Ejercicios de alineamiento de secuencias: CLUSTALW insertar secuencias de FASTA

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Regiones conservadas y variables en proteinas

Codones y aminoácidos

The 20 amino acids have overlapping properties Small change big change

Minor changes are more frequent Relative substitution frequency Relationship between physico-chemical difference and relative substitution frequency Minor changes are more frequent Relative substitution frequency Drastic changes are infrequent Physico-chemical difference Kimura (1983) The neutral theory of molecular evolution.

Pseudogenes as a paradigm of neutral evolution Pseudogenes show an extremely high rate of nucleotide substitution. Li, Gojobori and Nei (1981) Nature 292: 237-239

Conservation in a ‘typical’ gene Splice sites Start of translation Start of transcription Polyadenylation site On the basis of 3,165 human-mouse pairs MGSC Nature (2002) 420 520-562

Degeneracy of the Genetic Code nonsynonymous synonymous Colors represent amino acids Each of the 61 sense codons can mutate in 9 different ways 134 of the 549 possible changes are synonymous

Synonymous changes can be neutral mutations If most DNA changes were due to adaptive evolution than one would imagine that most changes would occur in the first and second codon positions. If DNA divergence includes neutral mutations, then the third position should change more rapidly because synonymous mutations are more likely to be neutral. King, J. L., and Jukes, T. H. 1969. Non-Darwinian evolution, Science 164, 788-798.

Preponderance of changes in the 3rd position The first 220 nucleotides of human and mouse renin binding protein The third position of all codons are marked Of the 31 changes: 4 - 1st position 4 - 2nd position 23 - 3rd position

Estimating separately the rate of synonymous change and non-synonymous change KS = number of Synonymous substitutions per synonymous site KA = number of non-synonymous (Altering) substitutions per non-synonymous site One way of estimating Ks and Ka would be to examine each change individually and check if it is synonymous or not. In the following we present a method for doing this in a systematic manner.

Nucleotide sites can be classified into 3 types of degenerate sites 2-fold Degenerate changes of this nucleotide relate to pairs of codons for the same AA 4-fold degenerate – changes of this nucleotide relate to 4 codons for the same AA 0-fold degenerate - no change at this nucleotide leaves coding for the same AA Synonymous - Altering (AA = amino acids)

4-fold degenerate sites are found in 32 of the 3rd position of 61 codon sites

2-fold degenerate sites are found in 25 of the 3rd positions and 8 of the 1st position

0-fold degenerate sites are found in 2nd position sites of all codons (61) and in of 53 of the 1st position sites

Classify each site in a sequence according to the degeneracy of the sites. 002 202 204 004 - - - 000

Counting the number of 4-,2-,0-fold sites Classify each site in a sequence according to the degeneracy of the sites. 000002002002204002004204002004000002002004004004002002204002004004004 000002002002204002004204002004000002002004004002002002204002004004002 Counting the number of 4-,2-,0-fold sites (taking the average between the two sequences) L0= (45+45)/2 = 45 L2= (13+15)/2 = 14 L4= (10+8)/2 = 9

S4 S2 S0 V4 V2 V0 4-fold 2-fold 0-fold transition transversion Classify the differences with another sequence as a. transition (S) or transversion (V) b. degeneracy (0,2,4) 4-fold 2-fold 0-fold S4 S2 S0 transition V4 V2 V0 transversion

S4 S2 S0 V4 V2 V0 Synonymous mutations Non-synonymous mutations 4-fold The key simplification is the special relationship between transition/transversion and degeneracy: Synonymous mutations 4-fold 2-fold 0-fold S4 S2 S0 transition V4 V2 V0 transversion Non-synonymous mutations (Exceptions: 1st position of arginine (CGA,CGG,AGA,AGG), last position of isoleucine (AUU, AUC, AUA)).

We distinguish between transitions and transversions according to the Kimura model

(~6 times more transitions than transversions) Use Kimura’s 2-parameter model to estimate the numbers of transitions (Ai) and transversions (Bi) per i-th type site. Calculate the proportions of transitional and transversional differences: Pi = Si/Li (12/70) Qi = Vi/Li (3/70) Kimura model is used to correct for multiple hits: (~6 times more transitions than transversions) The Kimura model is similar to the Jukes-Cantor model (from the previous lecture) but also takes into consideration that transitions and transversions occur at different frequencies Ai = (1/2) ln (1/(1- 2Pi – Qi)) – (1/4) ln (1/(1- 2Qi)) Bi = (1/2) ln (1/(1- 2Qi)) (0.242) (0.045)

The Molecular Clock of Viral Evolution Different rates Relationship between the number of nucleotide substitutions and the difference in the year of isolation for the H3 hemagglutinin gene of human influenza A viruses. All sequence comparisons were made with the strain isolated in 1968. Gojobori et al. 1990 PNAS 87 10015-10018