Mutation, DNA repair, and Evolution (with references to disease, including aging and cancer)

1. Mutation, DNA repair, and Evolution (with references to disease, including aging and cancer) Objectives: 1. Define the nature of genetic variation 2. Provide a context for understanding mutation (disease and aging) Define evolutionary principles, such as natural selection, mutation rate, molecular clocks Explain why rates of mutation/variation differ between genes, and between different regions of the same gene Reading: Krane and Raymer, pp. 57-65

2. What is implied here? 1995 statement by the National Association of Biology Teachers: “Evolution is an unsupervised, impersonal, unpredictable, and natural process of temporal descent with gradual modification”

3. How Do We Explain Adaptation? • Divine creation; intelligent design • Inheritance of acquired characteristics (Lamarck) • Natural selection (Darwin)

4. What is the keyboard upon which Natural Selection plays? --- and --- What is the ultimate source of genetic variation? --- or --- What is the pool of raw materials from which new variants are selected?

5. Why is everyone different? What are the three sources of genetic variation?

7. A human karyotype, or chromosome spread

9. 2N = 4 So, is it possible that no genetic information was inherited from the paternal grandfather (F1) and none from the maternal grandmother (M2)?

10. Meiosis in humans creates tremendous variety: 223 combinations of 23 chromosomes via meiosis = 8.4 million ways to assort 23 pairs of chromosomes Then,… 8.4 million (sperm) x 8.4 million (egg) = Every conception is one among 70 trillion unique possibilities

11. Crossing over between homologous chromosomes during meiosis adds even more variation Meiosis animation showing random assortment + crossing over

12. 3. Mutation: a change in the sequence of DNA “The Dwarf Francisco Lezcano” Normal DNA sequence 5’…A G C T A C G G G G T G G G C…3’ 5’…A G C T A C A G G G T G G G C…3’ Achondroplasia painted by Diego Velazquez c. 1642

13. How do gene mutations affect the structure of proteins? Point mutations can affect protein structure and function Type of point mutations: - substitutions (missense and nonsense mutations) - insertions and deletions (frameshift mutations) Wild-type sequence

14. Categorize three different types of point mutations 1 2 3 GAA TGC TAG … AAA GAA TGG GTT … … lys glu trp val … … TCG CCA TGG CCA … … ser pro trp pro… … ATA TAC GTA CAT … … ile tyr val his … glu cys X (stop) Chemical type: Transversion Transversion Transition Missense Missense Nonsense Informational type: Functional type: Deleterious Non-synonymous Nondegenerate site Deleterious Non-synonymous Nondegenerate site Deleterious Non-synonymous Nondegenerate site

15. Categorize three more types of point mutations 4 5 6 GTC TGC A … AAA GAA TGT GTT … … lys glu cys val … … TCG CCA TGG CCA … … ser pro trp pro … … ATA TAC GTA CAT … … ile tyr val his … … TCG CAC ATG GCC A … … ser his met gly … val cys Chemical type: Transversion Transition Indel (insertion) Missense Missense Frameshift Informational type: Functional type: Neutral (silent) Synonymous (4-fold degenerate site) Neutral Synonymous (2-fold degenerate site) Deleterious Non-synonymous Is a 4-fold degenerate site ever functionally constrained?

16. What is natural selection? “It may be said that natural selection is daily and hourly scrutinising, throughout the world, every variation, even the slightest; rejecting that which is bad, preserving and adding up all that is good; silently and insensibly working, whenever and wherever opportunity offers, at the improvement of each organic being in relation to its organic and inorganic conditions of life.” -The Origin of Species Charles Darwin

17. Ironically, natural selection alone did not explain how species originate, i.e., How Natural Selection can create a new species from a pre-existing one For Natural Selection to work, a new mechanism was required Enter Gregor Mendel Darwin’s concept of Natural Selection provided a mechanism by which species could adapt, become stronger, faster…..fitter

18. The monk,…and physicist,…and mathematician 7 pea traits, or characters, studied by Mendel Gregor Mendel: 1866 - first described a set of mathematical rules by which the appearance of an organism (its PHENOTYPE) could be related to its inherited genetic makeup (GENOTYPE)

19. DominantversusRecessive traits

20. How does natural selection occur? • Variation exists within populations • These variations are heritable • More are born than can survive (“the struggle for existence”) • Some variations are “better adapted” to their environment and, therefore, survive to produce more offspring than those “less well adapted.” • The frequency of these successful variations, therefore, increases over time • As the environment changes, different variations are favored.

21. Light and dark morphs of the moth Biston betularia

23. dd = peppered (light colored) Dd and DD = melanic (dark colored) Soot on lichens due to industrialization • At the genetic level, evolution is simply a change in the gene pool of a population over time.

24. Let’s look at the following example of two genetic diseases caused by mutations in the pathway for tyrosine metabolism, Alkaptonuria (AKU) and Phenylketonuria (PKU) Review of terms: • Homozygous • Heterozygous • Recessive • Dominant • Allele • Haplotype • Pedigree • - Functional constraint • - Heterozygote advantage

25. Relationship between genes and proteins Example of a monogenic, or single-gene disorder What is wrong with the mouse on the right?

27. Alkaptonuria, aka Black Urine disease, in humans • Symptoms: • urine and ear wax turns black as ink shortly after exposure to air • ochronosis: musculoskeletal effects, including progressive • degenerative arthritis of the large joints, esp. hip and shoulder - signs of ochronosis: -black deposits in the sclerae (white of eye) -blue colored auricles (ear lobes)

28. Inheritance pattern of Alkaptonuria, i.e., genetic basis of the disease A simple pedigree Incidence: rare, ~1/150,000 births

29. Highly toxic: Phenylketonuria (PKU) Mildly toxic: Alkaptonuria Highly toxic: Type 1 tyrosinemia

30. What is the gene defect, or molecular genetic basis, • in alkaptonuria? • four point mutations occur in the human HGO gene • - Blastp comparison of HGO gene from human and fungus Are any of the sites in these proteins functionally constrained?

31. Molecular basis of Alkaptonuria, 1898-1998 Mutations: Proline230Serine and Valine300Glycine account for most cases of the disorder Haplotypes: each of these two mutant alleles constitutes a haplotype, i.e., one haploid type. Progeny inherit one haplotype from each parent.

32. But wait: are all mutations deleterious or neutral? Can a mutation rarely be advantageous? And,….can a “deleterious” mutation ever be advantageous? Let’s examine the strange example of the PKU gene, defects in which cause a deadly disease, phenylketonuria

33. Postnatal testing for a common inherited disorder ……phenylketonuria (PKU)

34. Phenylketonuria (PKU) PKU is an inherited error of metabolism caused by a deficiency in the enzyme phenylalanine hydroxylase (PAH). Symptoms: Loss of the PAH enzyme results in mental retardation, organ damage, unusual posture and can, in cases of maternal PKU, severely compromise pregnancy. Inheritance: autosomal recessive disorder, caused by mutations in both alleles of the gene for phenylalanine hydroxylase (PAH), found on chromosome 12.

35. Biochemical basis of PKU: Mutations in both copies of the gene for PAH means that the enzyme is inactive or is less efficient, and the concentration of phenylalanine and phenylpyruvate in the body can build up to toxic levels.

37. What is the incidence of Alkaptonuria and Phenylketonuria? AKU: relatively mild, non-fatal & rare, ~1/100,000 to 1/200,000 PKU: severe and rapidly fatal* and common in some groups Caucasians: 1/10,000 Turks: 1/2600 Irish: 1/4500 Japanese 1/143,000 How many caucasians carry the defective trait? 1. 1/10,000 live births x 1/4 chance of homozygous offspring with disease when each parent is a carrier = 1/2500 marriages are between parents who are both carriers; and… 2. Square root of 1/2500 = 1/50 caucasians is heterozygous, i.e., carries one defective allele and one normal allele.

38. Mutations in the PAH gene have occurred independently in many human populations and spread due to evolutionary forces

39. A deleterious mutation should be eliminated from the gene pool, but the deadly PKU allele has instead survived and spread widely…..WHY? pku frequency Parents +/pku x +/pku 50% 1/4 1/2 1/4 X dies 33% Progeny +/+ +/pku +/pku pku/pku

40. Why is PKU, a devastating disease, common? And why is AKU, a milder disease, rare? • the answer is probably rooted in evolution (ultimate causes): • - two fungi, Aspergillus ochraceus and Penicillium viridicatum Ochratoxin A

41. PHE TYR PHE TYR PHE TYR Gene dosage determines how much phenylalanine will be present in the body +/+ +/pkupku/pku PKU genotype: Phenylalanine levels: “The dose makes the poison”

42. Does evolution explain the high incidence of PKU in humans? Carriers of PKU (+/pku heterozygotes) have elevated phenylalanine levels - --BUT they do not develop PKU symptoms Women who are PKU carriers have amuch lower-than-average incidence of miscarriage • Ochratoxin A is a fungal poison known to cause spontaneous abortion. The fungi that produce this toxin grow on grains (wheat, barley, rye, etc.). Hypothesis: Excess phenylalanine inactivates the fungal poison Rationale: If PKU carriers were more likely to have children than non-carriers because of the protective effects of the PKU gene, then over time, the disease-causing allele would increase and spread through the population.

43. Genes are selected for their reproductive fitness, even if the consequences for some individuals is disastrous pku frequency Parents +/pku x +/pku 50% 1/4 1/2 1/4 X X dies Progeny +/+ +/pku +/pku pku/pku Susceptibility to miscarriage reduces reproductive success

44. “Natural Selection, the blind, unconscious, automatic process which Darwin discovered, and which we now know is the explanation for the existence and apparently purposeful form of all life, has no purpose… ….It is the blind watchmaker” ---Richard Dawkins, The Blind Watchmaker

45. Arms Race without End

46. The Red Queen hypothesis explains how antibiotic resistant bacteria, and new diseases, emerge "...in our country," said Alice, still panting a little, "you'd generally get to somewhere else -- if you ran very fast for a long time as we've been doing." "A slow sort of country!" said the Queen. "Now here, you see, it takes all the running you can do to keep in the same place. If you want to get somewhere else, you must run at least twice as fast as that."

47. Antibiotics Antibiotics on filter disc

48. Emergence of antibiotic-resistance coincides with the appearance of antibiotics