1. Gene duplication and its role in evolution
Núria Rafel i Millan
Genomics
UAB, December 2018

2. How a bacteria genome with few primordial genes evolved to the more tan genes in higher organisms?
Variation in gene number among organism indicates a process of new gene origination
New genes contribute to the origin of adaptative evolutionary novelties
Evolution sutides:
How new structres were generate?
How become fixed?
How they evolved?
How genomes evolved from a few primordial genes to the more than 20,000 genes in higher organisms is an important question in evolution. The variation in gene number amons organisms indicate a process of gene origination.
The origin of novel gene has a huge significance to adaptative evolution and organismal diversity.
Multiple process to the creation of novel genes are now known and this includes gene duplication that is thought to play the most important role.
The process of birth of novelties genes have fascinated biologist and thanks to de recents advances of molecular techniques and and molecular evolution and population provide useful tools to analyze this process

3. Molecular mechanisms of new gene structure
Exon shuffling: Mosaic protein by illegitimate recombination and retroposed exons.
Two o more exons form different genes can be brought together ectopically to créate a new exón-intron structure. Is a way to créate mosaic proteins

4. Molecular mechanisms of new gene structure
Gene duplication: Duplicated gene can evolve new functions and diverge
This classical model creates duplicate gene that can evolve to new functions , whereas the ancestral copy maintains its original function.

5. Molecular mechanisms of new gene structure
Retroposition: Duplicate genes through reverse transcription from the RNAm
Creates duplicate genes in NEW genomic positions through reverse transcription of expressed parental genes. It has to recruit new regulatory sequence to be functional and normally originate a chimeric strcture.

6. Molecular mechanisms of new gene structure
Mobile elements: Integration of TE into nuclear genes.
Integretion of the TE into the host genes generating new functions genes

7. Molecular mechanisms of new gene structure
Lateral gene transfer: Prokaryotes horitzontal transfer to recruit new genes. Also in protozoan and flowering plants. Also TE transfered is observed.
In prokaryotes genes are transferred btw organisms. It can recruit new genes and provide new phenotypes. Gene transfer was observed in protozoan and in flowering plants indicating that might be important in the evolution of eukaryotic genes.

8. Molecular mechanisms of new gene structure
Gene fusion/fission: Genes fuse by readthrough transcription. Otherwise, a gene can be Split in two
Two adjecents genes can be fuse into a single gene by readthrough transcription. Conversley, a single gene can be Split into two seperate genes. Gene fusión have been reported in higher eukaryotes

9. Molecular mechanisms of new gene structure
Normally a combination of all these mechanism arise new gene function, as in the case of jingwei gene from D. Melanogaster (first young gene described)
It combined exón shuffling , retroposition and gene duplication.
We are going to focus into the two mechanisms that implies gene duplication

10. New evolutionary opportunities.
Gen(om)e duplication
Duplicaition have been discribed as a key process during evolution.
Genetic redundancy ROBUSTNESS against deleterious mutations
Relaxed constraint (for mutations) WINDOW OF EVOLVABILITY
Genetic opportunities for adaptation
New evolutionary opportunities.
Two levels of duplications
Gen(om)e duplication provides a period of genetic redundancy that confers robustness against deleterious mutations, relaxed constraint, and genetic opportunities for adaptation.
window of evolvability” due to relaxed constraint
duplicated gene provides a greater, less-constrained chance for natural selection to shape a novel function and that way have new evolutionary opportunitie to be adapted into the dynamic sorrounding enviornment
Whole genome duplication (WGD)
Segmental duplication

11. Molecular mechanisms of GD
We can divide the mechanisms depending the material source of duplication:
1) DNA
2) RNA

12. 1) DNA mechanisms of GD
A) Unequal crossing over B) Duplicative transposotion by
Non-homologous end joining (NHEJ)
Unequal crossing over is a type of gene duplication or deletion event that deletes a sequence in one strand and replaces it with a duplication from its sister chromatid in mitosis or from its homologous chromosome during meiosis. between homologous sequences that are not paired precisely. non-reciprocal recombination.[1] Unequal crossing over requires a measure of similarity between the sequences for misalignment to occur. The more similarity within the sequences, the more likely unequal crossing over will occur.
Duplicative transposition of DNA sequences can be accomplished by one of two main pathways: nonallelic homologous recombination (NAHR) or nonhomologous endjoining (NHEJ). The difference between the two pathways is based on whether homologous sequences are used as a template during double-strand-break repair. Recombination between these nonallelic homologous sequences can result in the duplication of the intervening sequences, which can then lead in turn to more duplications because of pairing between the new paralogues. The rejoining of the broken ends via NHEJ is associated with deletions of various sizes, but also insertions of sequences (filler DNA) that are often copied from sites close to the DSB. NHEJ does not require sequence similarities for the incorporation of filler DNA into the break.
Polyploidy is an evolutionary process whereby two or more genomes are brought together into the same nucleus. WGD events can be associated with important evolutionary transitions involving the origins of higher taxa.
C) Polyploidization (whole genome duplication)

13. 1) DNA mechanisms of GD C) Polyploidization (whole genome duplication)
S. Cerevisia  ancient tetraploid with 88% loss of paralogues
Significant factor in vertebrats  two rounds of WGD took place
WGD associated to increase evolvability and a reduced probabilities to extinct
Occured in70% of angiosperm and 95% of pteridophyts. Major source of gene duplication in plants. It is a process still undergoing in plants.
polyploidy appears to be one of the major processes that has driven and shaped the evolution of higher organisms. WGD followed by massive gene loss and specialization have long been postulated as a powerful mechanism of evolutionary innovation
Genome duplication is often followed by increased rates of evolution and directional selection on some genes, it has been seen A reduced probability of extinction after WGD.genome duplication events in vertebrates are preceded by multiple extinct lineages, When lineages diverge, the ones that experienced a WGD exhibit increased rates of evolution compared to nonduplicated lineages. BC genome duplication is associated with increased evolvability, which in turn contributes to reduced probabilities of extinction .
genome duplication has been recognized as a prominent factor in the evolution of eukaryotes whole-genome duplications also are thought to have been a significant factor in vertebrate evolution, two rounds of genome duplication had taken place in the evolution of vertebrates
Saccharomyces, experienced an ancient whole-genome duplication. It was an ancient tetraploid, gene loss after duplication that resulted in the loss of 88% of paralogous genes in the yeast Saccharomyces cerevisia ,
WGD is the primary source of gene duplicates in plants. genome duplication is ubiquitous in plants.
In plants, polyploidy was proposed to have occurred in the lineage of at least 70% of angiosperms (Masterson1994) and in 95% of pteridophytes
ANGIOSPERMS genome duplication in angiosperm genomes has been central for angiosperms evolution. The two major branches of
the angiosperms (eudicots and monocots show much more rapid structural evolution than vertebrates. This difference appears to be due largely to the tendency of angiosperms for chromosomal duplication and subsequent gene loss.
C) Polyploidization (whole genome duplication)

14. 2) RNA mechanism of GD A) Retroposition
RNAm reverse transcribed to cDNA
 Due to L1 LINE elements activity in mammals
Features
Lack of introns
Lack of regulatory elements
Poly-A sequence and flanking short direct repeats
Not linked with parental copy (normally in other chomosomes)
Retroposition is a process when a messenger RNA (mRNA) is reverse transcribed to complementary DNA (cDNA) and then inserted into the genome. This mechanisms creates duplicates genes in new genomic positions through reverse transcription of parental genes. To be heritable have to occur in the germ line (only genes expressed in the germ line can suffer this processs). A machinery that transcribe the RNAm to cDNA is required. In mammals the L1 retro element provides the machinery responsible of retroposing the nuclear genes
There are several molecular features of retroposition: lack of introns and regulatory sequences of a gene, presence of a poly-A sequence, and presence of flanking short direct repeats. A duplicated gene generated by retroposition is usually unlinked to the original gene, because the insertion of cDNA into the genome is more or less random.
Recent studies have found that retrogenes that are integrated near other coding regions or even in introns of expressed coding sequences are much more likely to be expressed than those that are integrated far from coding sequences

15. 2) RNA mechanism of GD A) Retrotranscription
Lack of expression potential Integreted near coding regions (even introns) rather than far
As a retroposed gene copy does not usually retropose a promotor copy it has to recruit a new regulatory region to be functional or it will die out as a pseudogene. They need to obtain a core promoter and probably other elements to regulate their expression
A ) Insert into an intronic sequence of host gene. The presence of splicing signal enable to form splicing variants and the retrocopy could be transcribed as a fusión transcript together with the host gene.
B) Insertes into open chromatin activly transcribed and facilitates the accessibility of the transcriptional machinery. The presence of enhancers from neighebhood genes and weak promotor sequence that evolve to a functional ones, streghten their transcriptional activity C)

16. 2) RNA mechanism of GD A) Retrotranscription
Lack of expression potential Integreted near coding regions (even introns) rather than far
C) Recruiment of distant promotors via acquisition of new intron/exón strucutre.
D) Recruitment of promotors from CpG proto-promotors
E) Inheritcance of parental promotors through alternative transcription start site
F) De novo promotor evolution by single nt substitution

17. Fate of duplicated genes
Most common fate
Family genes evolution
Paralogous genes
Orthologous genes
In some cases
The fate of duplicate genes
WGDs result in new gene copies of every gene in a genome
and, obviously, all the flanking regulatory sequences.
Non functionalization PSEUDOGENES
It is generally not advantageous for species to carry two identical genes. Duplication of a gene produces functionalredundancy. Pseudogenization, the process by which a functional gene becomes a pseudogene, usually occurs after duplication if the duplicated gene is not under any selection pseudogenization occur by mutations that disrupt structure and function of one of the two duplicate
Subfunctionalization
In general, a duplicate gene is deleterious for the genome. Two genes with identical functions are unlikely to be stably maintained in the genome unless the presence of an extra amount of gene product is advantageous. After duplication, both daughter genes are maintained in the genome for a period of time during
which they differentiate in some aspects of their functions. This can occur by subfunctionalization, in which each daughter gene adopts part of the functions of their parental gene.
For example, engrailed-1 and engrailed-1b are pair of transcription
factor genes in zebrafish generated by a chromosomal
segmental duplication. engrailed-1 is expressed in the
pectoral appendage bud, whereas engrailed-1b is expressed
in a specific set of neurons in the hindbrain/spinal cord.
On the other hand, the sole engrailed-1 gene of the mouse,
orthologous to both genes of the zebrafish, is expressed
in both pectoral appendage bud and hindbrain/spinal cord
(Force et al. 1999).
Neofunctionalization
one gene copy, or paralog, takes on a totally new function after a gene duplication event. Neofunctionalization is an adaptive mutation process; meaning one of the gene copies must mutate to develop a function that was not present in the ancestral gene. While the other duplicate retains its original function
It is now known that many multigene and supergene families exist in eukaryote genomes: multigene families with uniform copy members like genes for ribosomal RNA, those with variable members like immunoglobulin genes, and supergene families such as those for various growth factor and hormone receptors.
FAMILY GENES
Duplication of the genes could happen regularly and arise a set of family genes. gene conservation’. Was proposed by Ohno (1970) there are differnet models for why these duplicates would maintain the original
There are different mechanism of the evolution of family genes.
Divergent evolution, where different genes in the family adquire different funcitons (globines=
Concerted evolution; the different copies of the family genes are subjected to a freguents crossing over and gene conversion that way genes never divergence to much and mutations can be spread among the family genes in a process of homogenitiation.
Birth and death evolution; it happen in that genes that have a high rate of duplication but also have high rate of pseudogenes and inactivated genes having high rates of gene birth and death.
In both cases A strong purifying selection against mutations that modify gene function can also prevent duplicated genes from diverging.
PARALOGOUS/ ORTHOLOGOUS
Orthologs are genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution
Paralogs are genes related by duplication within a genome. Orthologs retain the same function in the course of evolution, whereas paralogs evolve new functions, even if these are related to the original one.

18. Evolution of GD Main forces shaping GD evolution
Two different hypothesis NOT mutually exlcusive
Classical Model: Postulated by Kimura
Rapid adquisition of novel function
The Waiting Model
Immediate Model
Partial duplication in conjucting with exon shuffling, gene fusión…
Main forces shaping GD evolution
>>1
DARWINIAN POSITIVE SELECTION
Kimura’s is based on an obvious intuition that new gene duplicates might provide redundant functions in their early stages and, therefore, the mutations in the duplicates are neutral or nearly neutral — we call this the ‘waiting model’, because it requires time to reach the moment of functional distinction.
A more straightforward conjecture is that adaptive evolution might have had a principal role throughout the creation and subsequent evolution of new genes — we call this the ‘immediate model’, because it requires no waiting time for the evolution of a new function
How gene duplicates could acquire new functions and ultimately be preserved in a lineage.
The first test for selection was to analyse the KA/KS ratio in new gene lineagesshowed that a new duplicate ribonuclease gene RNASE1B, which (the douc langur), has a KA/KS ratio that is significantly higher than unity (0.0310/ = 4.03), whereas its paralogous copy RNASE1 has no evolutionary changes indicates that there is strong Darwinian selection on the new gene. Furthermore, it was consistently found that, which predicts that there is an altered optimal pH for RNASE1B has a lower optimal pH value, which is correlated with a digestive system that has evolved.
A MCDONALD-KREITMAN TEST showed excess amino-acid replacement substitutions in compared to the artologous copy in other spicies and the paralagous copy in the same gene. this indicates that deleterious mutations is relaxed on one or both copies of the gene; this is attributable to the extra sheltering of recessive mutations when there are extra copies of a locus. Consequently,mutations that would normally be eliminated by selection could accumulate at one or both loci. sphinx has also undergone accelerated substitution since its formation: there are 18 substitutions in sphinx versus 2 substitutions in the parental gene ATP synthase F.
 But  Novel gene functions must evolve in response to changing environmental or ecological pressures. Genes encoding proteins that interact with the environment tend to be more frequently retained after duplication than those that interact with intracellular compartments
Positive selection and relaxation of negative selection lead to the rapid fixation of the new genes.
Positive Darwinian selection selection has been detected in many studies as the main fource to evolve novel genes . New genes are an outcome of adaptative evolution, natural selection may preserve beneficial novel genes functions

19. X chromosome bias and sexual chromosome origin
Retrogenes tend to be expressed in testes. Hypothesis:
Autosomal hypertranscription in spermatogenic cells
Insertion of retrocopies manily close to germline-expressed cells (open chromatin)
X-linked parental genes scaping to autosomes (Retrogenes out of X) more tan expected by random
Retrogenes studies have shwn that there is an overall propensity of retrogenes to be expressed in testes. To explain these expression releted process and natural selection have been postulated. In meitoic i post meiotic sparmetogenic cells the autosomal chromosomes appear to be in a state of hypertranscription. These allows transcription of DNA that is usually not transcribed and therefore facilitated the transcription of retrocopies that evolved to bona ide genes. Natural selection then further enchanced their promotors and refind testis expression.
Retrocopies might preferently insert to open chromatin, activley transcribed. Given htat to pass to next generation, retocopies have to be produced in the germ line they predominantly insert close to germ.exprssed genes.
It was found in mammals and drsosophila a disprportionaly large number of parental genes on the X chromosome that given rise to functional retrogenes on autosomes. These autosomes retrogenes are specifically expresse in testis whereas their X-linked parents are transcriptionally silenced during these stages due to MSCI.
Hyptohesis that retrogen that stem ou of X have been fixed during evolution and shape by natural selection to compensate for parental silencing in MSCI. This compensation hypothesis (loss retrogene function damage mice) . There fore MSCI may be the main forcé responsable for the preferential fixation of X-drived retrogenes.
Mitotic Sex Chromosome Inactivation (MSCI) may be the MAIN FORCE for preferential fixation of X-derived retrogenes

20. X chromosome bias and sexual chromosome origin
Gene export from X is an ongoing process
On eutherian and marsupial lineages
MSCI is the recombination barrier between X and Y
¿¿WHEN DID BEGIN??
Means that MSCI emerged then
these chromosomes where originated in the common ancestor of marsupials and eutherians
NOT in the common ancestor of all mammals

21. GD pathologies
Duplicate genes phenotypic effects mainly determined by protein imbalance
Dosage-sensitives genes (over- or under- expressed)
Relation of gene dosage-fitness
The phenotypic consequence of duplicating large regions or even whole chromosomes is at least in part determined by regulatory imbalances of the product. It is a priori expected that duplicate genes will exhibit an increase in mRNA expression.
Duplication of such dosage-sensitive genes, required at stoichiometrically precise levels, may be tolerated in whole-genome, but not in small-scale, duplications (20). As balance hypothesis posits, connected genes are particularly dosage sensitive, and tend not to be retained after local duplications.
The relationship between gene dosage and fitness (phenotype) is complex. 
Linear relationship that is often found for structural and regulatory proteins (Figure 3a);
second fits a “diminishing returns” principle typical of enzymes that function at limiting concentrations .
The third alternative corresponds to a diminished fitness for both increased and decreased gene dosage, indicating either multisubunit complexes with a single component that has a tight stoichiometry (46) (gene balance hypothesis), or specific regulatory imbalances as a consequence of under- and overexpression. Transcriptional regulators can cause phenotypes both when under- and overexpressed (84), a mechanism responsible for many developmental/malformation syndromes (Figure 3c) (Table 1).
A fourth alternative concerns proteins with a propensity to aggregate (Figure 3d). These proteins display a dual behavior. The wild-type protein aggregates in a dose-dependent manner once the diploid threshold dose is exceeded (e.g., gene duplication).
Genes encoding such proteins include α-synuclein (SNCA), responsible for one rare familial form of early-onset Parkinson disease, and the amyloid β precursor protein (APP) that causes one form of dominant early-onset Alzheimer disease

22. Conclusions
Gene duplication is one of the main source of new gene structure generation
There are different mechanism of GD (DNA or RNA) and different level (segmental duplication or whole genome duplication)
Gene(ome) duplication has been very important for the evolution of organisms and the main shaping force of new genes have been darwinian positive selection
Retrocopies have had a very important role in the origin of sexual chromosomes
GD can cause several pathologies

23. THANKS FOR YOUR ATTENTION!!

24. References
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Hoekstra HE, Coyne JA The locus of evolution: evo-devo and the genetics of adaptation. Evolution. 61(5): Review.
Katju V, Lynch M On the formation of novel genes by duplication in the Caenorhabditis elegans genome. Mol Biol Evol. 23(5):
Kaessmann H, Vinckenbosch N, Long M RNA-based gene duplication: mechanistic and evolutionary insights. Nat Rev Genet 10(1): Review.
Long M, Betrán E, Thornton K, Wang W The origin of new genes: glimpses from the young and the old. Nat Rev Genet. 4(11): Review.