1. 4: Genome evolution

2. Gene Duplication

3. Gene Duplication - History 1936: The first observation of a duplicated gene was in the Bar gene of Drosophila. 1950: Alpha and beta chains of hemoglobin are recognized to have been derived from gene duplication 1970: Ohno developed a theoretical framework of gene duplication 1995: Gene duplications are studied in fully sequenced genomes

4. Types of Genomic Duplications Part of an exon or the entire exon is duplicated Complete gene duplication Partial chromosome duplication Complete chromosome duplication Polyploidy: full genome duplication

5. Mechanism of Gene Duplication Genes are duplicated mainly due to unequal crossing over

6. Mechanism of Gene Duplication If these regions are complementary, it increases the chance of unequal crossing over. For example, if both of these regions are the same repeated sequence (microsatellite, transposon, etc ’… )

7. After a Gene is Duplicated Alternative fates: 1.It can die and become a pseudogene. 2.It can retain its original function, thus allowing the organism to produce double the amount of the derived protein. 3.The two copies can diverge and each one will specialize in a different function. Identical copiesOne copy diesDivergence

8. Invariant repeats If the duplicated genes are identical or nearly identical, they are called invariant repeats. Many times the effect is an increase in the quantity of the derived protein, and this is why these duplications are also called “ dose repetitions ”. Classical examples are the genes encoding rRNAs and tRNAs needed for translation. Invariant repeats

9. Variant repeats Some classic examples: Trypsin, the digestive enzyme and Thrombin (cleaves fibrinogen during blood clotting) were derived from a complete gene duplication. Lactalbumin, connected with lactose synthesis and Lysozyme, which degrades bacteria cell wall are also a result of an ancient gene duplication. Variant repeats

10. 4: Genome evolution

11. Dose Repetition

12. Gene duplication in mosquito as a response to insecticides Kingdom = Metazoa (humans are also Metazoa) Phylum = Arthropoda (humans are Chordata) Class = Insecta (humans are Mammalia) Order = Diptera (humans are Primates) Genus = Culex (humans are Homo) Species = pipiens (sapiens)

13. Organophosphorous insecticides Organophosphorous insecticides (e.g., parathion and malathion) interact with many enzymes and in particlar they inhibit the acetylcholinesterase (AChE) activity in the central nervous system, inducing lethal conditions.

14. Organophosphorous insecticides The acetylcholine is a is a neurotransmitter that, upon release from neurons, stimulates the opening of a Na+ and K+ channels. These channels regulate the function of the brain as well as the heart, lungs, and skeletal muscles. The acetylcholinesterase catalyzes the hydrolysis of acetylcholine to form inactive acetate and choline.

15. Acetylcholinesterase Acetyl-CoA + Choline Acetylcholine Postsynaptic tissue Cholinergic neuron Acetylcholinesterase

16. Acetyl-CoA + Choline Acetylcholine Postsynaptic tissue Acetylcholinesterase Cholinergic neuron Insecticide

17. Esterases Esterases are detoxifying carboxylester hydrolase which are responsible for the resistance to organophosphorous insecticides. These enzymes are none specific.

18. Detoxifying esterases Acetyl-CoA + Choline Acetylcholine Postsynaptic tissue Cholinergic neurone Insecticide Esterase

19. Esterases Culex pipiens typically has 2 genes encoding esterases: Est-3 and Est-2. These genes are separated by an intergenic DNA fragment varying between 2 – 6 kb. Est-3Est-2

20. Alignment of predicted est α2 and est β2 amino acid sequences of Culex quinquefasciatus ~47% similarity between the two sequences [Biochem.J.(1997) 325,359-365]

21. Esterases Resistance alleles correspond to an esterase over- production (which binds or metabolizes the insecticide) relative to basal esterase production of susceptibility alleles. Several resistance allele have been described.

22. Different allele show 85-90% of similarity Esterase starch gel

23. Esterases For most alleles, the over-production of esterase is the result of gene duplication. This concerns either one locus or both. Est-3Est-2 B A A B 47 % of similarity ~100 % of similarity

24. Nomenclature for the various resistance genes and their products at the Ester resistance locus Genetica 112–113: 287–296, 2001

25. Esterases The duplication of the two esterase loci, explains the tight statistical association of some electromorphs, like A2 and B2. Although, A4, A2 and A1 are coded by alleles of the Est-locus, and B2 and B4 by alleles at the Est-2 locus, A1, A4- B4 and A2-B2 are considered as alleles of a single superlocus (named Ester). Independent amplifications have occurred only a few times.

26. Esterases The level of gene duplication varies between the different alleles: Ester B1 could reach easily 100 copies in the field Ester 4 has never been found above few copies. It varies also within and among populations for a given amplified allele. Why the various amplified alleles have distinct limits of amplification is unknown.

27. Frequency of resistance allele in Montpellier (France) 111213141 km 111213141 km 111213141 km Treatment area A1

28. Esterases Resistance allele has a cost for the mosquito. In absence of insecticide in the environment non resistant-mosquitoes have the best fitness.

29. Geographic distribution of resistance allele Genetica 112–113: 287–296, 2001

30. Esterases The level of gene duplication varies between the different alleles: Ester B1 could reach easily 100 copies in the field Ester 4 has never been found above few copies. It varies also within and among populations for a given amplified allele. Why the various amplified alleles have distinct limits of amplification is unknown.

31. Gene Duplication in Aphids as a response for insecticide. Same story than the mosquitoes

32. Few Words About Aphids Kingdom=Metazoa (humans are also metazoa) Phylum=Arthropoda (humans are Chordata) Class=Insecta (humans are Mammalia) Order=Hemiptera (humans are Primates) Genus=Myzus (humans are Homo) Species=persicae (sapiens) Around 4,000 species, ~250 are pests.

33. Few Words About Aphids The Myzus persicae likes … lettuce. In fact, it is the most important aphid pest on lettuce

34. E4 & FE4 Myzus persicae has 2 genes encoding esterases E4 and FE4, which are responsible for the resistance to organophosphorous insecticides. These genes show 99% identity in nucleotide sequences, both have exactly the same exon- intron structure (same size and same positions).

35. Many copies of E4 and FE4 Resistance strains of the aphid were found to contain multiple copies of E4 and FE4. The sequences of all copies are 100% identical. It is believed that this duplication occurred within the last 50 years, with the introduction of the selective agent.

36. Take home message I: Increase in gene number can occur quite rapidly under selection pressure.

37. Take home message II: Mutations of gene duplication are not the limiting step (in evolution). It is selection that counts most.

38. 4: Genome evolution

39. Duplications of RNA-specifying genes

40. Ribosome Ribosome is a complex of proteins and RNA (called rRNA) on which proteins are built, based on the information in the mRNA. Ribosomes are always composed of two units – big and small.

41. Ribosome In prokaryotes the entire ribosome is 70S, and is composed of a 50S large subunit, and a 30S small subunit. In eukaryotes the entire ribosome is 80S, and is composed of a 60S large subunit and a 40S small subunit. Each subunit contain different rRNA. The S value is the sedimentation coefficient in ultracentrifuge.

42. rRNA There are also ribosomal genes coded by the mitochondrial genome. In fact, the mitochondrial ribosome is coded by both nuclear and mitochondrial genes.

43. Comparison of ribosome structure in Bacteria, Eukaryotes, and Mitochondria Mitochondrial (55S)Eukaryotic (80S)Bacterial (70S) 39S60S50SLarge Subunit 16S (1560 nts)28S (4700 nts)23S (2904 nts) rRNAs (1 of each) 5S (120 nts) 5.8S (160 nts) 48~4933Proteins 28S40S30SSmall Subunit 12S (950 nts)18S (1900 nts)16S (1542 nts)rRNA 29~3320Proteins 16S, 18S are the most commonly used genes in phylogenetic analysis

44. Eukaryotic rRNA genes 28S, 5.8S, and 18S rRNAs are encoded by a single transcription unit (45S) separated by 2 internally transcribed spacers (ITS) and bounded by externally transcribed spacers (ETS). 18S28S ITS 1ITS 2ETS 5.8 S

45. In Human the 45S rDNA is organized into 5 clusters (each has 30-40 repeats) These clusters are located on chromosomes 13, 14, 15, 21, and 22. These clusters are transcribed by the RNA polymerase I. Human rRNA genes 18S28S18S28S18S28S18S28S

47. 5SrRNA genes occurs in tandem arrays and there are about ~200-300 true 5S genes and many dispersed pseudogenes. In human there are two gene cluster on chromosome 1 (in dogs there is a single gene cluster). 5S rRNA is transcribed by RNA polymerase III. Human rRNA genes

48. Numbers of rRNA and tRNA genes per haploid genome in various organisms __________________________________________________________________________ Genome Source Number of Number ofApproximate rRNA sets tRNA genes a genome size (bp) __________________________________________________________________________ Human mitochondrion 1 222  10 4 Nicotiana tabacum chloroplast 2 372  10 5 Escherichia coli 7 ~ 1004  10 6 Neurospora crassa ~ 100 ~ 2,6002  10 7 Saccharomyces cerevisiae ~ 140 ~ 3605  10 7 Caenorhabditis elegans ~ 55 ~ 3008  10 7 Tetrahymena thermophila 1 ~ 800 c 2  10 8 Drosophila melanogaster 120-240 590-9002  10 8 Physarum polycephalum 80-280 ~ 1,0505  10 8 Euglena gracilis 800-1,000 ~ 740 2  10 9 Human~ 300 ~ 1,3003  10 9 Rattus norvegicus 150-170 ~ 6,5003  10 9 Xenopus laevis 500-760 6,500-7,8008  10 9 __________________________________________________________________________ Correlation between the number of rRNA genes and the genome size

49. Correlation between number of rRNA genes and genome size: an exception The general pattern: bigger genomes  more genes to transcribed  more rRNA needed. Numbers of rRNA and tRNA genes per haploid genome in various organisms __________________________________________________________________________ Genome Source Number of Approximate rRNA sets genome size (bp) __________________________________________________________________________ Human mitochondrion 1 2  10 4 Nicotiana tabacum chloroplast 2 2  10 5 Escherichia coli 7 4  10 6 Neurospora crassa ~ 100 2  10 7 Saccharomyces cerevisiae ~ 140 5  10 7 Caenorhabditis elegans ~ 55 8  10 7 Tetrahymena thermophila 1 2  10 8 Drosophila melanogaster 120-240 2  10 8 Physarum polycephalum 80-280 5  10 8 Euglena gracilis 800-1,000 2  10 9 Human~ 300 3  10 9 Rattus norvegicus 150-170 3  10 9 Xenopus laevis 500-760 8  10 9 __________________________________________________________________________

50. 4: Genome evolution

51. 51 Concerted Evolution

52. 18S rRNA tree Bos Homo Ornithorhyncus Gallus Xenopus Danio Tetraodon Branchiostoma Saccoglossus Strongylocentrotus Capitella Aplysia Lottia Tribolium Apis Trichinella Caenorhabditis Schmidtea Drosophila Anopheles Trichoplax Hydra Stylophora Nematostella Sycon Leucetta Caulophocus Walteria Chondrosia Chondrilla Negombata Amphimedon Biemna Monosiga Cryptococcus Ustilago Neurospora Schizosaccaromyces Kluyveromyces 0.1 Cnidaria Hexactinellida Bilateria Demospongiae Calcarea

53. Evolution of rRNA genes Although there are many copy of the same gene in the genome and the duplication is an ancient phenomena (since all organisms have many copies). All copies present in one genome are almost identical.

54. Divergent (classical) evolution Duplication Mutation Time Speciation

55. Divergent (classical) evolution vs. concerted evolution Divergent evolution Concerted evolution

56. Duplication Mutation Time Speciation

57. Question ? How is it possible that all the ribosomal copies remain identical ?? ????

58. (a) Stringent selection. (b) Recent multiplication. (c) Concerted evolution.

59. (a) Stringent selection. Refuted by the fact that the ITS regions are as conserved as the functional rRNA sequences.

60. (b) Recent multiplication. Refuted by the fact that the intraspecific homogeneity does not decrease with evolutionary time.

61. (c) Concerted evolution.

62. 62 CONCERTED EVOLUTION A member of a gene family does not evolve independently of the other members of the family. It exchanges sequence information with other members reciprocally or non- reciprocally. Through genetic interactions among its members, a multigene family evolves in concert as a unit.

63. CONCERTED EVOLUTION Concerted evolution results in a homogenized set of nonallelic homologous sequences.

64. 64 CONCERTED EVOLUTION REQUIRES: (1) the horizontal transfer of mutations among the family members (homogenization). (2) the spread of mutations in the population (fixation).

65. Mechanisms of concerted evolution 1. Unequal crossing-over 2. Gene conversion 3. Duplicative transposition.

66. Mechanisms of concerted evolution 1- Unequal crossing 1 2

67. 3 4

68. Gene conversion

69. Gene conversion (one possible origin) (a) Heteroduplexes formed by the resolution of Holliday structure or by other mechanisms.

70. (b) The blue DNA uses the invaded segment (e') as template to "correct" the mismatch, resulting in gene conversion. Gene conversion (one possible origin)

71. (c) Both DNA molecules use their original sequences as template to correct the mismatch. Gene conversion does not occur. Gene conversion (one possible origin)

73. Gene conversion has been found in all species and at all loci that were examined in detail. The rate of gene conversion varies with genomic location.

74. concerted evolution: Advantages of Gene Conversion over Unequal Crossing-Over 1. Unequal crossing-over changes the number of repeats, and may cause a dosage imbalance. Gene conversion does not change repeat number.

75. concerted evolution: Advantages of Gene Conversion over Unequal Crossing-Over 2. Gene conversion can act on dispersed repeats. Unequal crossing- over is severely restricted when repeats are dispersed.

76. deletion duplication

77. 77 concerted evolution: Advantages of Unequal Crossing-Over over Gene Conversion 1. Unequal crossing-over is faster and more efficient in bringing about concerted evolution. At the mutation level, UCO occurs more frequently than GC.

78. concerted evolution: Advantages of Unequal Crossing-Over over Gene Conversion 2. In a gene-conversion event, only a small region is involved.

79. 79 20,000 bp 1,500 bp In yeast, an unequal crossing-over event involves on average ~20,000 bp. A gene-conversion track cannot exceed 1,500 bp.