinetochores
The kinetochore is the spindle-fiber attachment site on the chromosome
Incorrect or unbalanced attachment to the spindle activates spindle checkpoint proteins, triggers a block in the separation of the sister chromatids by preventing activation of the anaphase-promoting complex (APC/C) 46

47. 47
Fig

48. Cancer
Cancer cells have a small number of mutations that prevent normal checkpoint function
Cancer is not one disease but rather many diseases with similar cellular attributes
All cancer cells show uncontrolled growth as a result of mutations in a relatively small number of genes
Cancer is a disease of somatic cells 48

49. Cancer
1% of cancer cases are familial: show evidence for segregation of a gene in pedigree
99% are sporadic: the result of genetic changes in somatic cells
Within an organism, tumor cells are clonal, which means that they are descendants from a single ancestral cell that became cancerous. 49

50. Cancer Cells vs. Normal Cells
In normal cells, cell-to-cell contact inhibits further growth and division, a process called contact inhibition
Cancer cells have lost contact inhibition: they continue to grow and divide, and they even pile on top of one another 50

51. Cancer Cells vs. Normal Cells
Even in the absence of damage, normal cells cease to divide in culture after about 50 doublings = cell senescence
Senescence of normal cells is associated with a loss of telomerase activity: the telomeres are no longer elongated, which contributes to the onset of senescence and cell death
Cancer cells have high levels of telomerase, which help to protect them from senescence, making them immortal 51

52. Key Mutational Targets
Many cancers are the result of alterations in cell cycle control, particularly in control of the G1-to-S transition
These alterations also affect apoptosis through their interactions with p53
The major mutational targets for the multistep cancer progression are of two types:
Proto-oncogenes
Tumor-suppressor genes 52

53. Key Mutational Targets
The normal function of proto-oncogenes is to promote cell division or to prevent apoptosis
The normal function of tumor-suppressor genes is to prevent cell division or to promote apoptosis 53

54. Familial Cancers
Mutations that predispose to cancer can be inherited through the germ line
The presence of this mutation predisposes the individual to cancer, because it reduces the number of additional somatic mutations necessary for a precancerous cell to progress to malignancy 54

55. Li–Fraumeni Syndrome
The Li–Fraumeni syndrome shows clear autosomal dominant inheritance. However, the affected individuals have a range of different tumors and often have more than one, including osteosarcoma, leukemia, breast cancer, lung cancer, soft-tissue sarcoma, and brain tumors
A large fraction of Li–Fraumeni families show segregation for a mutation in the p53 gene.
A situation analogous to the human Li–Fraumeni syndrome has been created in mice by experimental knockout (loss of function) of the p53gene via the germ-line transformation 55

56. 56
Fig