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EVOLUTION OF EUKARYOTIC
GENOME
GENE 342
Lecture 14 – Molecular evolution
MOLECULAR EVOLUTION I
• Ability to clone, amplify, manipulate and sequence DNA molecules from organisms
has had an enormous impact on the study of evolution
• Darwin’s focus of evolution was on the traits of organisms, which are passed on more
or less faithfully from parents to their offspring every generation, but which also undergo
modifications as the organisms adapt to changing conditions
• DNA molecules are passed from parents to offspring generation after generation.
However, this process of genetic transmission is not perfect as mutation occur and
modified DNA molecules are passed to the offspring
• Over long periods of time mutations accumulate and the DNA sequence is changed,
segments of DNA molecules may also be duplicated and re-arranged.
Molecules as “Documents of Evolutionary History”
• Fossils-mineralized remains of animals and plants long since dead, were avidly
collected in Darwin’s day. Fossils – evidence of organisms once lived on earth
• From detailed study of fossils, scientists could construct the phenotypes and behavior
of ancient organisms
MOLECULAR EVOLUTION I CONT…
Molecules as “Documents of Evolutionary History”
• Comparisons between currently living organisms and the fossilized remains of extinct
organisms stimulated the speculation about the origin of species
• DNA molecules, like fossils, contain information about life history. DNA molecules in
creatures today are derived from their ancestors-parents, grandparents and so on, to the
very first organisms
• Each DNA molecule is the end result of a long historical process involving mutation,
recombination, selection and genetic drift
• In Metaphorical terms, the sequence of nucleotides in a DNA molecule in current
version of an ancient text that has been altered (mutated), cut and pasted (recombined),
preserved fro its value (selected), and randomly disseminated (subject to genetic drift)
• Thus, DNA molecules are “documents of evolutionary history”. So, too, are protein
molecules. Polypeptides are encoded by genes, which are segments of DNA molecules
MOLECULAR EVOLUTION I CONT…
Molecules as “Documents of Evolutionary History”
• The analysis of DNA and protein sequences has several advantages over more
traditional methods of studying evolution based on comparative anatomy, physiology
and embryology:
1. DNA and Protein sequences follow simple rules of heredity. By contrast,
anatomical physiological, and embryological traits are subject to all the
vicissitudes of complex heredity
2. Molecular data sequence are easier to obtain and are amenable to
quantitative analysis framed in the context of evolutionary genetics
theory
3. The interpretation of quantitative analysis is usually much more straight
forward than the interpretation of analysis based on morphological data
4. Molecular sequence data allow researchers to investigate evolutionary
relationships among organisms that are phenotypical dissimilar, e.g. DNA and
protein sequences from bacteria, yeast, protozoa, and humans can be
compared to the study the evolutionary relationships among them
Molecular Evolution I Cont…
Molecules as “Documents of Evolutionary History”
• One problem with the molecular approach to evolution is that researchers usually
cannot obtain DNA or protein sequence data from extinct organisms
• In a few exceptional cases, such data have been obtained from fossils. However, in
none of these cases was the fossilized specimen more than a few tens of thousands of
years old
• Thus, truly ancient organisms are beyond the reach of any molecular investigation.
Molecular Evolution I Cont…
Molecular Phylogenies
• Evolutionary relationships among organisms are summarized in diagrams called
phylogenetic trees or phylogenies
• A phylogeny that only shows the relationships is an unrooted tree, whereas the one
that shows their derivation is a rooted tree
• The branches at the tips of the tree-called terminal branches lead to the organisms that
are under study
Molecular Evolution I Cont…
Molecular Phylogenies
• Each branch point in a tree represents a common ancestor of the organisms farther out
in the tree.
• Some molecular analysis of evolutionary relationships are based on a single gene or
gene product and other analysis combine data obtained by sequencing different genes
and gene products
• Descendants of an ancestral DNA or protein sequences are said to be homologous,
even if they have diverged significantly from their ancestor and are different from each
other
Molecular Evolution I Cont…
Molecular Phylogenies
• Two sequences that come to resemble each other even though they are derived from
entirely different ancestral sequences are said to be analogous
• The construction of phylogenies should always be based on the analysis of
homologous sequences.
• Methods currently available to construction of phylogenies from DNA and protein
sequence data have four features in common:
1. Aligning the sequences to allow comparisons among them
2. Ascertaining the amount of similarity between any two sequences
3. Grouping the sequences on the basis of similarity
4. Placing the sequences at the tip of a tree
Molecular Evolution I Cont…
Rates of Molecular Evolution
• By linking the branch points of a tree to a specific times in the evolutionary history of
the sequences, we can determine the rate at which the sequences have been evolving
• An example of this kind of analysis consider α-globin – one of the two peptide in the
blood protein hemoglobin. α-globin is 141 amino acid long
Number of dissimilar amino acids in the α-globin of representative vertebrates
Mouse
Human
Mouse
Chicken
Newt
Carp
16
Chicken
35
39
Newt
62
63
63
Carp
68
68
72
74
Shark
79
79
83
84
85
• The fossil records of these organisms provide information about key events in the
evolutionary history
Molecular Evolution I Cont…
Rates of Molecular Evolution
100
200
300
400
500
Million
years
ago
Tree constructed using the
evidence from fossil
records
Molecular Evolution I Cont…
Rates of Molecular Evolution
• The topology of the tree is consistent with the molecular data presented in the table
above
• The extent to which the amino acid sequences of these organisms differ can be used
to estimate the rate at which α-globin has been evolving
•To obtain this rate:
1. Need to determine the average number of amino acid changes occurred since
the split of any two lineages from a common ancestor
2. Start with the two closely related organisms (humans & mice), which are
different in 16 of the 141 amino acid sites in α-globin
 the proportion of different site in α-globin of these two species is 16/141 =
0.11 i.e., 0.11 is the average number of differences per amino acid site
3. Consider very distantly related organisms (Humans & Carps) – differ in 68 0f
141 amino acid sites = 68/141=0.48 i.e., almost half of the site have changed
during the evolution of the lineage that produced these species
 with such high frequency of changed site, we might expect that some of the
sites have changed multiple times
Molecular Evolution I Cont…
Rates of Molecular Evolution
• The observed proportion (0.48) must therefore underestimate the average number of
changes that have occurred during the long time since the human and carp lineages split
• observed proportions can be adjusted upwards to account for multiple amino acid
substitution at particular sites. The adjustment involves Poisson Correction
• Poisson corrected differences (values) estimates the average number of changes that
have occurred per amino acid site in α-globin during the time since the evolving lineages
split from a common ancestor
Number of dissimilar amino acids in the α-globin of representative vertebrates
Mouse
Chicken
Newt
Carp
Shark
Human 16/141= 0.11 35/141=0.24 62/141=0.44 68/141=0.48 79/141=0.56
0.25
0.45
Mouse
0.46
0.56
0.45
Chicken
0.51
0.58
Newt
0.52
0.59
Carp
0.60
Poisson-Corrected Average Number of Amino Acid Differences In The α-globin
of representative vertebrates and Associated Evolutionary Rates
Human
Mouse
Chicken
Newt
Carp
Mouse
Chicken
0.2
0.28
0.33
Newt
0.58
0.59
0.59
Carp
0.66
0.66
0.66
0.74
Shark
0.82
0.82
0.89
0.91
0.92
Molecular Evolution I Cont…
Rates of Molecular Evolution
• The observed proportion (0.48) must therefore underestimate the average number of
changes that have occurred during the long time since the human and carp lineages split
• observed proportions can be adjusted upwards to account for multiple amino acid
substitution at particular sites. The adjustment involves Poisson Correction
• Poisson corrected differences (values) estimates the average number of changes that
have occurred per amino acid site in α-globin during the time since the evolving lineages
split from a common ancestor
• Note that the human and Carp lineages, the average number of changes per amino
acid site is 0.66 (0.48), which is almost 1.4 times the observed proportion of amino acid
differences between the human and carp α-globins
• The rate of which α-globin has evolved can be calculated from the average number of
changes per amino acid site from each pair of organism
• This rate is the average number of changes per amino acid site divided by the total
time that the two lineages have been evolving
Calculation of the α-globin Evolving Rate
For example: the lineages that produced humans and mice from a common ancestor
80mya
100
200
300
400
500
Million
years
ago
Calculation of the α-globin Evolving Rate
For example: the lineages that produced humans and mice from a common ancestor
80mya
 the total time that these lineages have been evolving is therefore 2 x
80 million years = 160 mln yrs
 Using the Poisson corrected average number of amino acid (a.a.) changes
per site from the table above, we have:
Av. Number of a.a. changes per site
Total Evolution Time
=
0.12 a.a. changes per site
160 mln yrs
=
0.74 x 10-9 a.a. changes per year
• From this rate, it is clear that α-globin has been evolving at a little less than one amino
acid change per site every billion years
MOLECULAR EVOLUTION II
Variation In Evolutionary Rates
• Rates of amino acid sequence evolution range over three orders of magnitudes:
1. At extremes, fibrinopeptide, derived from a protein involved in blood
clotting evolves at a rate of greater than 8 a.a. substitutions per site every
billion years
2. Histones-interact intimately with DNA, evolve at a rate of only 0.01 a.a.
substitutions per site every billion years
3. Amino acids on the surface of α-globin change at a rate of about 1.3
substitutions per site every billion years where as a.a. in the interior of the
molecule change at a rate of only 0.17 substitution per site every billion years
• Observed variation in evolutionary rates is explained by the hypothesis: In more
evolving proteins, the exact a.a. sequence is not as important as it is in more slowly
evolving proteins
• The hypothesis speculate that in some proteins, amino acid changes can occur with
relatively impunity, whereas other, are rigorously selected against
Molecular Evolution II Cont..
Variation In Evolutionary Rates
• The rate of evolution depends on the degree to which are amino acid sequence of a
protein is constrained by selection to preserve that protein function
• Slowly evolving proteins are more constrained than rapidly evolving proteins
• Variation in evolutionary rates is therefore explained by the amount of functional
constraint on the a.a sequence
• e.g. the specific a.a. at or near the active site of enzymes might be expected to be
more rigorously constrained by selection than a.a. that simply less important ones
• Functionally more important proteins, or parts of proteins, evolve more slowly than
functionally less important ones
• Variation in the evolutionary rate is also seen when DNA sequences are examined
DNA sequences in pseudogenes-duplicated genes that do not encoded functional
products because they have sustained one or more lesions such as such as
frameshifting or nonsense mutations, have the highest evolutionary rate
Molecular Evolution II Cont…
Variation In Evolutionary Rates
• The nucleotides in a pseudogene are not constrained by selection because the
function of the pseudogene has already been destroyed
• However, the nucleotides in the first & second position of a codon in a functional gene
are re-constrained because changing them will almost always change the amino acid
specified by that codon
• Some of these changes will be conservative in the sense that the new amino acid will
be structural and functionally life the original amino acid
• e.g. if the first nucleotide in the codon CTT mutates to A, the amino acids specified by
this codon will change from leucine to isoleucin, which similar proteins
• However, other substitutions in this codon may cause a non-consrvative change in the
amino acid sequence. E.g. if CTT mutates, TTT, the a.a. specified the code will change
from leucine to phenyalanine, which has very duufferent chemical properties
• Nucleotides in the third position of codons within function genes evolve much faster
than nucleotides in either the he first or second position
Molecular Evolution II Cont…
Variation In Evolutionary Rates
• This is due to the genetic code being degenerate. Many amino acids are specified by
more than codon e.g. prolong by four different codons: CCT, CCC, CCA, and CCG
• As long as the two nucleotides in a codon are both C, any nucleotide can be present in
the third position and the codon will specify proline-that is, the third nucleotide position is
fourfold degenerate
• Changing the last nucleotide in a proline codon, should therefore be inconsequential
for the structure and the function of the poplypeptide encoded by a gene.
• The high level of degeneracy accounts for the faster evolutionary rate of third position
nucleotides
• A nucleotide substitution that does not change the a.a. specified by a codon is called
synonymous substitution otherwise non-synonymous substitution
Molecular Evolution II
Neutral Theory of Molecular Evolution
• Neutral Theory was developed to explain the evolution of DNA and protein sequences.
This theory focuses on three processes: mutation, purifying selection, and random
genetic drift
• Mutation is at the root of all nucleotide and amino acid substitution that occur during
evolution. Without mutation, and protein molecules could not evolve.
• Some of the mutations that occur spontaneously improve the fitness of organisms, i.e.,
they are beneficial mutations that might, over time, spread through a population and
become fixed
• Other mutations depress fitness and are eliminated from a population by the force of
purifying selection
• Many mutations, like random changes in a piece of a complex machinery, are likely to
impair function. However, some mutations may have little or no effect on fitness. Such
mutations are selectively neutral
• Synonymous nucleotide substitutions in the third position of codons and any other type
of nucleotide substitution in a pseudogene might be selectively neutral
Molecular Evolution II Cont…
Neutral Theory of Molecular Evolution
• The rate of a selectively neutral mutation depends completely on random genetic drift
• Most selectively neutral mutations are lost from a population shortly after they first
appear but a smaller fraction of them survive generations and ultimately spread
throughout the population and become fixed
• The rate of fixation of selectively neutral mutations in a population is equal to the rate
at which genes mutate to selectively neutral allelles.
• To see that the rate of fixation is equal to the neutral mutation rate:-
Calculation
- Suppose the population size is N, and that each generation a fraction µ
of the 2N copies of a gene mutate to a selectively neutral alleles
- Let us assume that each new mutant is unique;
 Given the large number of nucleotides that can mutate with a gene,
and the extremely low probability that exactly the same nucleotide
change will occur more than once
- Therefore, each generation the number of selectively neutral mutations
that appear in the population will be 2Nµ
 because each mutant is unique, its frequency will be 1/2N
- Therefore, the probability that a particular mutation will ultimately be fixed
is 1/2N
- The rate at which selectively neutral mutants of any sort are fixed is simply
the number of such mutants that appear each generation (2Nµ) x probability
that any one of them will ultimately be fixed (1/2N)
-Thus, the rate of fixation of selectively neutral mutants is
2Nµ x (1/2N) = µ
Molecular Evolution II Cont…
Neutral Theory of Molecular Evolution
• The rate of a selectively neutral mutation depends completely on random genetic drift
• Most selectively neutral mutations are lost from a population shortly after they first
appear but a smaller fraction of them survive generations and ultimately spread
throughout the population and become fixed
• The rate of fixation of selectively neutral mutations in a population is equal to the rate
at which genes mutate to selectively neutral alleles.
• To see that the rate of fixation is equal to the neutral mutation rate:See the calculation above
• Neutral Theory says that for selectively neutral mutations, the rate of molecular
evolution is equal to the rate at which these mutants occur in the population
• The rate of evolution does not depend on population size, the efficiency of selection, or
peculiarities, of the mating system.
Molecular Evolution II Cont…
Neutral Theory of Molecular Evolution
• Neutral Theory explains:1. Why substitution rates are approximately constant in many lineages over
long period of evolution time
2. Why substitution rate differ among proteins and DNA regions
3. The variation in eveolutionary rates that is observed among proteins and
DNA regions by revoking differences in functional conditions
Molecular Evolution II Cont…
Molecular Evolution and Phenotypic Evolution
• By definition, the Neutral theory has nothing to say about the evolution of traits that are
adaptive
• The giraffe’s long neck, the elephant trunk, and the camel’s hump are all adaptations
that enhance fitness. So to, is the large, highly convoluted brain of humans
• The phenotypic changes that occur during this process produce new varieties and
eventually new species
• Q? Can the evolution of adaptation and the diversification of organisms be explained
by the relentless accumulation of nucleotide and amino acid substitutions
• A Probaly in some case it can. However, change at the molecular level is not a
guarantee that phenotypic evolution will occur
-Nile Crocodiles (Crocoduylud nulotias
- great White Shark (Carcha rodon canrcharrs
-House shoe crab
Molecular Evolution II Cont…
Molecular Evolution and Phenotype Evolution
• e.g. Crocodiles, sharks, and horseshoe crabs have accumulated amino acid and
nucleotide changes at rates similar to highly diversified groups of animals such as birds,
mammals, and inserts
• Fossil records of these type organisms have a very little change in phenotype since
they first appeared hundreds of million of years ago
• Q? What sort of genetic changes might be responsible for the evolution of novel
phenotypes?
 Some possible answers are coming from the genome sequencing projects and
studies in developmental genetics
• A: Genes often become duplicated during evolution and that the duplicates sometimes
acquire different functions.
2. Portions of genes maybe be duplicated and recombined with other genes. Eukaryote
genes are segmented into exons and introns – Exones in a gene encodes a separate
functional domain in the gene’s polypeptide product
Molecular Evolution II Cont…
Molecular Evolution and Phenotype Evolution
• Exons from a gene could be combined with exons from another gene to create a
coding sequence that would specify a protein with some of the properties of each of the
original gene products
 therefore, novel proteins could be created by combining exons in modular fashion – a
process now called exon shifting
3. In addition to gene duplication and exon shifting, evolutionary diversification seems
to have benefited from spatial and temporal changes in the expression of genes,
especially those whose products regulate the expression of other genes