Fundamentals 1: 10:00 - 11:00Scribe: Brittany Paugh

Fundamentals 1: 10:00 - 11:00Scribe: Brittany Paugh

Wednesday, September 23, 2009Proof: Chris Bannon

Dr. PittlerGene and Gene MutationsPage1 of 9

**Slides 1-9 were covered 9/22. Some of this material will be covered quickly, some removed. He will tell us as he goes what to ignore from these slides. **

1. Genes and Gene Mutations [S1]:
2. Mutation [S2]
3. Mutation [S3]
4. There is such a thing called a good mutation.
5. Mutation [S4]
6. There are very few changes at the protein coding area. We know that most of our DNA is non-coding anyway so hopefully mutations that do arise won’t lead to detrimental effect on protein function.
7. Table 12.1 [S5]
8. Mutation [S6]
9. Mutation [S7]
10. Recombination must happen. It ensures variation. That s why none of us are exactly the same.
11. Somatic Mutation [S8]
12. Hemoglobin [S9]
13. We have been over the structure of hemoglobin previously
14. Its function is to carry oxygen and carbon dioxide to and from the lungs for delivery of oxygen and removal of waste/byproducts (carbon dioxide)
15. it has two beta chains and two alpha chains.
16. A single base change in the hemoglobin gene causes sickle cell anemia
17. Single base change in hemoglobin causes sickle cell anemia [S10]
18. Base changes in hemoglobin are the cause of sickle cell anemia.
19. Normal red blood cells (picture on the left, wild type phenotype) are smaller and move through capillaries easily
20. Mutant phenotype (right) is stretched out, which makes it more difficult to travel through capillaries. This causes a number of problems such as organ damage, when RBC’s become lodged and blood cannot flow freely.
21. There is a change in the glutamine to a valine so this is a single nucleotide change. You end up with molecule that is very stretched out.
22. Hemoglobin genotype causes sickle cell anemia phenotype [S11]
23. Mutation encodes valine in place of glutamic acid (VG)
24. Altered surface of hemoglobin allows molecules to link in low oxygen conditions and create sickle shape of red blood cells.
25. Sickling of red blood cells causes anemia, joint pain, and organ damage when RBCs become lodged in small blood vessels.
26. Different sites in a gene can mutate and cause distinct phenotypes [S12]
27. The effects are more or less severe depending on the site of the mutation
28. Promoter defects and defects in one of the hemoglobins causes anemia;
29. If you reduce the amounts of beta hemoglobin so ratio of alpha to beta is now going to change, it will now be higher for alpha, and it can lead to a number of problems.
30. Heterozygous mutation  milder thalassemia minor
31. Homozygous mutation  more severe thalassemia major
32. Collagen [S13]
33. Collagen is an important component of many cells (60% of protein in bone and cartilage.) it is also in the sclera.
34. Its structure is a triple helix. Procollagen molecule is clipped at specific sites at the ends to form the mature molecule; triple helix of multiple subunits.
35. Different collagen mutationsDistinct disorders [S14]
36. All different types of collagen disorders are explained here, he said we could read through this on our own
37. Eye and teeth disorders as a result of collagen mutations.
38. There are a number of collagen genes
39. Ehlers-Danlos Syndrome [S15]
40. This is an extreme example. Most of us cannot do this. (Picture of young boy stretching out his mouth) This is extreme elasticity of tissue most of us can’t stretch out the tissue like this. I hope for the dentists this is not your first patients’ teeth.
41. Alzheimer disease [S16]
42. Alzheimer disease is a complex disease, caused by one particular gene (presenillin1) and the mutation is autosomal dominant
43. What you see here is a number of different mutations that occur in transmembrane segments or in the segments that are bound within the membrane. Each can lead to the disorder of Alzheimer’s.
44. These proteins function to monitor the beta amyloidal usage
45. Accumulation of beta amyloidal defects (30+ missense mutations) Then you end up with the characteristic plaques
46. Genotype to distinct phenotype [S17]
47. Look at this slide on your own.
48. Shows four different diseases and the proteins that cause them
49. Genotype to distinct phenotype S18]
50. Huntington’s disease—(Huntingtin protein)
51. Mutation: Extra nucleotides in gene result in extra amino acids
52. Uncontrollable movements, personality changes
53. Marfan’s Syndrome---Lincoln’s Syndrome—(Fibillin)
54. Mutation: Too little elastic connective tissue protein
55. Phenotype: Long limbs, weakened aorta, spindly fingers, sunken chest, lens dislocation
56. Neurofibromatosis
57. Mutation: defect in protein
58. Phenotype: benign tumors of nervous tissue beneath skin (Neurofibromin)
59. Spontaneous mutation [S19]
60. Spontaneous mutations could be foundation for cancer, and a number of other different diseases
61. Can occur in individual cells, somatic tissues or during development
62. Not caused by exposure to known mutagen; doesn’t mean its not caused by them but its just not known
63. Thought to be due to errors in DNA replication
64. DNA bases have a slight chemical instability; little bit of dynamics (exists in alternating forms called tautomers) there are a number of repair mechanisms present to try to prevent this from leading to a disease
65. Spontaneous mutation rate [S20]
66. Spontaneous mutation rates differ for different genes, throughout the genome
67. Size dependence
68. Sequence dependence
69. Hot spots: those areas of the DNA that are repetitive in nature are harder for the polymerases to get through and copy properly
70. On average 1 in 100,000 chance of acquiring a mutation in a gene each round of replication. There are certain genes that are more susceptible to mutations. There are certain regions in genome that have mutations at about~2% and then some up to 6%.
71. Each individual has multiple new mutations. Most occur by chance and are not in coding regions of genes, reason for vast amount of noncoding dna.
72. Mutation rates of genes causing disease [S21]
73. This is not worth memorizing. The take home lesson is that there is a great difference in mutation rate at different genes, which seems to have a lot to do with the primary structure at that region
74. Everyone of us has mutations. Junk DNA that doesn’t seem to have any function may exist just because they are more likely to mutate and that’s okay.
75. Mutations in pathenogens [S22]
76. Mutations occur in bacteria and in viruses, often leading to more virulent
77. Becomes able to create diseases in individuals. It becomes worse with improper use of antibiotics. The result is super bacteria that cause a number of problems. The bacteria can mutate their DNA to create immune strands that are a problem. A number of viruses made worse by this.
78. See with many different viruses, AIDS, etc.
79. Mutational hot spots exist [S23]
80. We talked about the concept of a hotspot. What are some examples? They are: any short repetitive sequence pairing of repeats that may interfere with replication or repair enzymes
81. Palindromes are often associated with insertions or deletions
82. Duplications of larger regions and mispairings during meiosis. You can get a loss of a region due to a mispairing.
83. Mutations [S24]
84. More than 1/3 of the many mutations that cause alkaptonuria occur at or near one or more CCC repeats, even though these repeats account for only 9% of the gene (hot spot)
85. Mutations in the gene for clotting factor IX, which causes hemophilia B occur 10 to 100 times at any 11 sites in the gene that have direct repeats of CG (CGCGCG…..)
86. Repeat regions are more susceptible because polymerase have trouble getting through those properly

1. Small or large insertion or deletion [S25]
2. Here is example of the hairpin loops forming. Might happen when DNA opens up for replication so its now in single strand form. Sometimes there is false pairing that occurs and it has to be resolved. Hairpin will never be resolved and DNA is lost
3. Here is aPalindrome sequence that you can cut with a restriction enzyme
4. On the Right....a mispairing where there should be two alphas on each, but instead there are three one and one on the other. Chromosomes are not aligned so this leads to loss of material (1) or duplication of material (3)
5. Mutations [S26]

a. He said to go through this on your own here it is

b.The blood disorder alpha thalassemia illustrates the effect of direct repeats of an entire gene wild-type has four genes that specify alpha globin chains, two next to each other on chromosome 16

c. Homologs with repeated genes can misalign during meiosis when the first sequence on one chromosome lies opposite the second sequence on the homolog

d. If crossing over occurs, a sperm or oocyte can form that has one or three of the alpha globin genes instead of the normal two

e. A person with three alpha globin genes produces enough hemoglobin and is considered healthy

f. Individuals with only two copies of the gene are mildly anemic

g. Single alpha globin individuals are severely anemic, and a fetus lacking alpha globin does not survive

h. Alpha thalassemia is common because carriers have an advantage, they are protected against malaria

1. Example [S27]
2. This is the same example of chromosome 16 explained in slide 25
3. Basis for thalassemia
4. Induced mutation [S28]

a. Can be environmentally induced.

b. chemical that causes a mutagen that leads to cancer = carcinogen

c. Carcinogens can be mutagens, and mutagens can be carcinogens (not mutually exclusive).

1. Induced mutation [S29]

a. Alkylation makes a base no longer functional.

b. Alkylation agents remove a DNA base, which is replaced with any of the four bases-three of which creates a mismatch with the complimentary strand

c. Acridine dyes add or remove a single DNA base. Adding or deleting a single base destroys a gene’s information, altering the amino acid sequence of the encoded protein

d. Mutagenic chemicals alter base pairs, so that an A-T replaces G-C, or vice versa, thereby changing a genes DNA sequence

e. X-rays and other forms of radiation can delete a few bases or break chromosomes

f. Cigarettes(source of radiation) and airplanes cause radiation as well

1. Ames test [S30]
2. This is leading into the cancer lecture
3. Ames test is an in vitro test of the mutagenicity of a substance using Salmonella bacteria with a mutation in the gene for histidine. It is an oxotrope (some thing which requires a chemical/molecule present in media for it to grow) for histidine. It tests for agents that cause cancer. Any chemical can be tested.
4. Bacteria are exposed to test substance.
5. Growth of bacteria on media without histidine is recorded.
6. Bacteria only grow if mutations have occurred.
7. Rate of mutation is determined, as likelihood or mutation of gene requiring histidine is as likely as any other mutation, therefore can extrapolate from this the rate of histidine mutation is rate for any mutation.
8. Substance can be mixed with mammalian liver tissue prior to testing to mimic toxin processing in humans.
9. Mutagen/Source [S31]
10. These things containing mutagens are found in things we encounter every day. (Peanut butter-alphatoxin)
11. can lead to anaphylactic shock in some individuals
12. smoked meats contain nitrates
13. A limitation in…. [S32]

a. BYPASS THIS SLIDE 

1. Natural Exposure to mutagens [S33]
2. Natural environment sources of radiation include:
3. Cosmic rays, sunlight, and radioactive minerals in the earth’s crust
4. Medical x-rays and ionizing radiation hazards (affecting sugar backbone of DNA)
5. Weapons facilities, research laboratories, health care facilities, nuclear power plants, and certain manufacturing plants
6. Radiation exposure is measured in milligrams and the annual exposure in the northern hemisphere: 360 milligrams
7. Most of the radiation that we are exposed to are ionizing type which removes electrons from atoms
8. Ionizing radiation breaks the sugar-phosphate backbone DNA

XXXIV. Natural exposure to mutagens [S34]

a. Three types of ionizing radiation (alpha, beta, gamma)

b. Alpha is the least energetic and most short-lived (Most dangerous)

I. Absorbed by the skin

ii. Uranium and radium

1. Beta can penetrate deeper (Will cause significant tissue damage when ingested, but typically not that harmful)
2. Tritium (isotope of hydrogen), Carbon-14, and strontium-70
3. Both alpha and beta tend not to harm us
4. However if eaten or inhaled they will do damage
5. Gamma can penetrate all the way through the body therefore it damages our tissues
6. Plutonium and cesium isotopes used in weapons
7. This form of radiation is intentionally used to kill cancer cells

1. Natural exposure to mutagens [S35]
2. X-rays are non-ionizing radiation
3. Have less energy and do not penetrate the body to the extent that gamma rays do (NOT in gamma class of radiation)
4. The effects of radiation damage to DNA depends on the functions of the mutated genes
5. Mutations in oncogenes or tumor suppressor genes can cause cancer
6. Chemical mutagens
7. The risk that a chemical will cause a mutation is often less than the natural variability in susceptibility within a population. Different chemicals go different places in the body.
8. Table 12.6 (airplanes also in addition to listed sources)

1. Types of mutations [S36]
2. Point mutation [S37]

a. A point mutation is a change of a single nucleotide to one of the other three possible nucleotides… There are 2 types

b.Transitionpurine replaces purine A  G or G  A

pyrimidine replaces pyrimidine C  T or T  C

c. Transversion

Purine replaces pyrimidine A or G  T or C

Pyrimidine replaces purine T or C  A or G

XXXVIII.Missense mutation [S38]

a. A point mutation that exchanges one codon for another causing substitution of an amino acid

i, Missense mutations may affect protein function severely, mildly or not at all (ex. change ATG to TTG)

b. Hemoglobin mutation (previous example)

i. glutamic acid (CTC) -> valine (CAC)causes sickle cell anemia

c. The DNA sequence CTC encodes for mRNA GAG that specifies glutamic acid

d. There is a point mutation in sickle cell disease that changes (Transversion) the DNA sequence to CAC that encodes for mRNA GUG that specifies valine and this causes an alteration in function

XXXVIIII Nonsense mutation [S39]

1. Change from a codon encoding an amino acid to any one of 3 codons encoding a stop codon; leading to premature termination.
2. In 15% of people who have Becker muscular dystrophy (milder adult form of the condition) the muscle protein dystrophin is normal, but, its levels are reduced
3. The mutation causing the protein shortage is in the promoter for the dystrophin gene and thus slows the transcription process
4. The other 85% have Becker muscular dystrophy have shortened proteins, not a deficiency of normal-length proteins
5. Point mutations can disrupt (premature) trimming of long precursor molecules of collagen(A type of Ehlers-Danlos syndrome

1. Splice site mutation [40]
2. Point mutations can affect a gene’s product when it alters a site where introns would normally be removed from mRNA (GU on left side of intron and AG on the right side of the intron)
3. Anything that affects the function of a spliceosome can lead to disease—if intron is maintained then almost always you end up with a premature stop codon.
4. it can affect the phenotype if an intron gets translated into amino acids, or an exon is skipped instead of being translated
5. When we retain an intron the bases are added to the protein-coding portion of mRNA
6. Cystic fibrosis: Missense mutation alters an intron site so that it is not removed
7. Splice site mutation [41]
8. Insertion or deletion mutation [42]

a.Frameshift mutation is the addition or deletion of nucleotide thatis not divisible by 3 (genetic code is read in triplicate; codons). Almost always leads to a premature stop codon and an altered protein sequence. It probably leads to a loss of functional protein or a gain of function of the protein.

1. Insertion or deletion mutation [43]

a.It is worth knowing that the Muscular dystrophy gene is 2.5 million base pairs in length.

i. Its no surprise that there are large alterations that effects it. Often they are insertion. With a very large gene you can often see these tandem inversion (A tandem duplication is a particular form sequences of insertion in which identical sequences are found side by side..)

ii. often see tandom inversions occur, in which errors in replication lead to duplications and deletions both of which can lead to disease.

b. Deletion is the removal of sequences. Insertion is the addition of sequences.Two-thirds of Duchenne muscular dystrophy cases are large deletions.

c. Gaucher disease is caused by a single base insertion creating a frameshift

d. Charcot-Marie-Tooth disease is caused by a tandem duplication of 1.5 million bases

1. Table 12.7 [44]
2. Missense.. Single replacement
3. Nonsense. Stop codon is read prematurely and a truncated protein results
4. Deletion. Entire codon deleted
5. Frame Shift: insertion of a single extra nucleotide/ letter which shifts everything downstream of it
6. Expanding from generation to generation. There are a number of triple repeat diseases. There is a chance to increase severity in each generation. This is called Anticipation; that is a medical term for this condition. The reason for that it is due to continuous expansion of repeat regions, going through generations and can eventually eliminate the function of a particular gene.
7. Pseudogenes [45]

a.A pseudogene is a DNA sequence that is non-functional; reminiscent of a gene but which is not translated (may or may not transcribe RNA).

I. Counterpart to a pseudogene can in-fact encode for a gene and recombination between counterpart and pseudogene and can end up inactivating actual gene.

b. Pseudogenes may have evolved from original functional gene by duplication and acquired mutation.

c. Crossing over between a pseudogene and a bona fide gene can disrupt gene expression.

d. The problem is there is a counterpart that is a functional gene and recombination from that gene from it can occur.

e. Some of the nonfunctioning DNA is possible remnant of evolution

1. Expanding repeats [46]

a.Insertion of triplet repeats leads to extra amino acids.

b. Some genes are particularly prone to expansion of repeats. There are a number of diseases.

c. Number of repeats correlates with earlier onset and more severe phenotype (as with Anticipation discussed earlier).

d. Expansion of the triplet repeat and coincident increase in severity of phenotype occur with subsequent generations, phenomena termed anticipation.

i. Each generation can be more and more severe due to expansion or more and more repeats.