Invasion of a multitude of genetic niches by mobile endonuclease genes

Persistence of a mobile DNA element in a population reflects a balance between the ability of the host to eliminate the element and the ability of the element to survive and to disseminate to other individuals. In each of the three biological kingdoms, several families of a mobile DNA element have been identified which encode a single protein that acts on nucleic acids. Collectively termed homing endonuclease genes (HEGs), these elements employ varied strategies to ensure their survival. Some members of the HEG families have a minimal impact on host fitness because they associate with genes having self-splicing introns or inteins that remove the HEGs at the RNA or protein level. The HEG and the intron/intein gene spread throughout the population by a gene conversion process initiated by the HEG-encoded endonuclease called 'homing' in which the HEG and intron/intein genes are copied to cognate alleles that lack them. The endonuclease activity also contributes to a high frequency of lateral transmission of HEGs between species as has been documented in plants and other systems. Other HEGs have positive selection value because the proteins have evolved activities that benefit their host organisms. The success of HEGs in colonizing diverse genetic niches results from the flexibility of the encoded endonucleases in adopting new specificities.     

The recent transfer of a homing endonuclease gene

The myxomycete Didymium iridis (isolate Panama 2) contains a mobile group I intron named Dir.S956-1 after position 956 in the nuclear small subunit (SSU) rRNA gene. The intron is efficiently spread through homing by the intron-encoded homing endonuclease I-DirI. Homing endonuclease genes (HEGs) usually spread with their associated introns as a unit, but infrequently also spread independent of introns (or inteins). Clear examples of HEG mobility are however sparse. Here, we provide evidence for the transfer of a HEG into a group I intron named Dir.S956-2 that is inserted into the SSU rDNA of the Costa Rica 8 isolate of D.iridis. Similarities between intron sequences that flank the HEG and rDNA sequences that flank the intron (the homing endonuclease recognition sequence) suggest that the HEG invaded the intron during the recent evolution in a homing-like event. Dir.S956-2 is inserted into the same SSU site as Dir.S956-1. Remarkably, the two group I introns encode distantly related splicing ribozymes with phylogenetically related HEGs inserted on the opposite strands of different peripheral loop regions. The HEGs are both interrupted by small spliceosomal introns that must be removed during RNA maturation.     

The evolution of homing endonuclease genes and group I introns in nuclear rDNA

Group I introns are autonomous genetic elements that can catalyze their own excision from pre-RNA. Understanding how group I introns move in nuclear ribosomal (r)DNA remains an important question in evolutionary biology. Two models are invoked to explain group I intron movement. The first is termed homing and results from the action of an intron-encoded homing endonuclease that recognizes and cleaves an intronless allele at or near the intron insertion site. Alternatively, introns can be inserted into RNA through reverse splicing. Here, we present the sequences of two large group I introns from fungal nuclear rDNA, which both encode putative full-length homing endonuclease genes (HEGs). Five remnant HEGs in different fungal species are also reported. This brings the total number of known nuclear HEGs from 15 to 22. We determined the phylogeny of all known nuclear HEGs and their associated introns. We found evidence for intron-independent HEG invasion into both homologous and heterologous introns in often distantly related lineages, as well as the "switching" of HEGs between different intron peripheral loops and between sense and antisense strands of intron DNA. These results suggest that nuclear HEGs are frequently mobilized. HEG invasion appears, however, to be limited to existing introns in the same or neighboring sites. To study the intron-HEG relationship in more detail, the S943 group I intron in fungal small-subunit rDNA was used as a model system. The S943 HEG is shown to be widely distributed as functional, inactivated, or remnant ORFs in S943 introns.     

The spread of LAGLIDADG homing endonuclease genes in rDNA

Group I introns that encode homing endonuclease genes (HEGs) are highly invasive genetic elements. Their movement into a homologous position in an intron-less allele is termed homing. Although the mechanism of homing is well understood, the evolutionary relationship between HEGs and their intron partners remains unclear. Here we have focused on the largest family of HEGs (encoding the protein motif, LAGLIDADG) to understand how HEGs and introns move in rDNA. Our analysis shows the phylogenetic clustering of HEGs that encode a single copy of the LAGLIDADG motif in neighboring, but often evolutionarily distantly related, group I introns. These endonucleases appear to have inserted into existing introns independent of ribozymes. In contrast, our data support a common evolutionary history for a large family of heterologous introns that encode HEGs with a duplicated LAGLIDADG motif. This finding suggests that intron/double-motif HEG elements can move into heterologous sites as a unit. Our data also suggest that a subset of the double-motif HEGs in rDNA originated from the duplication and fusion of a single-motif HEG encoded by present-day ribozymes in LSU rDNA.     

The population genetics of using homing endonuclease genes in vector and pest management

Homing endonuclease genes (HEGs) encode proteins that in the heterozygous state cause double-strand breaks in the homologous chromosome at the precise position opposite the HEG. If the double-strand break is repaired using the homologous chromosome, the HEG becomes homozygous, and this represents a powerful genetic drive mechanism that might be used as a tool in managing vector or pest populations. HEGs may be used to decrease population fitness to drive down population densities (possibly causing local extinction) or, in disease vectors, to knock out a gene required for pathogen transmission. The relative advantages of HEGs that target viability or fecundity, that are active in one sex or both, and whose target is expressed before or after homing are explored. The conditions under which escape mutants arise are also analyzed. A different strategy is to place HEGs on the Y chromosome that cause one or more breaks on the X chromosome and so disrupt sex ratio. This strategy can cause severe sex-ratio biases with efficiencies that depend on the details of sperm competition and zygote mortality. This strategy is probably less susceptible to escape mutants, especially when multiple X shredders are used.     

Outcrossed sex allows a selfish gene to invade yeast populations.

Homing endonuclease genes (HEGs) in eukaryotes are optional genes that have no obvious effect on host phenotype except for causing chromosomes not containing a copy of the gene to be cut, thus causing them to be inherited at a greater than Mendelian rate via gene conversion. These genes are therefore expected to increase in frequency in outcrossed populations, but not in obligately selfed populations. In order to test this idea, we compared the dynamics of the VDE HEG in six replicate outcrossed and inbred populations of yeast (Saccharomyces cerevisiae). VDE increased in frequency from 0.21 to 0.55 in four outcrossed generations, but showed no change in frequency in the inbred populations. The absence of change in the inbred populations indicates that any effect of VDE on mitotic replication rates is less than 1%. The data from the outcrossed populations best fit a model in which 82% of individuals are derived from outcrossing and VDE is inherited by 74% of the meiotic products from heterozygotes (as compared with 50% for Mendelian genes). These results empirically demonstrate how a host mating system plays a key role in determining the population dynamics of a selfish gene.     

Evolution of <Emphasis Type="Italic">Pleopsidium</Emphasis> (Lichenized Ascomycota) S943 Group I Introns and the Phylogeography of an Intron-Encoded Putative Homing Endonuclease

The sporadic distribution of nuclear group I introns among different fungal lineages can be explained by vertical inheritance of the introns followed by successive losses, or horizontal transfers from one lineage to another through intron homing or reverse splicing. Homing is mediated by an intron-encoded homing endonuclease (HE) and recent studies suggest that the introns and their associated HE gene (HEG) follow a recurrent cyclical model of invasion, degeneration, loss, and reinvasion. The purpose of this study was to compare this model to the evolution of HEGs found in the group I intron at position S943 of the nuclear ribosomal DNA of the lichen-forming fungus Pleopsidium. Forty-eight S943 introns were found in the 64 Pleopsidium samples from a worldwide screen, 22 of which contained a full-length HEG that encodes a putative 256-amino acid HE, and 2 contained HE pseudogenes. The HEGs are divided into two closely related types (as are the introns that encode them) that differ by 22.6% in their nucleotide sequences. The evolution of the Pleopsidium intron-HEG element shows strong evidence for a cyclical model of evolution. The intron was likely acquired twice in the genus and then transmitted via two or three interspecific horizontal transfers. Close geographical proximity plays an important role in intron-HEG horizontal transfer because most of these mobile elements were found in Europe. Once acquired in a lineage, the intron-HEG element was also vertically transmitted, and occasionally degenerated or was lost. [Reviewing Editor: Dr. Manyuan Long]     

Adaptation for horizontal transfer in a homing endonuclease

Selfish genes of no function other than self-propagation are susceptible to degeneration if they become fixed in a population, and regular transfer to new species may be the only means for their long-term persistence. To test this idea we surveyed 24 species of yeast for VDE, a nuclear, intein-associated homing endonuclease gene (HEG) originally discovered in Saccharomyces cerevisiae. Phylogenetic analyses show that horizontal transmission has been a regular occurrence in its evolutionary history. Moreover, VDE appears to be specifically adapted for horizontal transmission. Its 31-bp recognition sequence is an unusually well-conserved region in an unusually well-conserved gene. In addition, the nine nucleotide sites most critical for homing are also unusually well conserved. Such adaptation for horizontal transmission presumably arose as a consequence of selection, both among HEGs at different locations in the genome and among variants at the same location. The frequency of horizontal transmission must therefore be a key feature constraining the distribution and abundance of these genes.     

Sexual mating of Botrytis cinerea illustrates PRP8 intein HEG activity

Strains of Botrytis cinerea are polymorphic for the presence of an intein in the Prp8 gene (intein +/?). The intein encodes a homing endonuclease (HEG). During meiosis in an intein +/? heterozygote, the homing endonuclease initiates intein ‘homing’ by inducing gene conversion. In such meioses, the homing endonuclease triggers gene conversion of the intein together with its flanking sequences into the empty allele. The efficiency of gene conversion of the intein was found to be 100%. The extent of flanking sequence affected by the gene conversion varied in different meioses. A survey of the inteins and flanking sequences of a group B. cinerea isolates indicates that there are two distinct variants of the intein both of which have active HEGs. The survey also suggests that the intein has been actively homing during the evolution of the species and that the PRP8 intein may have entered the species by horizontal transfer.     

A parasitic selfish gene that affects host promiscuity

Selfish genes demonstrate transmission bias and invade sexual populations despite conferring no benefit to their hosts. While the molecular genetics and evolutionary dynamics of selfish genes are reasonably well characterized, their effects on hosts are not. Homing endonuclease genes (HEGs) are one well-studied family of selfish genes that are assumed to be benign. However, we show that carrying HEGs is costly for Saccharomyces cerevisiae, demonstrating that these genetic elements are not necessarily benign but maybe parasitic. We estimate a selective load of approximately 1–2% in ‘natural’ niches. The second aspect we examine is the ability of HEGs to affect hosts'' sexual behaviour. As all selfish genes critically rely on sex for spread, then any selfish gene correlated with increased host sexuality will enjoy a transmission advantage. While classic parasites are known to manipulate host behaviour, we are not aware of any evidence showing a selfish gene is capable of affecting host promiscuity. The data presented here show a selfish element may increase the propensity of its eukaryote host to undergo sex and along with increased rates of non-Mendelian inheritance, this may counterbalance mitotic selective load and promote spread. Demonstration that selfish genes are correlated with increased promiscuity in eukaryotes connects with ideas suggesting that selfish genes promoted the evolution of sex initially.     

Evolution of divergent DNA recognition specificities in VDE homing endonucleases from two yeast species

Homing endonuclease genes (HEGs) are mobile DNA elements that are thought to confer no benefit to their host. They encode site-specific DNA endonucleases that perpetuate the element within a species population by homing and disseminate it between species by horizontal transfer. Several yeast species contain the VMA1 HEG that encodes the intein-associated VMA1-derived endonuclease (VDE). The evolutionary state of VDEs from 12 species was assessed by assaying their endonuclease activities. Only two enzymes are active, PI-ZbaI from Zygosaccharomyces bailii and PI-ScaI from Saccharomyces cariocanus. PI-ZbaI cleaves the Z.bailii recognition sequence significantly faster than the Saccharomyces cerevisiae site, which differs at six nucleotide positions. A mutational analysis indicates that PI-ZbaI cleaves the S.cerevisiae substrate poorly due to the absence of a contact that is analogous to one made in PI-SceI between Gln-55 and nucleotides +9/+10. PI-ZbaI cleaves the Z.bailii substrate primarily due to a single base-pair substitution (A/T₊₅ → T/A₊₅). Structural modeling of the PI-ZbaI/DNA complex suggests that Arg-331, which is absent in PI-SceI, contacts T/A₊₅, and the reduced activity observed in a PI-ZbaI R331A mutant provides evidence for this interaction. These data illustrate that homing endonucleases evolve altered specificity as they adapt to recognize alternative target sites.     

Insect population control by homing endonuclease-based gene drive: an evaluation in Drosophila melanogaster

Insects play a major role as vectors of human disease as well as causing significant agricultural losses. Harnessing the activity of customized homing endonuclease genes (HEGs) has been proposed as a method for spreading deleterious mutations through populations with a view to controlling disease vectors. Here, we demonstrate the feasibility of this method in Drosophila melanogaster, utilizing the well-characterized HEG, I-SceI. In particular, we show that high rates of homing can be achieved within spermatogonia and in the female germline. We show that homed constructs continue to exhibit HEG activity in the subsequent generation and that the ectopic homing events required for initiating the strategy occur at an acceptable rate. We conclude that the requirements for successful deployment of a HEG-based gene drive strategy can be satisfied in a model dipteran and that there is a reasonable prospect of the method working in other dipterans. In characterizing the system we measured repair outcomes at the spermatogonial, spermatocyte, and spermatid stages of spermatogenesis. We show that homologous recombination is restricted to spermatogonia and that it immediately ceases when they become primary spermatocytes, indicating that the choice of DNA repair pathway in the Drosophila testis can switch abruptly during differentiation.     

Optimising Homing Endonuclease Gene Drive Performance in a Semi-Refractory Species: The Drosophila melanogaster Experience

Homing endonuclease gene (HEG) drive is a promising insect population control technique that employs meganucleases to impair the fitness of pest populations. Our previous studies showed that HEG drive was more difficult to achieve in Drosophila melanogaster than Anopheles gambiae and we therefore investigated ways of improving homing performance in Drosophila. We show that homing in Drosophila responds to increased expression of HEGs specifically during the spermatogonia stage and this could be achieved through improved construct design. We found that 3′-UTR choice was important to maximise expression levels, with HEG activity increasing as we employed Hsp70, SV40, vasa and βTub56D derived UTRs. We also searched for spermatogonium-specific promoters and found that the Rcd-1r promoter was able to drive specific expression at this stage. Since Rcd-1 is a regulator of differentiation in other species, it suggests that Rcd-1r may serve a similar role during spermatogonial differentiation in Drosophila. Contrary to expectations, a fragment containing the entire region between the TBPH gene and the bgcn translational start drove strong HEG expression only during late spermatogenesis rather than in the germline stem cells and spermatogonia as expected. We also observed that the fraction of targets undergoing homing was temperature-sensitive, falling nearly four-fold when the temperature was lowered to 18°C. Taken together, this study demonstrates how a few simple measures can lead to substantial improvements in the HEG-based gene drive strategy and reinforce the idea that the HEG approach may be widely applicable to a variety of insect control programs.     

Distribution, sequence homology, and homing of group I introns among T-even-like bacteriophages: evidence for recent transfer of old introns

Self-splicing group I introns are being found in an increasing number of bacteriophages. Most introns contain an open reading frame coding for a homing endo-nuclease that confers mobility to both the intron and the homing endonuclease gene (HEG). The frequent occurrence of intron/HEG has raised questions whether group I introns are spread via horizontal transfer between phage populations. We have determined complete sequences for the known group I introns among T-even-like bacteriophages together with sequences of the intron-containing genes td, nrdB, and nrdD from phages with and without introns. A previously uncharacterized phage isolate, U5, is shown to contain all three introns, the only phage besides T4 found with a "full set" of these introns. Sequence analysis of td and nrdB genes from intron-containing and intronless phages provides evidence that recent horizontal transmission of introns has occurred among the phages. The fact that several of the HEGs have suffered deletions rendering them non-functional implies that the homing endonucleases are of no selective advantage to the phage and are rapidly degenerating and probably dependent upon frequent horizontal transmissions for maintenance within the phage populations. Several of the introns can home to closely related intronless phages during mixed infections. However, the efficiency of homing varies and is dependent on homology in regions flanking the intron insertion site. The occurrence of optional genes flanking the respective intron-containing gene can strongly affect the efficiency of homing. These findings give further insight into the mechanisms of propagation and evolution of group I introns among the T-even-like bacteriophages.     

Comparative multivariate analysis of codon and amino acid usage in three Leishmania genomes

Multivariate analysis of codon and amino acid usage was performed for three Leishmania species, including L. donovani, L. infantum and L. major. It was revealed that all three species are under mutational bias and translational selection. Lower GC 12 and higher GC 3S in all three parasites suggests that the ancestral highly expressed genes (HEGs), compared to lowly expressed genes (LEGs), might have been rich in AT-content. This also suggests that there must have been a faster rate of evolution under GC-bias in LEGs. It was observed from the estimation of synonymous/non-synonymous substitutions in HEGs that the HEG dataset of L. donovani is much closer to L. major evolutionarily. This is also supported by the higher d N value as compared to d S between L. donovani and L. major, suggesting the conservation of synonymous codon positions between these two species and the role of translational selection in shaping the composition of protein-coding genes.     

Homing endonuclease mediated gene targeting in Anopheles gambiae cells and embryos

Homing endonuclease genes (HEGs) are ‘selfish’ genetic elements that combine the capability to selectively disrupt specific gene sequences with the ability to rapidly spread from a few individuals to an entire population through homologous recombination repair events. Because of these properties, HEGs are regarded as promising candidates to transfer genetic modifications from engineered laboratory mosquitoes to wild-type populations including Anopheles gambiae the vector of human malaria. Here we show that I-SceI and I-PpoI homing endonucleases cleave their recognition sites with high efficiency in A. gambiae cells and embryos and we demonstrate HEG-induced homologous and non-homologous repair events in a variety of functional assays. We also propose a gene drive system for mosquitoes that is based on our finding that I-PpoI cuts genomic rDNA located on the X chromosome in A. gambiae, which could be used to selectively incapacitate X-carrying spermatozoa thereby imposing a severe male-biased sex ratio.     

Intermolecular interaction between a branching ribozyme and associated homing endonuclease mRNA

RNA tertiary interactions involving docking of GNRA (N; any base; R; purine) hairpin loops into helical stem structures on other regions of the same RNA are one of the most common RNA tertiary interactions. In this study, we investigated a tertiary association between a GAAA hairpin tetraloop in a small branching ribozyme (DiGIR1) and a receptor motif (HEG P1 motif) present in a hairpin structure on a separate mRNA molecule. DiGIR1 generates a 2', 5' lariat cap at the 5' end of its downstream homing endonuclease mRNA by catalysing a self-cleavage branching reaction at an internal processing site. Upon release, the 5' end of the mRNA forms a distinct hairpin structure termed HEG P1. Our biochemical data, in concert with molecular 3D modelling, provide experimental support for an intermolecular tetraloop receptor interaction between the L9 GAAA in DiGIR1 and a GNRA tetraloop receptor-like motif (UCUAAG-CAAGA) found within the HEG P1. The biological role of this interaction appears to be linked to the homing endonuclease expression by promoting post-cleavage release of the lariat capped mRNA. These findings add to our understanding of how protein-coding genes embedded in nuclear ribosomal DNA are expressed in eukaryotes and controlled by ribozymes.     

PRP8 inteins in species of the genus Botrytis and other ascomycetes

The mobile elements termed inteins have a sporadic distribution in microorganisms. It is unclear how these elements are maintained. Inteins are intervening protein sequences that autocatalytically excise themselves from a precursor. Excision is a post-translational process referred to as 'protein splicing' in which the sequences flanking the intein are ligated, reforming the mature host protein. Some inteins contain a homing endonuclease domain (HEG) that is proposed to facilitate propagation of the intein element within a gene pool. We have previously demonstrated that the HEG of the PRP8 intein is highly active during meiosis in Botrytis cinerea. Here we analysed the Prp8 gene status in 21 additional Botrytis species to obtain insight into the mode of intein inheritance within the Botrytis lineage. Of the 21 species, 15 contained a PRP8 intein whereas six did not. The analysis was extended to closely related (Sclerotiniaceae) and distantly related (Ascomycota) taxa, focussing on evolutionary diversification of the PRP8 intein, including their possible acquisition by horizontal transfer and loss by deletion. Evidence was obtained for the occurrence of genetic footprints of previous intein occupation. There is no compelling evidence of horizontal transfer among species. Three distinct states of the Prp8 allele were identified, distributed over different orders within the Ascomycota: an occupied allele; an empty allele that was never occupied; an empty allele that was presumably previously occupied, from which the intein was precisely deleted. The presence of the genetic footprint identifies 20 species (including Neurospora crassa, Magnaporthe oryzae and Fusarium oxysporum) that previously contained the intein but have lost it entirely, while only 18 species (including Podospora anserina and Fusarium graminearum) appear never to have contained a PRP8 intein. The analysis indicates that inteins may be maintained in an equilibrium state.