1 Patterns of Substitution and Replacement

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6 Pattern of Substitution * in Pseudogenes * Based on a sample of 105 mammalian retropseudogenes.

7 The sum of the relative frequencies of transitions is ~68% If all mutations occur with equal frequencies the expectation is 33%

8 In the absence of selection, DNA will tend to become AT-rich 59.2% of all substitutions are from G and C 56.4% of all substitutions are to A and T In comparison to the 50% expectation, 59.2% of all substitutions are from G and C, and 56.4% of all substitutions are to A and T.

9 ( CG dinucleotides excluded)

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13 Pattern of Substitution * in mtDNA * Based on 95 sequences from human and chimpanzee.

14 * Based on 95 sequences from human and chimpanzee. The sum of the relative frequencies of transitions is ~ 94 % If all mutations occur with equal frequencies the expectation is 33%

15 Mutations: Strand (Leading and Lagging) Effects

16 Possible inequalities between strands A change from G to A actually means that a G:C pair is replaced by an A:T pair. This can occur as a result of either a G mutating to A in the one strand or a C to T mutation in the complementary strand. Similarly, a change from C to T can occur as a result of either a C mutating to T in one strand or a G mutating to A in the other.

17 Detection of Strand Inequalities in Mutation Rates If G  A on leading strand, then C  T on lagging strand If G  A on lagging strand, then C  T on leading strand If G  A on leading = G  A on lagging, then G  A = C  T

19 If there are no differences in the mutation pattern between the two strands, then

20 pyrimidines (C, T) purines (G, A) The transitional rate between pyrimidines (C, T) is much higher than that between purines (G, A), suggesting different patterns and rates of mutation between the two strands. Is G  A = C  T?

21 Pattern of amino-acid replacement

22 Physicochemical distances = measures for quantifying the dissimilarity between two amino acids.

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25 Grantham’s physicochemical distances between pairs of amino acids

26 The most similar amino acid pairs are leucine and isoleucine (Grantham's distance = 5) and leucine and methionine (Grantham's distance = 15).

The most dissimilar amino acid pairs

28 A replacement of an amino acid by a similar one (e.g., leucine to isoleucine) is called a conservative replacement. A replacement of an amino acid by a dissimilar one (e.g., glycine to tryptophan) is called a radical replacement.

29 Empirical findings: amino acids are mostly replacedsimilar During evolution, amino acids are mostly replaced by similar ones.

30 Similar amino acids Dissimilar amino acids A little A lot

31 SimilarDissimilar

32 Kimura 1985

33 Exchanges between similar structures occur frequently. Exchanges between dissimilar structures occur rarely. Nothing happens, but if it does, it doesn’t matter.

34 Amino-acid exchangeability Numbers in parentheses denote codon family for amino acids encoded by two codon families 60-90% of the amino- acid replacements involve the nearest or second nearest neighbors in the ring Argyle’s exchangeability ring

35 What protein properties are conserved in evolution? Protein specific constraints: Protein specific constraints: The evolution of each protein-coding gene is constrained by the specific functional requirements of the protein it produces. General constraints: General constraints: Are there general properties that are constrained during evolution in all proteins?

36 degree of conservation lowhigh bulkiness (volume)

37 degree of conservation lowhigh hydrophobicity

38 degree of conservation lowhigh polarity

39 degree of conservation lowhigh optical rotation

40 degree of conservation lowhigh charge optical rotation surprise!

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47 Amino-acid composition may be an important factor in determining rates of nucleotide substitution.

48 Most conserved amino acids: Glycine Glycine is irreplaceable because of its small size. Lysine Lysine is irreplaceable because of its involvement in amidine bonds that crosslink polypeptide chains Cysteine Cysteine is irreplaceable because of its involvement in cystine bonds that crosslink polypeptide chains Proline Proline is irreplaceable because of its contribution to the contortion of proteins.

49 Does the frequency of amino acids in proteins reflect “functional need” or “availability”?

50 The frequencies of nucleotides in vertebrate mRNA are 22.0% uracil, 30.3% adenine, 21.7% cytosine, and 26.1% guanine.

51 The expected frequency of a particular codon can be calculated by multiplying the frequencies of each of the nucleotides comprising the codon.

52 The expected frequency of the amino acid can be calculated by adding the frequencies of each codon that codes for that amino acid.

53 For example, the codons for tyrosine are UAU and UAC, so the random expectation for its frequency is: 1.057[(0.220)(0.303)(0.220) + (0.220)(0.303)(0.217)] = Since 3 of the 64 codons are stop codons, this frequency for each amino acid is multiplied by a correction factor of

54 By plotting the expected frequency against the observed frequency, we can see if some amino acids are occurring more or less often than expected by chance. If the observed and expected frequencies are close to equal, we would expect a regression line with a slope = 1.

55 Excluding arginine, the correlation between observed and expected frequencies was highly significant (r = 0.9). Arginine frequency seems to be affected by selection acting on one or more of its codons.

56 Conclusions (?) Amino acid frequencies are not determined by functional requirements. Amino acid frequencies are determined by nucleotide composition and the number of codons for for each amino acid.