1. Adaptive Molecular Evolution Nonsynonymous vs Synonymous

2. Reading for today Li and Graur chapter (PDF on website) Evolutionary EST paper (PDF on website) Page and Holmes pp. 231 - 243

3. Predictions of neutral theory There is an inverse correlation between rate of substitution and degree of functional constraint. Patterns of base composition and codon usage reflect mutational rather than selective pressures. There is a constant rate of molecular evolution. The level of within species variation is the product of population size and mutation rate and is correlated with levels between species.

4. The neutral theory of molecular evolution

5. Well accepted “rule”: Evolutionarily conservation of genes and regions implies functional importance

7. What molecular changes are different between species?

8. % Amino Acid Sequence Divergence 10 20 30 40 50 60 70 1000 800 600 400 200 NUMBER OF PAIRS 948 483 238 138 57 12 2 Rodent x Human 1880 Orthologous Sequence Pairs (~4% of genes) Makalowski & Bogusti, PNAS 95, 9407 (1998) 1.Genes involved in immune response 2.Genes involved in olfaction 3.Genes involved in reproduction 4.Genes implicated in human disease?

9. Potential causes of rapid evolution Lack of constraint: Selectively neutral evolution Adaptive value for change: Positive “Darwinian” selection Compare cDNA sequences.

10. 2 types of changes in codons Synonymous = silent change (amino acid stays the same) Nonsynonymous = replacement change (changes amino acid) Val GTC Val GTG Ala GCC Synonymous Change Nonsynonymous Change

11. Rates of synonymous changes is similar to pseudogenes Synonymous changes

13. Synonymous and nonsynonymous sites are both in coding regions. Synonymous sites are considered selectively neutral. Therefore, we can use synonymous sites as a “ruler” for nonsynonymous substitutions. When nonsynonymous changes exceeds synonymous changes, infer positive selection. Prot. 1: IleCysIleLysAlaLeuValLeuThr DNA1: ATATGTATAAAGCGAGTCCTGTTAACA DNA2: ATATGTATAAAGCGAGTCCTGTTAACA Prot. 2: IleCysIleLysAlaLeuValLeuThr There are more nonsynonymous than synonymous sites in coding DNA

14. d N = # nonsynonymous substitutions/# nonsynonymous sites d S = # synonymous substitutions/# synonymous sites Test for selection by comparing d N and d S d N /d S = 1: Neutral evolution d N /d S < 1 : Purifying selection d N /d S > 1 : Positive selection The d N /d S ratio (  ) measures the selective pressure

15. Multiple methods for calculating d N /d S “Counting” methods –Nei and Gojobori –Li et al. Maximum likelihood methods (model of codon evolution) –Muse and Gaut –Neilsen and Yang

16. Codon degeneracy Non-degenerate –All mutations produce nonsynonymous change Two-fold degenerate –one of the three possible changes is synonymous Four-fold degenerate –all mutations produce synonymous change

17. When counting sites: Non-degenerate (1) –nonsynonymous Two fold degenerate (2) –1/3 synonymous and 2/3 nonsynonymous Four fold degenerate (4) –synonymous Note: Three fold degenerate treated as two-fold.

18. Example: Degeneracy 1 AspThrAlaVal Sequence 1GACACAGCGGTT How many synonymous sites in sequence 1? First, assign degeneracy to each codon position.

19. Example: Degeneracy 11 AspThrAlaVal Sequence 1GACACAGCGGTT

20. Example: Degeneracy 111 AspThrAlaVal Sequence 1GACACAGCGGTT

21. Example: Degeneracy 1112 AspThrAlaVal Sequence 1GACACAGCGGTT

22. Example: Degeneracy 11121 AspThrAlaVal Sequence 1GACACAGCGGTT

23. Example: Degeneracy 111211 AspThrAlaVal Sequence 1GACACAGCGGTT

24. Example: Degeneracy 1112114 AspThrAlaVal Sequence 1GACACAGCGGTT

25. Example: Degeneracy 11121141 AspThrAlaVal Sequence 1GACACAGCGGTT

26. Example: Degeneracy 111211411 AspThrAlaVal Sequence 1GACACAGCGGTT

27. Example: Degeneracy 1112114114 AspThrAlaVal Sequence 1GACACAGCGGTT

28. Example: Degeneracy 11121141141 AspThrAlaVal Sequence 1GACACAGCGGTT

29. Example: Degeneracy 111211411411 AspThrAlaVal Sequence 1GACACAGCGGTT

30. Example: Degeneracy 1112114114114 AspThrAlaVal Sequence 1GACACAGCGGTT

31. Example: Degeneracy 1112114114114 AspThrAlaVal Sequence 1GACACAGCGGTT How many nonsynonymous sites in sequence 1?

32. Example: Degeneracy 1112114114114 AspThrAlaVal Sequence 1GACACAGCGGTT How many nonsynonymous sites in sequence 1? 8 nondegenerate sites 1 two fold degenerate site = 8.66 nonsynonymous sites

33. Example: Degeneracy 1112114114114 AspThrAlaVal Sequence 1GACACAGCGGTT How many synonymous sites in sequence 1?

34. Example: Degeneracy 1112114114114 AspThrAlaVal Sequence 1GACACAGCGGTT How many synonymous sites in sequence 1? 3 four fould degenerate sites, 1 two fold = 3.33 synonymous sites.

35. Example: Degeneracy 1112114114114 AspThrAlaVal Sequence 1GACACAGCGGTT Sequence 2 GCCACTTCGGTT AlaThrSerVal Degeneracy 2114114114114 Sequence 2 has 8 nonsynonymous sites and 4 synonymous sites. For this comparison, we average number from both sequences. Nonsynonymous sites = (8.66 + 8)/2 = 8.33 Synonymous sites = (3.33 + 4) = 3.67

36. Example: Degeneracy 1112114114114 AspThrAlaVal Sequence 1GACACAGCGGTT Sequence 2 GCCACTTCGGTT AlaThrSerVal Degeneracy 2114114114114 There are 2 nonsynonymous changes, So dn = 2/8.33 = 0.24 There is 1 silent change, So ds = 1/3.67 = 0.27 dn/ds = 0.23/0.27 = 0.88 < 1 despite having more nonsynonymous changes.

37. Other factors can effect calculation of d N /d S Transition/transversion ratio –Transitions typically more frequent Pathway of substitution Codon bias

38. Nearly all counting methods assume all pathways are equally likely.

39. Codon Bias Unequal codon usage results in reduced number of effective codon sites. Ignoring codon bias leads to underestimate of ds.

40. Maximum likelihood methods incorporate models of codon evolution / bias. Transition

41. Problems with dn/ds for detecting selection Positive selection acting only on a few sites (binding cleft). Burst of positive selection followed by purifying selection (lineage specific events). Positive selection in promoter and non-coding regions. Positive selection for post-translational modification (glycosylation).