1. 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

3. 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
mo = vT fo
Neutral Theory Predicts k = mo :
Rate of substitution
So,
k = vT fo

4. 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!

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

7. Also expect pattern of substitution to vary within genes
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
Schematic of Eukaryotic Protein-Coding Locus

9. 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.

10. How do we explain variation in rates of nonsynonymous
substitution among genes?

11. What are the Patterns Of Nucleotide Substitution Within Coding and
Non-coding Gene Regions?
Mouse vs Human
Time of divergence ~ 80,000 mya
rate = substitutions / site / 109 years

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

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

14. (3) Overall rate of substitution is lowest for non-
degenerate sites, highest for 4-fold degenerate sites.
Table 7.2

15. # nonsyn. substitutions
Positive Selection
KA/NA > KS/NS
# nonsyn. substitutions
nonsyn. site
# syn. substitutions
syn. site
>
Examples
Immunity (immunoglobulins, major histocompatibility)
Self / non-self recognition
Sex-related genes

16. Map of the Human Leukocyte (HLA)
Loci on Chrom. 6
Class I
Class II
Gene
DPB1
DPA1
DQB1
DQA1
DRB1
DRB3
DRA B C A 21 4 17 12 47 4 1 50 14 32
Number of alleles
HLA loci encode glycoproteins that present pieces of
bacteria and viral protein on the surface of cells for
possible recognition by the immune system

17. (1) Expressed on surface of all nucleated somatic cells
Structure of
MHC Class I Molecule
(1) Expressed on surface of all
nucleated somatic cells
(2) Presents peptides
to cytotoxic T-cells

19. Why is the rate of substitution at 4-fold degenerate sites lower
than the rate within pseudogenes?
Sub/Site/106
Synonymous substitutions
are not selectively neutral!
4 fold deg
Codon Usage is non-random:
species-specific, and
patterns may vary among
genes within a genome.
pseudo

20. No matter what nucleotide is substituted, the codon specifies
Four fold degenerate site
CUG
CUA
CUC
CUU
No matter what nucleotide is
substituted, the codon specifies
the same amino acid
Leucine

21. Frequency of
tRNA species
Frequency of codon
usage for highly
expressed genes
Frequency of codon
usage for lowly
expressed genes

22. Highly Expressed Genes
Gene Expression and Codon Bias in Unicellular Organisms
Highly Expressed Genes
Strong selection for translational efficiency
Restricted tRNAs used
Strong Codon Bias
Low rate of synonymous
substitution (fewer
neutral mutations)
Lowly Expressed Genes
Weak selection for translational efficiency
More tRNAs used
Weak Codon Bias
High rate of synonymous
substitution (more
neutral mutations)

23. Subs / Syn Site Prediction: If there is purifying selection against
synonymous substitutions, there should be
a lower rate of evolution in genes with high codon
bias.
Subs / Syn Site
Codon Bias
Low
High

24. What about Multicellular Organisms?
High Codon Bias
Low Codon Bias