Using a strain of maize in which one member of a chromosome pair exhibited the knob but its homologue did not, the scientists were able to show that some alleles were physically linked to the knobbed chromosome, while other alleles were tied to the normal chromosome. McClintock and Creighton then followed these alleles through meiosis, showing that alleles for specific phenotypic traits were physically exchanged between chromosomes.
Evidence for this finding came from the fact that alleles first introduced into the cross on a knobbed chromosome later appeared in offspring without the knob; similarly, alleles initially introduced on a knobless chromosome subsequently appeared in progeny with the knob Figure 1. Recombination also occurs in prokaryotic cells, and it has been especially well characterized in E. Although bacteria do not undergo meiosis, they do engage in a type of sexual reproduction called conjugation , during which genetic material is transferred from one bacterium to another and may be recombined in the recipient cell.
As in eukaryotes, recombination also plays important roles in DNA repair and replication in prokaryotic organisms. Figure 2: Structure of the Holliday junction. A Electron-microscope image of a recombination intermediate. In this image, the Holliday junction was partially denatured to assist its visualization. B Two possible configurations for the Holliday junction, with the DNA shown in the parallel left or antiparallel configuration right. Potter, H. DNA recombination: in vivo and in vitro studies.
Cold Spring Harb. All rights reserved. Liu, Y. Happy Hollidays: 40th anniversary of the Holliday junction. Nature Reviews Molecular Cell Biology 5 , Figure Detail.
Although common, genetic recombination is a highly complex process. It involves the alignment of two homologous DNA strands the requirement for homology suggests that this occurs through complementary base-pairing , but this has not been definitively shown , precise breakage of each strand, exchange between the strands, and sealing of the resulting recombined molecules.
This process occurs with a high degree of accuracy at high frequency in both eukaryotic and prokaryotic cells. The basic steps of recombination can occur in two pathways, according to whether the initial break is single or double stranded.
In the single-stranded model , following the alignment of homologous chromosomes, a break is introduced into one DNA strand on each chromosome, leaving two free ends.
Each end then crosses over and invades the other chromosome, forming a structure called a Holliday junction Figure 2. The next step, called branch migration , takes place as the junction travels down the DNA. The junction is then resolved either horizontally, which produces no recombination, or vertically, which results in an exchange of DNA. In the alternate pathway initiated by double-stranded breaks, the ends at the breakpoints are converted into single strands by the addition of 3' tails.
These ends can then perform strand invasion, producing two Holliday junctions. From that point forward, resolution proceeds as in the single-stranded model Figure 3. Note that a third model of recombination, synthesis-dependent strand annealing [SDSA], has also been proposed to account for the lack of crossover typical of recombination in mitotic cells and observed in some meiotic cells to a lesser degree. No matter which pathway is used, a number of enzymes are required to complete the steps of recombination.
The genes that code for these enzymes were first identified in E. This research revealed that the recA gene encodes a protein necessary for strand invasion. Meanwhile, the recB , recC , and recD genes code for three polypeptides that join together to form a protein complex known as RecBCD; this complex has the capacity to unwind double-stranded DNA and cleave strands. Two other genes, ruvA and ruvB , encode enzymes that catalyze branch migration , while Holliday structures are resolved by the protein resolvase , which is product of the ruvC gene.
In eukaryotes, recombination has been perhaps most thoroughly studied in the budding yeast Saccharomyces cerevisiae. Many of the enzymes identified in this yeast have also been found in other organisms, including mammalian cells. Such studies reveal that the Rad genes named for the fact that their activity was found to be sensitive to radiation play a key role in eukaryotic recombination. In particular, the Rad51 gene, which is homologous to recA , encodes a protein called Rad51 that has recombinase activity.
This gene is highly conserved, but the accessory proteins that assist Rad51 appear to vary among organisms. For example, the Rad52 protein is found in both yeast and humans, but it is missing in Drosophila melanogaster and C. RPA has a higher affinity for ssDNA than Rad51, and it therefore can inhibit recombination by blocking Rad51's access to the single strand needed for invasion.
Once access has been gained, Rad51 polymerizes on the DNA strand to form what is called a presynaptic filament, which is a right-handed helical filament containing six Rad51 molecules and 18 nucleotides per helical repeat.
The search for DNA homology and formation of the junction between homologous regions is then carried out within the catalytic center of the filament. In addition to proteins that assist Rad51 activity, there are also some proteins that inhibit it. It is thought that these proteins play a role in preventing recombination during DNA replication when it is not needed. Individuals who are heterozygous for BRCA2 are subject to increased risk for breast and ovarian cancer ; loss of both alleles causes Fanconi's anemia, a genetic disease characterized by predisposition to cancer, among other defects.
As previously described, the enzymes and mechanisms that carry out the process of homologous recombination are fairly well delineated. Not so well understood is the important question of how homologous sequences come to be in proximity so that recombination can proceed.
In their review, Barzel and Kupiec describe two alternate hypotheses, one of which they call the null model. This model proposes that homologues find one another through a passive process of diffusion, in which the DNA sequence at the broken end of a strand is sequentially compared to all of the other potential end sequences in the genome.
An alternate hypothesis proposes that homologous chromosomes reside in pairs constitutively. Acting against this hypothesis is the finding that in induced recombination experiments, the broken ends of strands recombine with what are called ectopic homologues areas of fortuitous sequence identity as frequently as they recombine with their true homologous chromosomes.
Furthermore, although homologous pairing has been observed in somatic cells of some organisms e. Transformation is a type of prokaryotic reproduction in which a prokaryote can take up DNA found within the environment that has originated from other prokaryotes. Transduction is a type of prokaryotic reproduction in which a prokaryote is infected by a virus which injects short pieces of chromosomal DNA from one bacterium to another.
Conjugation is a type of prokaryotic reproduction in which DNA is transferred between prokaryotes by means of a pilus. Key Terms transformation : the alteration of a bacterial cell caused by the transfer of DNA from another, especially if pathogenic transduction : horizontal gene transfer mechanism in prokaryotes where genes are transferred using a virus binary fission : the process whereby a cell divides asexually to produce two daughter cells conjugation : the temporary fusion of organisms, especially as part of sexual reproduction pilus : a hairlike appendage found on the cell surface of many bacteria.
Reproduction Reproduction in prokaryotes is asexual and usually takes place by binary fission. In a transformation, the cell takes up prokaryotic DNA directly from the environment. In b transduction, a bacteriophage injects DNA into the cell that contains a small fragment of DNA from a different prokaryote. In c conjugation, DNA is transferred from one cell to another via a mating bridge that connects the two cells after the pilus draws the two bacteria close enough to form the bridge.
Provided by : Boundless. The mutational deterministic hypothesis requires the rate of deleterious mutations per genome replication U to be greater than 1. It also requires these deleterious mutations to be subject to synergistic epistasis , so that their combined effect on fitness is greater than that expected from their individual effects However, although deleterious mutations occur frequently, there is no good evidence for the occurrence of frequent synergistic epistasis in RNA viruses, as most of the described epistatic interactions are antagonistic 87 , 88 , 89 , A high frequency of antagonistic epistasis is expected in RNA viruses because of the lack of genetic redundancy in their highly constrained genomes, such that individual proteins often perform multiple functions.
Additional evidence against the mutational deterministic theory is that the burden of deleterious mutations is seemingly high in all RNA viruses, indicating that it does not depend on genome structure or, hence, the propensity for recombination In summary, it seems unlikely that recombination has evolved as a means by which RNA viruses can purge deleterious mutations.
Rather, the population sizes of RNA viruses may be so large that sufficient viable progeny are produced every generation to guarantee survival. In addition, large population sizes mean that the accumulation of deleterious mutations can be offset by frequent back and compensatory mutations. RNA viruses may therefore possess a population-scale robustness that protects them from the accumulation of deleterious mutations Finally, it is important to note that there are also evolutionary costs associated with recombination in RNA viruses, as it is likely to increase both the degree of competition within a host 92 and the extent of complementation.
Complementation is particularly important because it enables defective viruses to parasitize the fully functional viruses that co-infect the same host cell, thereby allowing deleterious mutations to remain in viral populations for longer time periods. Co-infection is a prerequisite for both recombination and complementation.
Interestingly, protection from co-infection has been described for numerous plant viruses 93 , and mechanisms that limit the co-infection of individual cells by multiple viruses have been documented in a number of other viruses, including retroviruses 94 , pestiviruses 95 and alphaviruses 96 , 97 , some of which recombine frequently. In contrast to the theories described above, various other theories of the evolution of recombination in RNA viruses do not consider it a form of sexual reproduction.
Repair of genetic material. One theory states that the adaptive value of recombination comes from its ability to repair genetic damage Indeed, early work on recombination in retroviruses suggested the existence of a 'forced copy choice' model of recombination, in which the template switch occurs when a break in the RNA template forces the RT to seek an alternative and functional template However, replication occurs equally often on unbroken and on broken templates , and experimentally induced genetic damage has not been clearly associated with higher recombination rates Moreover, if genetic damage is the driving force behind recombination, the common exposure of viruses to oxidative stress is at odds with the strong disparities in their rates of recombination.
In theory, recombination as a form of repair could be a potent mechanism in viruses with diploid or pseudodiploid genomes, such as HIV. However, the process clearly relies on high rates of multiple infection, and as this is a feature that cannot be guaranteed for all viruses, it is unlikely that there would be sufficient pressure for recombination to be selected as a repair mechanism.
Recombination as a by-product of genome organization. Several theories for the evolution of recombination in RNA viruses argue that rather than being selected for its direct fitness benefits, as is proposed by all theories in which recombination functions as a form of sex, recombination is in fact a by-product of the processivity of RNA polymerases.
Under this hypothesis, the observed variation in the recombination rates is largely determined by differences in the genome organization and life cycles of RNA viruses Table 1. The basis of this theory is the notion that many aspects of genome organization in RNA viruses aim to control gene expression. Specifically, a major challenge for all RNA viruses is to control the levels of each protein that they produce.
Translation often results in a single large polyprotein that then needs to be proteolytically cleaved into individual proteins. Although this replication strategy is efficient and allows the naked RNA that is extracted from virions to be infectious in the absence of a co-packaged viral polymerase, it also means that equal amounts of each protein are produced; any difference in protein abundance must be achieved through differential polyprotein cleavage.
This constraint can be overcome by dividing the viral genome into separate 'transcriptional units' that offer greater control over gene expression 1. In the case of segmented viruses, reassortment would then occur through the normal packaging mechanism and, hence, as a by-product of co-infection by two segmented viruses, although this does not preclude the production of selectively beneficial genetic configurations.
Similarly, the existence of polycistronic mRNAs in bacteria, in contrast to the monocistronic mRNAs of eukaryotes, may explain why fewer segmented viruses of bacteria have been described to date, although this may change with an increased sampling of the virosphere. In some respects, the existence of these viruses is puzzling, as they need to go through an additional transcription step before they can translate their proteins, and they also need a viral transcriptase RdRP to enter the host cell in addition to the RNA.
However, such a life cycle opens up a powerful means to control gene expression at the level of transcription; transcription produces multiple mRNAs, which can form individual transcriptional units. Furthermore, the negative genome orientation removes the problem of having to use the same template for both translation and replication. This gene order results in a strong gradient of transcription such that more N protein is produced than L protein, probably because the enzymatic function of the L protein requires fewer copies of the protein than the number required for the structural nucleocapsid.
Such a conserved gene order, and one that correlates with the amount of protein product required, strongly suggests that natural selection is operating at the level of gene expression. Specifically, the genomic and antigenomic RNA molecules in viruses of this type are quickly bound to multiple nucleoprotein subunits, as well as to other proteins, to form ribonucleoprotein RNP complexes from which viral replication and transcription can proceed.
However, this tight complex of RNA and proteins lowers the probability of hybridization between complementary sequences in the nascent and acceptor nucleic acid molecules, and it is this hybridization that is required for the template switching that occurs during copy choice recombination. Intriguingly, 'illegitimate' recombination with a cellular mRNA has also been described in influenza A virus 4. Interestingly, the strategy of gene expression that is used by coronaviruses — discontinuous transcription — relies entirely on the template-switching property of the viral RdRP.
Discontinuous transcription of the large unsegmented genomes of MHV and similar coronaviruses leads to the production of subgenomic negative-sense RNAs through a copy choice mechanism; these RNAs serve as templates for the production of mRNA This suggests that the RdRP of MHV is selected to efficiently mediate template-switching events and that the very high rates of recombination observed are a direct consequence of this particular strategy for controlling gene expression.
Last, in some retroviruses, most notably HIV, recombination rates are extremely high. The pseudodiploidy of these viruses facilitates recombination because two RNA molecules must be packaged in the same virion, thus increasing the likelihood of template switching owing to the physical proximity of the RNAs during replication.
Furthermore, template switching is also an intrinsic component of the replication strategy of retroviruses. This generation of the dsDNA genome is not a straightforward conversion of positive-sense RNA into negative-sense DNA, followed by the synthesis of a positive-sense DNA complement; instead, two template switches, known as strong-stop strand transfers, are required to join and duplicate the long terminal repeats at the boundaries of the provirus. However, whereas strong-stop strand transfers occur only at specific positions, copy choice template switching may occur at any position in the retroviral genome.
The occurrence of pseudodiploidy and frequent template switching might suggest that these processes have been selected for to increase recombination rates in retroviruses, but recombination rates in fact differ greatly between retroviruses in a manner that reflects other aspects of viral biology.
For example, genome dimerization for HIV probably occurs randomly in the cytoplasm. By contrast, genome dimerization for MLV takes place in the nucleus, close to the transcription sites, and leads mostly to self-associations rather than the associations between genetically different parental molecules that would be needed for recombination More generally, both diploidy and complementation allow deleterious mutations to be masked in the case of diploidy, as recessive mutations , and this may provide an explanation for the evolution of diploidy , , Understanding the evolution of recombination remains one of the most challenging problems in biology.
We suggest that it is optimistic to believe that a single explanation applies to all organisms and that the precise mechanisms of recombination must be understood in each case. In particular, although it is clear that recombination is a key aspect of sexual reproduction in most cellular species, such that its evolution can be discussed in terms of the generation and removal of specific types of mutation, we argue that this does not seem to be the case in RNA viruses.
Indeed, it is striking that high levels of recombination appear to be a sporadic occurrence in RNA viruses, such that they cannot be universally advantageous, whereas theories for the evolution of sexual reproduction in eukaryotes attempt to explain recombination and clonality on the assumption that these are common and sporadic, respectively Rather, a review of the available data suggests that the differing rates of recombination and reassortment that characterize RNA viruses may reflect the mechanistic constraints that are associated with particular genome structures and viral life cycles.
If this hypothesis is upheld, then recombination should be considered a mechanistic by-product of RNA polymerase processivity a trait that varies according to the genomic architecture of the virus in question and not as a trait that is optimized by natural selection for its own selective value, although it may on occasion generate beneficial genotypes Box 3.
It is important to note that RNA viruses produce large numbers of progeny and that this, rather than recombination, is more likely to be the key to their evolutionary survival, as it buffers them from the adverse effects of the accumulation of deleterious mutations and regularly produces advantageous mutations.
However, it is equally clear that our knowledge of recombination and its determinants remains patchy for most RNA viruses, such that far more data are needed for a definitive understanding of the evolution of recombination. For example, it will be important to accurately measure recombination rates in viruses that differ markedly in their strategies for controlling gene expression. Fortunately, the development of next-generation sequencing methods is likely to facilitate the acquisition of data that will lead to important new insights into the causes and consequences of recombination in this major class of infectious agent.
If viral RNA polymerases harbour limited processivity and template switching may generate dysfunctional genomes 29 , , the switching process might represent a liability for RNA viruses. Could specific features of some RNA viruses limit the potential risks that are associated with frequent template switching? Copy choice RNA recombination depends on sequence similarity, thus reducing the probability of illegitimate recombination and the chance of recombination among distantly related strains, both of which present high risks of generating deleterious genomes.
However, sequence similarity is less relevant to copy choice recombination when the viral genome is covered by nucleoproteins, and this might explain why defective interfering particles are frequently found in negative-stranded RNA viruses. Similarly, although the process of dimerization in HIV does not favour the homodimerization of genomes, the compatibility of sequences at the dimerization initiation site is crucial to the formation of heterozygous particles and, hence, recombinants This may also explain, in part, the absence of recombination between HIV-1 and HIV-2, despite many cases of co-infection , and a similar process could contribute to the nonrandom pattern reassortment that is observed in some segmented viruses Finally, the risk of producing deleterious mutations by recombination is lowest when recombination breakpoints fall between rather than within genes, proteins or protein domains The observation that most intergenic and interprotein domains of HIV exhibit strong RNA structure is particularly striking in this respect, as these structures favour template switching in genomic regions where recombination is less likely to be disruptive Similarly, the conserved motifs that contribute to the specific template-switching events for discontinuous transcription in coronaviruses are also likely to enhance template switching in these intergenic regions, in turn limiting the chances of intraprotein recombination in a phenomenon that is reminiscent of reassortment.
Recombination rates in RNA viruses have been measured in two different ways: the intrinsic rate of template switching that occurs during replication, and the recombination rate that can be inferred at the population level.
Unfortunately, large-scale comparative studies of recombination rates using either approach have yet to be undertaken. To accurately measure the frequency of template switching, recombinant products need to be accessed soon after their generation, before any form of selection can take place.
Although they were initially measured in vitro , recombination rates are now obtained from single-cycle experiments in co-infected cells in culture. These experiments provide valuable insights into the frequency and mechanisms of recombination, but they do not necessarily mimic natural systems, in which the frequency of co-infection is unknown.
The frequency of co-infection depends on many factors, including the size of the viral population, the frequency of different variants in the population and the likelihood of mixed infection. Recombination rates may also be estimated in live hosts , as has been carried out in natural HIV infections Methods that estimate the recombination rate at the population level are based on gene sequence analysis 8 and necessarily exclude deleterious recombinant forms that have been removed by purifying selection.
Some of these approaches explore linkage disequilibrium in population-wide genetic data sets , , whereas others rely on coalescent approaches to provide estimators of the population recombination rate The power of these approaches is that they allow a standardized framework for comparing recombination rates. However, they are limited in that the signal of recombination is sometimes difficult to distinguish from particular mutation patterns or from other factors such as geographical structure in the data, and that they rely heavily on the sample of sequences used in the analysis, which may be both small and biased.
For most RNA viruses, cross-species transmission is the most common way for a virus to enter a new host. Recombination could assist in this process because it enables viruses to explore a greater proportion of the sequence space than is accessible by mutation at any one time, thereby increasing the likelihood of finding a genetic configuration that facilitates host adaptation.
Notably, many recently emerged human diseases are caused by RNA viruses that display active recombination or reassortment. Recombination and reassortment are also powerful ways for emerging viruses to acquire new antigenic combinations that may assist the process of cross-species transmission. The continual shuffling of genes that encode the haemagglutinin and neuraminidase envelope proteins of influenza A virus a virus that is commonly associated with the occurrence of human pandemics represents a powerful example of the benefits of recombination and reassortment for the virus 50 , However, in most cases the emergence of a specific virus cannot be directly attributed to its ability to recombine.
For example, although HIV-1 recombines at a high rate, there is no evidence that recombination assisted the cross-species transfer of the virus from the chimpanzee reservoir population into humans.
In fact, there are few cases in which recombination seems to have directly resulted in viral emergence. One of the few examples is the alphavirus Western equine encephalitis virus, which was generated through recombination between a Sindbis-like virus and an Eastern equine encephalitis virus-like virus Similarly, a new coronavirus that emerged in turkeys is a recombinant infectious bronchitis virus that acquired a spike protein-encoding gene from another coronavirus Finally, the retrovirus Rous sarcoma virus is likely to have acquired pathogenicity through the recombination-mediated acquisition of a cellular oncogene In summary, the available data suggest that although recombination is sometimes directly helpful to the process of cross-species transmission, it is not a necessary precursor to successful viral emergence.
He received his B. During his Ph. Edward C. His research focuses on the evolutionary genetics of RNA viruses, with special emphasis on the main mechanisms of viral evolution, the molecular epidemiology of important human pathogens, and the evolutionary processes that underpin viral emergence.
Holmes's homepage. National Center for Biotechnology Information , U. Nat Rev Microbiol. Published online Jul 4. Etienne Simon-Loriere 1 and Edward C. Holmes 1, 2. Author information Copyright and License information Disclaimer. Holmes, Email: ude. Corresponding author. All Rights Reserved. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source.
This article has been cited by other articles in PMC. Abstract Recombination occurs in many RNA viruses and can be of major evolutionary significance. Main Populations of RNA viruses habitually harbour abundant genetic variability, which is in large part due to a combination of high mutation rates and large population sizes 1.
Open in a separate window. Figure 1. Generation of recombinant and reassortant RNA viruses. Figure 2. Potential consequences of a disassociation event during viral transcription. Figure 3. Evolutionary consequences of recombination. Table 1 Differing genome organizations and replication strategies of RNA viruses. Box 1 Are some viruses better adapted to deal with inevitable template switching?
Box 2 Measuring recombination rates in RNA viruses Recombination rates in RNA viruses have been measured in two different ways: the intrinsic rate of template switching that occurs during replication, and the recombination rate that can be inferred at the population level. Box 3 Recombination and viral emergence For most RNA viruses, cross-species transmission is the most common way for a virus to enter a new host. Acknowledgements E. RNA viruses can possess either a single segment such that they are unsegmented or multiple segments.
Those with multiple segments may experience reassortment. Defective interfering particles Defective viruses usually possessing long genome deletions that compete, and hence interfere, with fully functional viruses for cellular resources. Virions The final mature virus particles containing the RNA genome and the full set of proteins. Processivity A measure of the average number of nucleotides added by a polymerase enzyme per association—disassociation with the template during replication.
Packaging The process by which the nucleic acid genome and other essential virion components are inserted in the structural core or shell of a virus particle. Complementation The process by which a defective virus can parasitize a fully functional virus that is infecting the same cell; the defective virus 'steals' the proteins of the functional virus to restore its own fitness.
Multiplicity of infection MOI. The ratio of viruses to the number of cells that are infected. Breakpoint The site in the genome sequence at which a recombination event has occurred. Phylogenetic trees are incongruent on either side of the breakpoint. Linkage disequilibrium LD. The nonrandom association between alleles at two or more loci, being indicative of a lack of recombination.
Recombination reduces LD. Clonal interference The process by which beneficial mutations compete, and hence interfere, with each other as they proceed toward fixation. Epistasis An interaction between mutations such that their combined effect on fitness is different to that expected from their stand-alone effects. Depending on the nature of the deviation, epistasis can be either antagonistic positive or synergistic negative.
Genetic redundancy The situation in which a specific phenotype is determined by more than one gene, such as members of multigene families. Robustness The constancy of a phenotype in the face of pressure from a deleterious mutation. Provirus The DNA form of a retroviral genome that is integrated into the genetic material of a host cell.
Genome dimerization A non-covalent process by which retroviruses carry two RNA genomes in the virion. Sequence space All possible mutational combinations that are present in DNA or amino acid sequence data. Competing interests The authors declare no competing financial interests. References 1.
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