1. 4: Genome evolution

2. Exon Shuffling

3. 3 types of exon shuffling exon duplication = the duplication of one or more exons within a gene (internal duplication) exon insertion = exchange of domains between genes or insertions into a gene exon deletion = the removal of a segment from a gene.

4. Mosaic (or chimeric) protein = a protein encoded by a gene that contains regions also found in other genes. The existence of such proteins provides evidence of exon shuffling.

5. exon shuffling  mosaic proteins

6. 4: Genome evolution

7. Blood Clotting

8. Clotting - History 1863: Joseph Lister showed that blood is a fluid inside an ox, and undergoes clotting when put on a test glass.

9. Clotting – The end reaction thrombin fibrinogen fibrin

10. After the wound is healed … plasmin thrombin fibrinogen fibrin Clotting no fibrin (peptides)

11. After the wound is healed … plasminogen Plasmin should only be activated when the wound is healed. Plasmin is created from plasminogen by an enzyme called plasminogen activator (TPA). Tissue type plasminogen activator (TPA) fibrin no fibrin plasmin

12. Heart attacks plasminogen If TPA is given 1h after a heart attack it significantly increases the chance of surviving. ( If you run out of TPA chewing aspirin will also do ) Tissue type plasminogen activator (TPA) fibrin no fibrin plasmin

13. After the wound is healed … plasminogen Urokinase catalyzes the same reaction as TPA. urokinase fibrin no fibrin plasmin tissue type plasminogen activator (TPA)

14. After the wound is healed … plasminogen prourokinase urokinase fibrin no fibrin plasmin tissue type plasminogen activator (TPA) Prourokinase is the precursor of urokinase

15. After the wound is healed … Prourokinase and TPA are very similar. They both catalyze the same reaction (prourokinase only after it is cleaved to urokinase). But, the difference is that TPA interacts with fibrin and urokinase – does not. plasminogen prourokinase urokinase fibrin no fibrin plasmin tissue type plasminogen activator (TPA)

16. Prourokinase and TPA – the domains The difference is that TPA has another domain, the F1 domain (43 amino acids) that is missing in prourokinase. F1 = fibronectine type 1 module. F1 is responsible for the affinity of TPA to fibrin. KRProteaseEGKR ProteaseF1EGKR Prourokinase TPA

17. Prourokinase and TPA – the domains What is the origin of the F1 domain? KRProteaseEGKR ProteaseF1EGKR Prourokinase TPA

18. Prourokinase and TPA – the domains It probably came from another protein, called fibronectin. F5F3 F2 F2 = Collagen binding domain F3 = Heparin biding domain F4 = Cell binding domain 25 F1 6 Six repeats of F1 F1 3 F3 9 F4F3F1 3 S-S F5F3 F2 25 F1 6 3 F3 9 F4F3F1 3

19. Fibronectin Fibronectin can connect (F4) fibroblasts to fibrin (F1) to repair site of injury. F5F3 F2 F2 = Collagen binding domain F3 = Heparin biding domain F4 = Cell binding domain 25 F1 6 Six repeats of F1 F1 3 F3 9 F4F3F1 3 S-S F5F3 F2 25 F1 6 3 F3 9 F4F3F1 3

20. Much more complicated In fact, there are other domains in the clotting system that resemble each other. It looks like a big complicated puzzle of domain shuffling.

21. AP = apple module; EG = epidermal growth-factor; F1 & F2 = fibronectin type-1 & type-2; GA =  -carboxy-glutamate domain; KR = kringle

22. TPA acquired its exons from other genes …

23. There are also many computer programs that analyze a given sequence, and search for homology in known existing domains.

24. 4: Genome evolution

25. Phase limitations on exon shuffling

26. The phase of an intron ATGGGATTC GTTAG CCATTT Exon Intron of phase 0: lies between two codons Exon

27. The phase of an intron Exon Intron of phase 1: lies between the first and second positions of a codon Exon ATGGGAT GTTAG TCCCATTT

28. The phase of an intron Exon Intron of phase 2: lies between the second and third positions of a codon Exon ATGGGATT GTTAG CCCATTT

29. The class of an exon Exon of class 0-0. Starts at the beginning of a codon, and ends at the end of a codon. GTTAG CCATTT GTT

30. The class of an exon Exon of class 0-1. Starts at the beginning of a codon, and ends between positions 1 and 2 of a codon. GTTAG CCATTTG TT

31. The class of an exon Exon of class 2-1. Starts between positions 2 and 3 of a codon and ends between positions 1 and 2 of a codon. GTTA GCCATTTG TT Exon of class?

32. Symmetrical exons. GTTA GCCATTTGT TT Symmetrical exons are those that are multiples of 3 nucleotides? (Otherwise, they are asymmetrical).

33. Tandem duplication of symmetrical exons GTTA GCCATTTGTGCCATTTGT TT Tandem duplication of symmetrical exons will not cause a frameshift mutation. GTTA GCCATTTGT TT

34. Deletion of symmetrical exons Deletion of symmetrical exons will not cause a frameshift mutation. GC GTTA GCCATTTGT TT ATTT

35. What about exons insertion? Only symmetrical exons can be inserted without causing a frameshift mutation. GTTA GCCATTTGT TTACCG ATTTCAC

36. What about exons insertion? But not all symmetrical exons can enter. For example, a 0-0 exon will cause a frameshift if entered. GTTA GCCATTTGT TTA GGTACG CCG ATTTCAC GGTACG GTTA GCCATTTGT TTACCG ATTTCAC

37. What about exons insertion? 0-0 exons can only be inserted in phase 0 introns 1-1 exons can only be inserted in phase 1 introns 2-2 exons can only be inserted in phase 2 introns GTTA GCCATTTGT TTA GGTACG CCG ATTTCAC GGTACG GTTA GCCATTTGT TTACCG ATTTCAC

38. Prourokinase and TPA – exon classes All recruited exons are of class 1-1. It might be a “ frozen accident ” : if the first one was 1-1, all the rest should also be 1-1 … KRProteaseEGKR ProteaseF1EGKR Prourokinase TPA

39. 4: Genome evolution

40. exonization and exon lost

41. Splicing In splicing, introns are removed. There are signals in the DNA (in the mRNA) that direct the excision of introns. exon1exon2 protein exon1exon2 exon1exon2 mRNA mature mRNA exon1exon2 DNA

42. Exonization Mutations in the DNA that encode signals for intron excision might result in exonization of the intron. protein mRNA mature mRNA exon1 DNA exon1 exon2 mutation in the splicing signal

43. Exon lost Of course, in a similar vain, exons can also be removed due to such mutations. protein mRNA mature mRNA DNA exon1 exon2 mutation in the splicing signal exon1

44. 4: Genome evolution

45. Principal biochemical reactions in the synthesis of fatty acids from malonyl CoA in eukaryotes and eubacteria _____________________________________________________________________________________________________________________ Reaction Enzyme _____________________________________________________________________________________________________________________ 1. acetyl CoA + condensing-enzyme domain  acetyl-condensing enzymeacetyl transferase 2. malonyl CoA + acyl-carrier peptide  malonyl-acyl-carrier peptide malonyl transferase 3. acetyl-condensing enzyme + malonyl-acyl-carrier peptide   -ketoacyl-carrier peptide  -ketoacyl synthase  -keto-acyl carrier peptide + NADPH + H +   -hydroxyacyl-carrier peptide + NADP +  -ketoacyl reuctase 5.  -hydroxyacyl-carrier peptide  2-butenoyl-acyl-carrier peptide + H 2 O  -hydroxyacyl dehydratase 6. 2-butenoyl-acyl-carrier peptide + NADPH + H +  butyryl-acyl-carrier peptide + NADP + enoyl reductase 7. butyryl-acyl-carrier peptide + condensing-enzyme domain  butyryl-condensing enzyme + acyl-carrier peptide thioesterase _____________________________________________________________________________________________________________________ 7 enzymatic activities + 1 acyl carrier protein Multi-domain gene assembly

46. Principal biochemical reactions in the synthesis of fatty acids from malonyl CoA in eukaryotes and eubacteria _____________________________________________________________________________________________________________________ Reaction Enzyme _____________________________________________________________________________________________________________________ 1. acetyl CoA + condensing-enzyme domain  acetyl-condensing enzymeacetyl transferase 2. malonyl CoA + acyl-carrier peptide  malonyl-acyl-carrier peptide malonyl transferase 3. acetyl-condensing enzyme + malonyl-acyl-carrier peptide   -ketoacyl-carrier peptide  -ketoacyl synthase  -keto-acyl carrier peptide + NADPH + H +   -hydroxyacyl-carrier peptide + NADP +  -ketoacyl reuctase 5.  -hydroxyacyl-carrier peptide  2-butenoyl-acyl-carrier peptide + H 2 O  -hydroxyacyl dehydratase 6. 2-butenoyl-acyl-carrier peptide + NADPH + H +  butyryl-acyl-carrier peptide + NADP + enoyl reductase 7. butyryl-acyl-carrier peptide + condensing-enzyme domain  butyryl-condensing enzyme + acyl-carrier peptide thioesterase _____________________________________________________________________________________________________________________ In most bacteria, these functions are carried on by discrete monofunctional proteins. Multi-domain gene assembly

47. Principal biochemical reactions in the synthesis of fatty acids from malonyl CoA in eukaryotes and eubacteria _____________________________________________________________________________________________________________________ Reaction Enzyme _____________________________________________________________________________________________________________________ 1. acetyl CoA + condensing-enzyme domain  acetyl-condensing enzymeacetyl transferase 2. malonyl CoA + acyl-carrier peptide  malonyl-acyl-carrier peptide malonyl transferase 3. acetyl-condensing enzyme + malonyl-acyl-carrier peptide   -ketoacyl-carrier peptide  -ketoacyl synthase  -keto-acyl carrier peptide + NADPH + H +   -hydroxyacyl-carrier peptide + NADP +  -ketoacyl reuctase 5.  -hydroxyacyl-carrier peptide  2-butenoyl-acyl-carrier peptide + H 2 O  -hydroxyacyl dehydratase 6. 2-butenoyl-acyl-carrier peptide + NADPH + H +  butyryl-acyl-carrier peptide + NADP + enoyl reductase 7. butyryl-acyl-carrier peptide + condensing-enzyme domain  butyryl-condensing enzyme + acyl-carrier peptide thioesterase _____________________________________________________________________________________________________________________ In fungi, the activities are distributed between two proteins encoded by two unlinked intronless genes, FAS1 and FAS2. FAS1 encodes 3 of the 8 activities (  - ketoacyl synthase,  -ketoacyl reductase, and acyl- carrier protein). FAS2 encodes the rest of the five enzymatic activities. Multi-domain gene assembly

48. In animals, all functions are performed by one polypeptide, fatty-acid synthase. The gene product has 8 modules, including one that performs a dual function and another whose function is unrelated to fatty-acid synthesis but may determine the 3D structure of this protein.