Gene duplication & Exon shuffling

Question 1

How can a genome acquire a new gene?

Answer 1

• Horizontal gene transfer
• Exon shuffling
• Duplication and divergence
  o 1% chance for 1 gene to duplicate in 1 million years

Question 2

Function of genes

Answer 2

• Promiscuous = Side reaction has no biological function
• Bifunctional = both activities have a biological function
• Over evolution, 2 functions diverge → enzymes pick up different mutations → specialise → become better at catalysing one reaction or what was originally a side reaction

Question 3

How is DNA duplicated by recombination?

Answer 3

1. Unequal crossing over (meiosis)
   o Only requires certain lengths of similar sequences
   o Can get recombination between sets of repeats that are inappropriately lined up
   o One chromosome has duplication; other has deletion → have different daughter gametes → if have selective advantage will survive through evolution
2. Unequal sister chromatid exchange (mitosis)
   o Involves exchange between two chromatids
   o Paired up on repeat sequence → one chromatid duplication, one deletion
   o Depending on species will not be passed on to progeny
3. DNA amplification during replication
   o In haploid organisms (e.g. bacteria)
   o Unequal recombination during replication → ‘replication bubble’: DNA splits up in replication forks → homologous DNA but inappropriate lining up so one strand has duplication of region, other gets deletion
4. Replication Slippage
   o For short DNA sequences e.g. microsatellites, CAG triplet, poly-Q Huntington’s disease
   o Not common for genes
   o DNA loops out one repeat and starts to re-pair-up downstream → added DNA repeat as part of replication cycle
   o Other end has looped out → priming in wrong place → deleting the sequence
   o Can get insertions or deletions
   o Partial duplication of genetic material that codes for protein
5. Retrotransposition
   o Retrotransposons can reverse transcribe RNA copies back into DNA and spread across genomes over evolutionary time

Question 4

Successful gene duplication

Answer 4

• Successful = gene survives
• Successful outcome #1 → gene originally w/one copy duplicated → hypothesis: 2 copies should double synthesis rate if everything else is equal
  o If beneficial → retain that
  o If second copy does not provide dosing advantage → can pick up random mutations → will eventually inactive random mutation → over evolutionary time accumulate mutations → get pseudogenes (no longer fully functional gene)
• Successful outcome #2 → getting new function
  o “neofunctionalization” or sub-function of parental copies
• If selection pressure just for dosage → genes stay similar
• If no selection pressure for second copy → one copy either degrades entirely (pseudogene) or gets a new function if it provides advantage

Question 5

Gene neofunctionalization example

Answer 5

• Trypsin vs chymotrypsin
  o Evolved to be different proteases
  o Trypsin → cuts at Arg & Lys
  o Chymotrypsin → cuts at Phe, Trp & Tyr
  o Not structurally identical but similarities in proportion of strand/helices and nature of active site

Question 6

Pseudogenes

Answer 6

• Copies of functional genes → altered/missing regions
• Often have stop codons/frameshifts/missense mutations → kill reading frame of protein
• May have regulatory role → often producing RNA
• Increase genome size (cost/benefit)

Question 7

Types of pseudogenes

Answer 7

• “non-processed” pseudogenes:
  o Tandem duplication of genomic region
  o Inactivating mutations/incomplete duplications
  o Part of genome missing regulatory regions → no promoter, enhancers in correct place but does have original intron/exon structure
• “processed” pseudogenes:
  o Undergoes reverse transcriptase activity (LINE, retrovirus) → mRNA to cDNA → genome integration to make second duplicated gene copy
  o Lacks regulatory regions e.g. introns
  o Can have different combinations of exons
  o Loses most of promoter region except 5’ untranslated region at front of gene
  o Could contain poly(A) tail
  o Can integrate into same or different chromosome

Question 8

Examples of pseudogenes

Answer 8

• Ribosomal proteins
  o Highly duplicated across different species and highly conserved (essential for protein synthesis machinery)
  o Associated w/ L1 retrotransposon
  o May have functional role as have high expression rate
• Humans have 20,000 pseudogenes → most are ribosomal
  o 2/3 of these also in chimpanzee genome
  o Less than 12 shared w/mouse genome
  o Not clear what these genes are doing

Question 9

Multigene families

Answer 9

• If duplication is beneficial, multigene family can be formed.
• E.g. rRNA (v. important so highly conserved)
• Tandem gene families = clustered on same chromosome
• Dispersed gene families = on different chromosome

Question 10

Globin superfamily

Answer 10

Example of duplication & divergence
Carry out different functions in different tissues
Mixture of co-localised gene sin clusters and dispersal of these across the whole genome on different chromosomes → tandem & dispersed
Can trace evolution over different organisms → compare genes within/between species
Globins are v. common → present in all 3 domains of life
Haem-containing protein domain → v. diverse
Used for oxygen transport, storage, sensing & detoxification
Haemoglobin: tetramer (2α, 2ß)
Myoglobin: monomer
Different structures because changes the property of which they can load/take off oxygen
Others include: neuroglobin, androglobin, cytoglobin, globin E, globin X, globin Y

Question 11

Haemoglobin

Answer 11

• Cooperativity in binding:
  o Difficulty when oxygen initially tries to bind haem at low concentration
  o Each subsequent oxygen binding cooperatively helps the next one within tetramer → get non-linearity in binding curve → sigmoidal curve as haem requires high levels of oxygen to bind oxygen

Question 12

Myoglobin

Answer 12

• Found in muscles
• Has simpler binding curve → no cooperativity
• Higher affinity for oxygen
• Having different proteins for oxygen storage and transport w/different binding affinities is useful

Question 13

Genome duplication

Answer 13

• Larger duplication than genes/segments is possible → can affect genome structure
• Whole chromosome duplication → trisomy 21 → ‘down syndrome’
  o Gene product imbalance
  o Reduced life expectancy
• Genome sequencing suggested major metazoan lineages have undergone whole genome duplications (WGD)

Question 14

Polyploidy

Answer 14

• Multiple complete sets of chromosomes
• Useful in agriculture to make bigger cells → bigger fruit
• ~80% of flowering plants: oats, cotton, potatoes, bananas, coffee, etc
• Common in invertebrates, fish & amphibians; rare in mammals

Question 15

Autopolyploid

Answer 15

• Multiplication of identical species within single species
• Meiosis error within single species
• Fertilization of unreduced gametes
• Accidental production of diploid gametes not v. rare (1-40%)
• Can induce disease symptoms:
  o ‘Genomic shock’ → widespread activation of transposons, gene expression, recombination (short-term effect)
  o These can then stabilise over time → produce fertile gametes and pass down duplications
• Need to have even/paired up number of chromosomes to align properly during metaphase
• Autopolyploids can reproduce successfully but cannot breed with parent species → introduces speciation

Question 16

Allopolyploidy

Answer 16

• Hybridisation between 2 reproductively compatible species
• One-step model:
  o Fertilization of unreduced gametes from 2 diploid species
• Two-step model:
  o Hybridisation between haploid gametes followed by somatic doubling of chromosomes in zygote
  o In plants, pollen from 1 species germinates on stigma of 2nd → endoreduplication in zygote
• Triploids:
  o Tetraploid + diploid parents → triploid paired up zygotes
  o Triploid is viable but makes unbalanced gametes (odd #) so cannot segregate in meiosis II

Question 17

Triploid example: wheat-rye hybrid

Answer 17

• Cross good traits: high yield of wheat + disease tolerance of rye
• Wheat (n=28) + rye (n=14) = Triticale (n= 21) → not fertile
• To overcome this:
  o Treatment w/colchicine (chemical) interferes w/spindle machinery of cells → doubles chromosomes in germ cells
  o Now have 42 chromosomes → fertile Triticale

Question 18

The effects of WGD

Answer 18

• Cytogenetics = chromosome counts
  o Use dyes; do karyograms
• Detect multivalent formation = chromosomes line up and undergo homologous recombination
  o Can undergo more diversity and local gene duplication
  o Genome size comparison, etc
  o Difficult to discern ‘auto’ vs ‘allo’-ploidy
• Saccharomyces cerevisiae → brewer’s yeast
  o Compare every gene to every other gene
  o Duplicated sets of genes → can compare to ancestors, related yeast species, etc
  o Long time ago so evidence lost but estimate 10% of genes derive from WGD

Question 19

Genome duplication in multicellular organisms

Answer 19

• Genome duplication drives metazoan expansion
• Increase in organisational complexity
• Main controller of body counts = Homeobox gene (Hox genes)
  o Encode for ‘homeodomain’ → DNA binding proteins (~60 amino acids long) → transcription factors that regulate genes
• Studied a lot in fruit flies
  o E.g single homeotic mutation doubles number of wings in Drosophila (bithorax)

Question 20

Hox gene family

Answer 20

• V. well organised
  o Spatial and temporal collinearity
  o Order of genes on chromosome reflects expression order
• Expressed in different regions of developing embryo
• Blueprint same across many different species
  o Insect only 1 Hox cluster
  o Vertebrates e.g. mouse have many Hox cluster (usually 4)
  o Number of segments corresponds to number of clusters and components within them

Question 21

2R/3R hypothesis of WGD

Answer 21

• “Complexity in fish and vertebrate formation probably driven by WGD”
• Evidence: looking at Hox clusters
• B. lanceolatum → 1 cluster w/15 genes
  o Thought to be last common ancestor of all vertebrates
• Sea lamprey (fish-like parasite) → 4 clusters before increase in body plan complexity
• Hagfish (has spinal cord) → 4 clusters
• Sharks → even more clusters

Question 22

WGD benefits

Answer 22

• Raw material for evolutionary diversification
• Potential for neofunctionalization, divergence, pseudogene formation, etc for single genes → large amount of substrate for WGD
• Debate how beneficial it is in short-term → can get genomic shock from too much DNA
• Extra copies of genes provides some protection against environment and extinction
• Defence against mutation because have spare copy of every gene
  o Allows to do new things e.g. colonise new environments
• Fitness consequences:
  o Increased cell size (polyploidy)
  o Increased organ size
  o Faster growth (more metabolic components)
  o Have to evolve dosage regulated gene expression
• In allopolyploidy get heterosis (hybdrid vigour) → when unrelated sets of genes coming together give healthier, longer-lived, more robust offspring than highly-inbred species → providing larger combinations of wild-type and non-specialised genes

Question 23

Eukaryotic gene structure

Answer 23

• Evolution = Increasing complexity → gene number; protein number; functions
• Genes are split
• By Walter Gilbert in 1978
• Invented terms intron/exon
• Predicted existence of:
  o Alternative splicing = when RNA inside cell gets matured in different ways and introduce different exons so same gene can make different proteins within same cell
  o Exon shuffling = evolutionary process to increase/decrease number of introns/exons and swapping them around

Question 24

Exon shuffling theory

Answer 24

• Introns/exons often border particular subfunctions within proteins
• Eukaryotic proteins → ‘mosaic of motifs’
  o Domains 40-100aa = small motif building blocks for stabilization, binding, catalysis, etc
  o Discrete and modular → amenable for evolution
• Primordial exons correspond to domains:
  o Duplication, permutation, rearrangement when in new genome positions could generate new genes and proteins w/diverse functions
• Repetition in original gene has different outcomes:
  o Affect (increase?) stability, catalysis and modifies functions

Question 25

Illegitimate non-homologus recombination

Answer 25

Illegitimate non-homologus recombination
Can get unequal crossing between repeat sequences
For short motifs can get replication slippage
Microhomology can drive this illegitimate N-HR
E.g. αA-crystallin gene (hamster) transfected into mouse
Heat shock protein → topoisomerase I nicks DNA and ligates non-homologouse ends → end result: shuffled domain with duplicated gene
Over evolutionary time, this process could happen w/v. low probability but non-zero chance; if domain provide selective advantage, it would become fixed in population
Domain shuffling:
Structural domains from different genes joined together
Mechanisms include illegitimate N-HR → rare process but higher rate of shuffling in some organisms suggests retrotransposition

Question 26

LINES

Answer 26

• In exon shuffling:
  1. Gene w/domains I, II and III → downstream have a LINE between 2 exons
  2. When LINE is transcribed might take bit of adjacent exon w/ it
  3. After retro-transposition converted to cDNA jumps into genome disrupting gene
  4. Get chimeric transcript → potentially makes new protein different exons (adding one, replacing one, etc)
  5. Over time LINE is deleted; might undergo retro-transposition elsewhere in genome
  6. In evolution → get new gene
• LINES also induce ds breaks → can lead to domain shuffling and DNA repair

Question 27

Transposons

Answer 27

• Transposon carries exon w/it
• Mutator-like transposable elements (MULEs) → longer and can carry more things; contain flanking exons/introns
  o Found in plants (e.g. rice has 3000 MULEs in genome)
• As MULEs move around the genome they collect sequence → can form internal hybrid genes
  o Exons lack translation initiation/termination signals; not full genes, just coding DNA w/o stop codon

Question 28

Phases

Answer 28

• If shuffle exons in incompatible reading frames → end up adding extra domain in middle of protein → disrupts correct reading frame of all exons downstream
  o Perfect system needs exact multiples of 3 and needs to be in perfect frame to maintain protein shuffling compatibility during evolution
  o Need intron phases/classes to be the same
• Phase 0 = introns lie between 2 codons; perfect set of codons in exons 1 and 2; 0 phase shift
• Phase 1 = introns located after first nucleotide
• Phase 2 = introns located after 2nd nucleotide
  o Have extra 1/2 base in exon so need corresponding number of bases on other exon to restore reading frame
• Not all intron/exon classes are compatible w/each other → problem for shuffling
• Not all shuffling event is successful; further evolution required to purify mutated frame shift

Question 29

Splice frame rule

Answer 29

• ‘Following a successful shuffling event a newly acquired exon will be flanked by 2 introns of the same phase, otherwise it will produce a frameshift in the resulting coding sequence’
• Incompatible splicing → negative selection acts on gene to mutate further

Question 30

Evidence for exon shuffling

Answer 30

• Multicellular structure:
  o Extracellular matrix
  o Cell adhesion
  o Cellular receptors
• Shuffling essential for multicellular metazoans
• 6.4% of human genes show evidence for exon shuffling
• Phase 0 → most common form of exon
• Excess of symmetric exons in simpler organisms
• Over evolution, increase in 1-1 exon shuffling since the first animals
  o Could be because extra base Gly necessary for multi-domain linkage

Question 31

Evidence of 1-1 exon shuffling

Answer 31

• Phase 0 introns (most common)→ more ancient → higher frequency in earlier stages of eukaryotic evolution → explains prevalence in non-metazoan lineage
• 1-1 associated w/emergence of necessary features of multi-cellularity
  o After divergence of metazoan, shuffling began
  o Acquisition of protein domain must overcome structural limitations
  o Small & flexible domains fold independently → must be linked
  o Phase 1 introns v. frequently interrupt glycine codons → common in linker regions

Question 32

Shuffling evidence

Answer 32

1. Exon duplication: protein example α2 Type 1 collagen
   
   • Highly repetitive sequence
   • Tripeptide → Gly – X (often Pro) –Y (often hydroxyproline)
   • Gly-Pro typical in linker regions → destroys α/β structures
   • Chicken gene has 52 exons → 42 of them have Gly-X-Y repeats
   2. Tissue plasminogen activator (TPA)
      * Found in vertebrate blood → blood clotting
      * 4 exons
      * Upstream exon encodes ‘finger module’ → fibronectin on top of cascade interacts with plasminogen activator → plasminogen and EGF (epidermal growth factor)
      -Each subdomain coded by individual exons enable 1 protein to interact via partial dimerization between modules → easy way to build complicated protein-protein interaction network
      * Blood clotting cascade:
      -Lots of clotting factors
      - Exons spread through entire family → some for function (e.g. proteases); some for interaction between members (e.g. kringle domain)
      - Evolution has duplicated exons, shuffled them together for right compatibility, correct introns between them to get spliced and form different variants → get large/complicated split genes

Question 33

Protein-protein interaction

Answer 33

• Evolution of new protein has domains of other proteins; interacts with itself and other proteins and becomes hub of PPI networks
• Shuffling promotes self-interaction capacity
  o Many human PPI networks self-interact between components in network
  o Allow formation of dimeric proteins
  o Positive natural selection fixing this
  o Some domain types have v. specific type of interactions or v. promiscuous interactions → increase diversity in network → e.g. polyglutamines found in everything (sticky and unstructured)

Question 34

PPI example: Amyloid precursor protein (APP)

Answer 34

• From Alzheimer’s disease
  
  • Has different isoforms w/different exons from alternative splicing
  • APP undergoes protease processing:
  • α-secretase cuts immature APP → soluble APP
  • Or β/γ-secretase gives mixture of soluble APP and β-amyloid protein (bad because it forms v. stable b-sheet structure; intermediates are toxic to cell → drives neurodegeneration)
  • Amyloid plaque formation and different proteases due to different alternative splicing and expression of different variants of ‘Kunitz-type protease inhibitor’ (KPI) domain and different secretase forms
  • Processing determined by:
  • KPI domain presence → inhibits α-secretase (binds to trypsin domain)
  • If inherited → more likely to get one type of familial related Alzheimer’s
  • Evidence for KPI domain being gained by shuffling:
  • Flanked by introns
  • Has homology w/other related proteins in genome also embedded in exons