What are the Patterns Of Nucleotide Substitution Within Coding and Non-coding Gene Regions? Mouse vs Human T = 80,000 my rate = substitutions / site / 109 years

(1) Rates of substitution are extremely variable, more so for nonsynonymous substitutions. NSyn Rate Syn Rate # Codons

(2) In the majority of genes, the synonymous substitution rate greatly exceeds the nonsynonymous rate. NSyn Rate Syn Rate # Codons

(3) Overall rate of substitution is lowest for non- degenerate sites, highest for 4-fold degenerate sites. (4) Rate of transversional substitution is lower at 2-fold vs 4-fold degenerate sites.

(5) Rate of transitional substitution is higher than transversional substitution at 4-fold sites. (6) At nondegenerate sites, rate of transitional and transversional substitution are similar.

Schematic of Eukaryotic Protein-Coding Locus 5’ UTR 3’ UTR Intron 1 Intron 2 5’ 3’ Exon 1 Exon 2 Ex 3 GT AG GT AG Signal Sequences Initiation codon Stop codon

Percent divergence between cow and goat b- and g-globin genes and between cow and goat b-globin pseudogenes. Region K 5’ flanking 5.3+1.2 5’ untranslated 4.0+2.0 4-fold degenerate 8.6+2.5 Introns 8.1+0.7 3’ flanking region 8.8+2.2 3’ untranslated 8.0+1.5 Pseudogenes 9.1+0.9

Causes of Variation in Substitution Rates Rate of Substitution is determined by: (1) Mutation rate Among genes Among gene regions (2) Probability of fixation Neutral, advantageous, deleterious

Functional constraint: Range of alternative nucleotides that is acceptable at a site without negatively affecting the function or structure of a protein. Total Mutation Rate per Unit Time Fraction of Selectively Neutral Mutations Rate of Neutral Mutation V0 = vT fo Neutral Theory Predicts k = V0 : Rate of Substitution So, k = vT fo

k = vT fo i.e. Highest Rate of Substitution is Expected in So, rate of substitution will be greatest when fo is 1.0 i.e. Highest Rate of Substitution is Expected in Sequence That Does Not Have A Function Pseudogenes!

Expect an inverse relationship between the intensity of the functional constraint and the rate of neutral evolution

Also, expect higher rates of substitution for Given this relationship: Also, expect higher rates of substitution for synonymous vs nonsynonymous sites. Logic: (1) Mutations that result in amino acid replacements have a higher probability of causing a deleterious effect on the structure/function of the protein. (2) Accordingly, the majority of nonsynonomous mutations will be eliminated from the population by purifying selection. (3) As a result, there will be a reduction in the rate of nonsynonymous substitution vs synonymous substitution.

Causes of Variation in Substitution Rates Rate of Substitution is determined by: (1) Mutation rate Among genes Among gene regions (2) Probability of fixation Neutral, advantageous, deleterious

Functional constraint: Range of alternative nucleotides that is acceptable at a site without negatively affecting the function or structure of a protein. Total Mutation Rate per Unit Time Fraction of Selectively Neutral Mutations Rate of Neutral Mutation V0 = vT fo Neutral Theory Predicts k = V0 : Rate of Substitution So, k = vT fo

k = vT fo i.e. Highest Rate of Substitution is Expected in So, rate of substitution will be greatest when fo is 1.0 i.e. Highest Rate of Substitution is Expected in Sequence That Does Not Have A Function Pseudogenes!

Expect an inverse relationship between the intensity of the functional constraint and the rate of neutral evolution

Also, expect higher rates of substitution for Given this relationship: Also, expect higher rates of substitution for synonymous vs nonsynonymous sites. Logic: (1) Mutations that result in amino acid replacements have a higher probability of causing a deleterious effect on the structure/function of the protein. (2) Accordingly, the majority of nonsynonomous mutations will be eliminated from the population by purifying selection. (3) As a result, there will be a reduction in the rate of nonsynonymous substitution vs synonymous substitution.

Why is the rate of substitution at 4-fold sites lower than the rate within pseudogenes? Synonymous substitutions are not selectively neutral! Codon Usage is non-random: species-specific, and patterns may vary among genes within a genome.