1. Ejercicios de alineamiento de secuencias: CLUSTALW insertar secuencias de FASTA

2. Pedir alineamiento múltiple

3. Analizar resultado

5. Regiones conservadas y variables en proteinas

7. Codones y aminoácidos

8. The 20 amino acids have overlapping properties
Small change
big change

9. 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.

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

11. 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)

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

13. 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 Non-Darwinian evolution, Science 164,

14. 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:
st position
nd position
23 - 3rd position

15. 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.

16. 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)

17. 4-fold degenerate sites are found in 32 of the 3rd position of 61 codon sites

18. 2-fold degenerate sites are found in 25 of the 3rd positions
and 8 of the 1st position

19. 0-fold degenerate sites are found in 2nd position sites of all codons (61) and in of 53 of the 1st position sites

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

21. Counting the number of 4-,2-,0-fold sites
Classify each site in a sequence according to the degeneracy of the sites.
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

22. 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

23. 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)).

24. We distinguish between transitions and transversions according to the Kimura model

25. (~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)

26. 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 PNAS