Transcript Lecture PPT - Carol Lee Lab

Discussions – Optional
I. Wednesday 3:30-4:20 p.m. Noland 342
II. Friday 1:20-2:10 p.m. Noland 539
Examples: Adaptation or
not?
• After high altitude training athletes have
increased number of red blood cells (RBC)
• Tibetans and Sherpas have higher RBC than
lowland (<2000 m) people (Yi et al. 2010, Science
329:75-78)
Examples: Adaptation or not?
• Weeds from a cornfield have been found to
grow taller than those from soybean fields
when both populations are reared in
common-garden conditions
• Taller weeds from the cornfields survive at a
greater rate and leave more offspring
EPIGENETICS
WHAT IS EPIGENETICS?
• Epigenetics – gene regulation changes that
does not involve a change in DNA sequence
• Epigenetic changes can be INHERITED!!
EPIGENETICS
Common mechanisms may include but not limited to:
-DNA methylation
-Histone modifications
(De)Acetylation (De)Methyaltion,
Ubiquitination, Phosphorylation
-Regulatory non-coding RNAs
DNA methylation
Transgenerational inheritance of mothering style and stress in rat
Youngson and Whitelaw
(2008)
Mechanisms of Adaptation
Need Genetic variation upon which
selection could act
This variation could occur at many
hierarchical levels: At different structural
levels
And at different steps leading to protein
expression
OUTLINE
• The origin of genetic variation
• Examples of structural and regulatory
change by mutations
• Detection of selection (adaptation)
The origin of genetic variation
Sources of Variation
• Point Mutations
nucleotide substitution
• Insertions or Deletions
Insertions or deletions of nucleotides
Gene duplications (insertions) or loss
• Chromosomal Duplications
• Whole Genome Duplications
Where does the polymorphism
(genetic variation) come from?
• Mutations: change in genetic code
• Recombination (sex):
Intragenic recombination
Gene conversion
Unequal crossing over – gene duplication
• Changes by Transposable Elements
Mutations
Any change in the genetic code,
including errors in DNA replication or errors in DNA
repair
Mutations
Mutations that matter, in an evolutionary sense, are
that get passed on to the next generation:
i.e., those that occur in the cells that produce
gametes (the “germ line”)
Point Mutations
Point Mutations:
mistakes during DNA replication, or DNA repair
RATE OF MUTATIONS
In most species, mutation rate is LOW
Mutation rate
Base pairs
per base per replication
pair per
per haploid
replication
genome
per replication
per effective
genome
per sexual generation
per effective
genome
Organism
in haploid
genome
in effective
genome
T2, T4 phage
1.7*105
-
2.4*10-8
0.0041
E. coli
4.6*106
-
5.4*10-10
0.0025
S. cerevisiae
1.2*107
-
2.2*10-10
0.0026
C. elegans
8.0*107
1.8*107
2.3*10-10
0.0184
0.0041
0.036
D. melanogaster
1.7*108
1.6*107
3.4*10-10
0.0578
0.0054
0.140
Mouse
2.7*109
8.0*107
1.8*10-10
0.4860
0.0144
0.900
Human
3.2*109
8.0*107
5.0*10-11
0.1600
0.0040
1.600
Mutations: Double-Edged Sword
 Most mutations are ‘neutral’ with no effect on fitness
 Most mutations that arise within functional genes are harmful
 Mildly deleterious mutations persist longer in a population
because it takes longer to select them out
 Recessive mutations remain longer because they are eliminated
when homozygous, not when heterozygous
 Selection for favorable mutations leads to adaptation.
Where does the polymorphism
(genetic variation) come from?
Unequal crossing over – gene duplication
Gene Duplications
Gene Duplications
Duplication of genes due to DNA replication
error or recombination error (unequal
crossing over)
Lynch and Connery 2000
• 0.01 duplications per gene per million years
• Half life for a gene is 3-8 million years
Crossing over
Unequal crossing over
Gene Duplications
• Duplicate genes in Eukaryotes are
continuously created, tested, and discarded
• Duplicated genes either degenerate into
pseudogenes (no function), become new
genes, or subfunctionalize with an existing gene
mutations
Pseudogene
New gene
Each gene taking on
subfunctions of the original gene
Examples: Gene Families resulting from
gene duplications
•
•
•
•
•
•
•
•
•
Olfactory receptors
Steroid hormone receptors
Heat shock proteins
Ion uptake enzymes
Hemoglobins
Opsins
Melanins
Detoxification enzymes (cytochrome P450s)
Hox genes
Hierarchical processes that are affected by Mutations
STRUCTURAL
• Primary: Amino Acid composition (Amino
Acid substitutions)
• Secondary, Tertiary, Quaternary structure
REGULATORY
• Protein expression (transcription, RNA
processing, translation, etc)
• Protein activity (allosteric control,
conformational changes)
Diagram of eukaryotic gene
Each gene is composed of
regulatory and coding region
Eukaryotic
gene (DNA)
Futuyma D.J. (2009)
Hierarchical processes that are affected by Mutations
REGULATORY
Protein expression
• Transcription: Mutations at promoters, enhancers, (CIS)
transcription factors (TRANS), etc
•
RNA Processing: Mutations at splice sites, sites of
polyadenylation, sites controlling RNA export
•
Translation: Mutations in ribosomes, regulatory regions,
etc
Protein activity (allosteric control, conformational
changes)
Once these mutations have occurred
creating genetic variation, selection could
act on genes, gene expression, and on
genetic architecture (allelic and gene
interactions)
OUTLINE
• The origin of genetic variation
• Examples of structural and regulatory
changes by mutations
• Detection of selection (adaptation)
Example: temperature adaptation in
Fundulus heteroclitus
LDH
LDH is a glycolytic enzyme which catalyzes the reaction
between Pyruvate and Lactate
Protein function
STRUCTURE
• Amino acid composition (AA substitutions)
• Secondary, Tertiary, Quaternary structure
REGULATORY
• Protein expression (transcription, translation, etc)
• Protein activity (allosteric control, conformational
changes, receptors)
Fundulus heteroclitus
Populations in Maine and Georgia have different
proportions of alleles (isozymes) at LDH-B
Difference in alleles (isozymes) in North vs South
North: LDH-B b allele (cold-adapted)
South: LDH-B a allele (warm-adapted)
The two alleles have a difference of 2 amino
acids
Place and Powers, PNAS 1979
1° latitude change
= 1°C change in
mean water
temperature
Place and Powers, PNAS 1979
Catalytic efficiency (kcat/km) is higher for the b allele at low
temperature, and higher for the a allele at higher temperature
a allele homozygote
b allele homozygote
Place and Powers, 1979
Catalytic efficiency (kcat/km) is higher for the b allele at low
temperature, and higher for the a allele at higher temperature
• The two allele products
(the enzymes) show genetic
differences in catalytic
efficiency (adaptive
differences)
• They also show genotype
by environment interaction:
they differ in the their
optimal environments
(differences in plasticity)
Place and Powers, 1979
Protein function
STRUCTURAL
• Amino acid composition (AA substitutions)
• Secondary, Tertiary, Quaternary structure
REGULATORY
• Protein expression (transcription, translation, etc)
• Protein activity (allosteric control, conformational
changes, receptors)
activity
protein
mRNA
Crawford and Powers, 1989
Common Garden Experiment:
The Northern isozyme has
BOTH higher activity and
higher level of expression in
fish at constant lab conditions
(20°C temperature)
Higher Gene Expression of
LDH-B in the Northern
Maine population
Maine Florida Georgia New Jersey
Schulte et al. 2000
Transcriptional control
• What controls differences in gene expression of LDH in F. heteroclitus?
• Mutations within Promoter or Enhancer?
Quick Time™ and a
Photo - JPEG decompressor
are needed to s ee this pic ture.
Doug Crawford: Promoter
Patricia Schulte: Enhancer
Gene expression
• Transcription
Cis-regulation (at or near the gene)
Examples:
– RNA polymerase and promoter
– Enhancers
Trans-regulation (somewhere else in the genome)
Examples:
– Gene regulatory proteins (transcription factors)
TEMPERATURE ADAPTATION
in F. heteroclitus
•Cis-acting sequence ~ 500 bp upstream of the start site of
transcription of LDH-B
•S-population - a 7-bp site identical to a mouse mammary tumor
virus glucocorticoid responsive element (MTV-GRE) repressor
•N-population - this site differs from S population sequence by 1
bp and does not repress expression of LDH gene
•MTV-GRE repressor inhibits transcription in the absence of
stress hormones.
•When stress hormone levels are high, the repression is
removed and transcription increases
•The putative element within the F. heteroclitus LDH-B gene
might behave in a similar way.
Schulte et al. 2000
GRE present
control (GRE absent)
Transgenic Fish
GRE
present
Regulatory sequence (an
enhancer) was injected into
Northern and Southern Fish
control
(GRE absent) An enhancer, located
within a 500 base pair
sequence, significantly
increased gene expression
of LDH
Protein function
STRUCTURE
• Amino acid composition (AA substitutions)
• Secondary, Tertiary, Quaternary structure
REGULATORY
• Protein expression (transcription, translation, etc)
• Protein activity (allosteric control, conformational
changes)
Gillichthys seta
High rocky intertidal
Gulf of California
5° - 41°C
Gillichthys mirabilis
sloughs and estuaries
Gulf of California
to Tomales Bay (38.16°N)
9–30 °C
Fields and Somero, 1997, Fields et al. 2002
• A4-LDHs from Gillichthys seta and G. mirabilis have identical amino
acid sequences (no structural differences)
• But show potentially adaptive differences in substrate affinity for
Pyruvate (Km) and thermal stability
Pyruvate Km
(mmol/l)
G. seta more tolerant of a
broad temperature range;
LDH less sensitive to
temperature
Temperature °C
OUTLINE
• The origin of genetic variation
• Examples of structural and regulatory
change by mutations
• Detection of selection (adaptation)
Detection of Selection
How does one detect genetic
signatures of Natural Selection?
Neutral Theory
Kimura (1968, 1983)
Motoo Kimura (1924-1994)
Ph.D. University of Wisconsin in 1956
Under James Crow
Kimura argued that the great majority of evolutionary
changes at the molecular level are not caused by selection
but by random genetic drift.
Neutral Theory: Evidence
Molecular evolution takes
place at a relatively
constant rate, simply
through random genetic
drift, enough to provide a
“molecular clock” of
evolution.
Selection-Neutral Debate
• Kimura’s work spawned a heated debate on
the relative importance of neutral evolution
(genetic drift) versus genetic variation that is a
result of natural selection.
• Probability of fixation of neutral mutation:
1

2Ne
Neutral Theory
• Now considered the “null model” against
which evidence for selection should be
tested
Detecting Natural Selection
There are many statistical tests for detecting Natural
selection (Selective Sweeps)
The approach is to test for deviations from a null neutral
model (where genetic variation arises only from genetic
drift)
Null hypothesis: Neutral, no selection
Deviation from Neutral: selection
Inferences regarding selection provide a powerful tool
for the prediction of possible disease-related genomic
regions
Methods for Detecting Selection:
A. MacDonald-Kreitman Type Tests
B. Site Frequency Spectrum Approaches
C. Linkage Disequilibrium (LD) and Haplotype
Structure
D. Population Differentiation: Lewontin-Krakauer
Methods
These tests could be applied to single genes,
or across the whole genome.
Codon Bias in Amino Acid Substitutions
• Synonymous
substitutions:
Mutations that do not cause
amino acid change (usually
3rd position)
“silent substitutions”
• Nonsynonymous
substitutions:
Mutations that cause amino
acid change (1st, 2nd position)
“replacement substitutions”
QuickTime™ and a
Photo - JPEG decompressor
are needed to see this picture.
A. MacDonald-Kreitman Type Tests
(1) Ka/Ks Test
Nonsynonymous substitutions
Synonymous substitutions
Ka
Ks
>1
•
Need coding sequence (sequence that codes proteins)
•
Ks is used here as the “control”, proxy for neutral evolution so
Ka/Ks = 1
neutral evolution
•
A larger nonsynonymous substitution rate (Ka) than synonymous
(Ks) is used as an indication of selection (Ka/Ks >1)
•
Ka/Ks < 1 ?
(2) MacDonald-Kreitman Test
Need coding sequence
Need two species to determine divergence (D)
Under neutral scenario we would expect:
Dn (nonsynonymous substitutions) = Pn (nonsynonymous polymorphism)
Ds (synonymous substitutions)
Ps (synonymous polymorphism)
•Dn/Ds > Pn/Ps indicates adaptive substitutions
MK test at the ADH locus in 3 Drosophila species
McDonald and Kreitman, 1991. Nature, 351:652-654
Fn
Fs
68 sites of ADH
locus in total
compared
Fixed difference
Polymorphic
Nonsynonymous
7
2
Synonymous
17
42
Fn
Fs
7
17
 0.41 Pn
Ps
2
42
Fn
 0.05
Fs

Pn
Ps
p<0.01
B. Site Frequency Spectrum
• Selection affects the distribution of alleles within
populations, typically reducing allele frequency
• Method examines site frequency spectrum and
compares to neutral expectations
• Could be applied to a single locus. Now used often for
genomic scans for selective sweeps
• Lactose gene in humans, disease alleles
• Domestication alleles (corn, rice)
The frequency spectrum: an example
count of number of mutations
3529
4424
4961
5286
7019
G
G
G
G
A
C
C
T
C
C
T
T
T
T
C
T
C
C
C
C
A
G
A
A
G
A
A
C
A
A
A
A
G
G
A
Frequency class:
1
2
1
1
1
4
2
1
3
2
3
4
singleton
doubleton
1
The frequency spectrum
5
4
Count
2188
G
T
T
G
G
Sequence
tripleton
1972
5
A
A
G
A
A
Site
singleton
975
Derived
163
Ancestral
3
2
1
1
2
3
Frequency class
4
Site Frequency Spectrum
count of number of mutations
Excess of rare
alleles
0.6
0.5
Frequency
0.4
selective sweep
positive selection (2Ns=5)
negative selection (2NS=-5)
neutral(no selection, constant
population size, no subdivision)
0.3
0.2
Tests:
Tajima’s D
Fu’s Fs
Excess of
common
alleles
0.1
Fay and Wu’s H
0
1
3
5
7
9
11
13
15
17
Number of copies of derived allele
19
C. Linkage Disequilibrium (LD)
• The nonrandom association of alleles from
different loci, where they are found more or less
frequently together than expected
• Selection increases levels of linkage
disequilibrium during the process of selection
D. Population Differentiation: LewontinKrakauer Methods
• Selection would often increase the degree of
genetic distance between populations
• Compute pairwise genetic distances (FST) for many
loci between populations
• When a locus shows extraordinary levels of genetic
distance relative to other loci, this locus is a candidate
for positive selection
Estimates of adaptive evolution
% substitutions fixed by selection:
• ~50% in Drosophila
• ~56% E. coli, Salmonella
• ~75% env gene in HIV in a patient
• ~85% hemagluttin gene in human influenza virus
• But only 0.08-6% in Humans
• Species with large effective population size (smaller
organisms) evolve faster
-More mutations arise in the population because there are more
individuals generating more mutations… more opportunity on which
selection could act
-faster generation time
Examples
• Human Lactase gene: frequency spectrum, LD
• Corn from Teosinte: frequency spectrum
Evolution of the gene encoding lactase (LCT) in
humans (Tishkoff et al. 2007)
• Mutations in LCT is associated with the ability to digest
milk in adults
• This ability is prevalent in North Africa and Europe, and is
largely absent throughout the world
• The mutant C/T-13910 confers lactase persistence in Europeans. A
study of 470 Tanzanians, Kenyans, and Sudanese found three mutants
associated with lactase persistence (G/C-14010, T/G-13915, C/G-13907)
The mutant C/T-13910 confers lactase persistence in Europeans. A study
of 470 Tanzanians, Kenyans, and Sudanese found three mutants
associated with lactase persistence (G/C-14010, T/G-13915, C/G13907)
Evidence for Selection
• Evidence of a selective sweep; high
frequency of C-14010 allele
• Extensive LD on chromosomes with the C14010 allele, with haplotype homozygosity
extending > 2 kilobases
Signatures of Selection in Corn
(Maize)
Evolution of Corn from
Teosinte
Domesticated about 7000 yrs
ago in Southern Mexico
Selection for changes in a
few developmental genes
John Doebley
Yum Kaax: Mayan god of corn
http://www.wisc.edu/teosinte/index.htm
Morphological differences between teosinte and maize
• Maize with tb1
knocked out
• Has branching
patterns like
teosinte
maize
teosinte
Corn
F1 Hybrid
Teosinte
Genes selected for in Corn
Major morphological differences are
due to directional selection
on 5 genes
Genes:
• Teosinte branched1 (tb1): single mutation
affects branching and inflorescence
• Regulator of tb1
• tga glume (outer coating) reduction
on chromosome X
• teosinte – ~8-12 kernels
F1 hybrid 8 rows, corn 20+rows
Evidence for selection in 2-4%
of genes, ~1200 genes
Evidence –Teosinte and corn
• tb1 has greater allelic
variation in teosinte than in
corn
• Reduction in genetic
diversity in domesticated
corn