* Department of Biological Sciences, Biological Computation and Visualization Center, Louisiana State University
Department of Human Genetics, University of Utah Health Sciences Center
Correspondence: E-mail: mbatzer{at}lsu.edu.
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Abstract |
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Key Words: mobile elements parallel insertions homoplasy free
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Introduction |
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L1 elements have had significant effects upon the overall architecture of the human genome, such as expanding the size of the genome (Salem et al. 2003), altering gene expression, disrupting coding sequences and splice sites, and providing areas of sequence identity for both gene conversion and recombination events (Kazazian et al. 1988; Yang et al. 1998; Rothbarth et al. 2001). In addition, L1 elements have demonstrated an ability to shuffle regions of the genome by a process termed three-prime transduction (Boeke and Pickeral 1999; Moran, DeBerardinis, and Kazazian 1999; Goodier, Ostertag, and Kazazian 2000; Kazazian and Cotton 2001). L1 elements have also been implicated in DNA repair processes and in overall genomic stability, as they have demonstrated an ability to repair double-strand breaks in DNA by an endonuclease-independent insertion mechanism (Morrish et al. 2002). The TPRT-based mobilization and subsequent insertion of L1 elements have also been shown to generate genomic variation through the creation of deletions and duplications (Gilbert, Lutz-Prigge, and Moran 2002).
A limited number of L1 elements have been active during primate evolution and are believed to have amplified according to a model known as the "master gene model" (Deininger et al. 1992). According to this model, L1 elements were created from a series of "source genes" that gradually accumulated diagnostic base changes. These changes and subsequent amplification from source genes have resulted in a series of L1 sequences that contain shared sequence variants that define them as a subfamily. In addition, because of the time frame over which these changes occur within source genes, individual L1 subfamilies appear to be of different genetic ages (Deininger et al. 1992; Batzer, Schmid, and Deininger 1993; Deininger 1993; Smit et al. 1995). Due to their mode of amplification, L1 elements have created interspecies as well as intraspecies genetic differences. L1 insertions have properties similar to other mobile element insertions because they are known to be relatively stable upon insertion, have known ancestral states, and are thought to be identical by descent (IBD) characters for the study of population genetics and phylogenetic relationships (Sheen et al. 2000; Myers et al. 2002; Ovchinnikov, Rubin, and Swergold 2002; Roy-Engel et al. 2002; Mathews et al. 2003; Salem et al. 2003).
The most recently integrated subfamilies of human L1 elements were identified as a result of their sequence identity to known retrotransposition competent elements and disease-causing de novo inserts (Kazazian et al. 1988; Woods-Samuels et al. 1989; Schwahn et al. 1998; Meischl et al. 2000). We have previously completed a detailed study of the youngest known subfamilies of L1 elements, termed Ta (Transcribed, subset a) (Myers et al. 2002) and preTa (Salem et al. 2003). Collectively these L1 subfamilies are composed of several hundred elements, with many representing insertion polymorphisms in diverse human genomes (Boissinot, Chevret, and Furano 2000; Myers et al. 2002; Salem et al. 2003). Mobile element insertions are thought to be largely homoplasy free characters aside from the rare independent parallel insertion of two mobile elements into identical target sites in multiple genomes (Cantrell et al. 2001; Roy-Engel et al. 2002). However, the homoplasy free nature of mobile element insertion polymorphisms has been questioned (Hillis 1999). Here, we report an examination of 254 orthologous L1 Ta insertion sites in nonhuman primates to directly determine the levels of gene conversion and insertion site homoplasy associated with LINE elements.
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Materials and Methods |
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PCR Design and Analysis
PCR primer design and analysis were performed as previously described (Myers et al. 2002). A complete list of L1 Ta loci examined in this study, along with primer sequences, and annealing temperatures can be found as online Supplementary Material at the journal's Web site and at http://batzerlab.lsu.edu.
PCR amplification of the L1HS337 locus was accomplished using the Roche Expand Long Template PCR. These consisted of 50 µl reactions prepared according to the manufacturer's instructions, including 200 to 500 ng of template DNA and 0.3 µM of each primer as listed previously (Myers et al. 2002). The reaction cycle consisted of an initial denaturation period at 94°C for 120 s, 30 cycles of 10 s denaturation at 94°C, 30 s annealing at 60°C, and 180 s elongation at 68°C, followed by 10 min final extension at 68°C. For analysis, 10 µl of each sample and 10 µl of RediLoad (Research Genetics) were fractionated on a 1% agarose gel with 0.05 µg/ml ethidium bromide. This method was also used in an attempt to amplify preintegration sites for the following elements: L1HS327, L1HS405, L1HS469, L1HS498, and L1HS564.
Cloning and Sequence Analysis
Gel purified L1-related PCR products were cloned using Invitogen's TOPO TA Cloning Kit according to the manufacturer's instructions. DNA sequence analysis of the cloned PCR products was accomplished by the chain-termination method using an automated ABI Prism 3100 sequencer (Sanger, Nicklen, and Coulson 1977). One hundred thirteen DNA sequences were assigned GenBank accession numbers AY246432 to AY246544. The sequences AF461364 to AF461389 from previous studies were also used in this analysis (Morrish et al. 2002). Sequence alignments were generated using MegAlign software (DNAStar version 5.0) and can be found as online Supplementary Material and at http://batzerlab.lsu.edu.
Computational Analysis
L1HS Ta loci that experienced parallel insertion events in other nonhuman primate genomes (L1HS45, L1HS363, L1HS413, L1HS480, L1HS521, and L1HS561) were analyzed and annotated for sequence composition and proximity to coding sequence using BLAT searches (UCSC Genome Bioinformatics) of the human genome "November 2002 assembly of sequence," using the default parameters. In addition, 25,000 bp of sequence flanking the L1 Ta insertions were also analyzed for GC content using EditSeq software (DNAStar version 5.0).
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Results |
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The preintegration sites for five elements (L1HS327, L1HS405, L1HS469, L1HS498, and L1HS564) did not amplify in any nonhuman primate species. Previously, the insertion of L1 elements has been shown to be associated with large genomic deletions (Gilbert, Lutz-Prigge, and Moran 2002). Thus, one possible explanation for the absence of preintegration PCR products would be that a large deletion (>1 kb) occurred at each of these loci during L1 integration. If a deletion occurred during the integration of the L1 elements in the human genome, then the preintegration product sizes calculated computationally would be underestimates of the true size of each locus. To investigate this possibility, we utilized long template PCR reactions of these loci that would facilitate the amplification of larger (up to 25 kb) products. Unfortunately, PCR amplicons were not generated by any of these loci, suggesting that the retrotransposition of these L1 elements in humans may have generated deletions greater than 25 kb in size. Alternately, the orthologous loci in nonhuman primate genomes may have undergone sequence changes at the primer sites, preventing PCR amplification.
We have also isolated five smaller potential L1-mediated deletion events in the human genome, including L1HS337 (> 300 bp), L1HS178 (3 bp), L1HS242 (8 bp), L1HS443 (1 bp), and L1HS513 (1 bp). The two definitive examples of very small deletions (L1HS178 and L1HS242) resulting from the L1 insertion event identified in this study were discovered through sequence analysis that was performed due to another suspected variation within the locus in nonhuman primates and/or in humans possessing an empty site. There are likely many more instances of 1-bp to 10-bp deletions that result in shifts between observed and expected preintegration product sizes that are too small to detect upon gel electrophoresis and UV visualization.
The L1HS337 locus (GenBank accession numbers AY246490 to AY246494) has a deletion of approximately 375 bp that occurred when the Ta L1 element integrated at this locus (fig. 1A and B). PCR analysis of the locus in humans and nonhuman primates generated a preintegration site amplicon in humans and in nonhuman primates that was about 375 bp larger than the predicted product size. In addition, the endonuclease cleavage site for this locus as derived from the GenBank sequence appeared to be an atypical TTTA/T, which may also be indicative of an L1 locus involved in a genomic deletion. Sequence analysis confirmed the deletion of 372 bp of unique, nonrepetitive sequence that is found at the integration site in all nonhuman primates as well as in the empty human allele.
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The L1HS480 locus (GenBank accession numbers AY246523 to AY246528) has been particularly receptive to multiple independent mobile element insertions over the course of primate evolution (figs. 3, 4A and 4B). A PCR assay was performed on an extensive nonhuman primate panel incorporating 14 different species to more accurately delineate the mode and tempo of these insertion events. The results of the PCR analysis indicate the presence of an Alu S/Y mosaic element exclusively in the owl monkey genome about 40 bp from the human L1 Ta element integration. In addition, the Old World monkeys (green monkey, pig-tailed macaque, and rhesus macaque) share an independent Alu Y insertion that integrated 16 bp from the L1 Ta target site. These two independent Alu insertions as well as the L1 Ta insertion in the human lineage are distinct, and they are easily distinguished from one another by their subfamily-specific mutations and unique target site duplications. This region on chromosome 4q may be particularly susceptible to mobile element insertions as a result of some undefined unique chromatin structure. However, detailed sequence analysis of 25 kb of flanking sequence on either side of this locus did not yield any coding sequences or any other distinguishing sequence features that would suggest that this locus provides a hotspot for mobile element insertion. The L1HS480 locus illustrates an extreme example of the potential pitfalls associated with assessing phylogenetic relationships based on a mobile element locus, since the two independent Alu insertions would not be distinguishable based upon size. The Alu-based insertion homoplasy of this locus is only evident after detailed sequence analysis.
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Discussion |
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Despite convincing examples of these retroelements' utility in deciphering evolutionary relationships with a complete lack of homoplasy, incidences of multiple independent insertions being found at identical or nearly identical sites in different species do exist. For example, two independent mys LTR-containing retrotransposon insertions have been identified at the same locus in rodent species (Cantrell et al. 2001). This phenomenon of parallel independent insertions has also resulted in the existence of an element belonging to the B1 family of SINEs at the same insertion site in two different rodent species (Kass, Raynor, and Williams 2000). Three additional parallel independent Alu insertions have occurred at orthologous loci in the human and owl monkey genomes (Roy-Engel et al. 2002). Thus the homoplasy free nature of mobile elements has come into question (Hillis 1999).
It may be postulated that these examples of parallel independent retroelement insertions at orthologous loci combined with the instances reported here indicate that hotspots for mobile element insertion may exist at particular loci in related species. In order to explore this possibility, it was necessary to calculate whether the number of parallel insertion events found at L1 Ta integration sites differed significantly from the number of parallel insertion events that would be expected to occur by chance. Considering an average target site sequence length of 175 (225 minus an average of 50 bp consisting of the flanking primer binding sequences), a total of 233,800 bp of potential target site DNA was successfully screened in seven nonhuman primate species, including the common chimpanzee, pygmy chimpanzee, gorilla, orangutan, green monkey, owl monkey, and galago. Assuming a total genome size of approximately 3 x 109 bp and an average target site of 175 bp, we would expect about 17 million target sites per primate genome if we also assume that mobile element integration is entirely random. Combining the estimated L1 copy number of about 516,000 and estimated Alu copy number of 1.09 million in the human genome, we arrive at 1.606 million total mobile element insertions distributed among 17 million target sites (International Human Genome Sequencing Consortium 2001). The probability of finding either an L1 or Alu element within any randomly chosen 175 bp target site within the human genome would be about 9.45% (1.606/17). Using this figure, we can predict that two independent insertions would share the same target site at orthologous loci of primate genomes of comparable size and repeat copy number approximately 0.89% (0.09452) of the time due to chance alone. This figure is in good agreement with the average percentage of parallel Alu insertion events at L1 Ta element integration loci over seven nonhuman primate species of 1.04%, a figure calculated after discarding the potentially paralogous green monkey locus (L1HS521). Therefore, it appears that the observed frequency of independent parallel mobile element insertions at the orthologous loci of L1 Ta elements is not substantially greater than what would be expected to occur by chance alone.
The observation of three independent insertion events occurring at the L1HS480 locus calls into question the randomness of independent parallel insertion events, since the likelihood of this happening by chance alone is extremely low (0.084%). Perhaps this locus falls within an area prone to genomic instability related to retrotransposition events due to chromatin configuration, allowing the retrotransposition machinery comparatively easy access to this sequence region. However, the overall level of mobile element insertion site homoplasy will vary both as a result of the relative rates of retrotransposition in different genomes and the length of time since the divergence of species. Thus the longer the evolutionary time frame involved, the greater the opportunity for insertion site homoplasy.
It is also important to note that none of the cases of insertion homoplasy involved multiple L1 insertions that occurred in parallel in different primate genomes. The reason for this difference in insertion site homoplasy between Alu and L1 elements is unclear. However, it may be related to genome structural constraints imposed on L1 insertion events as a result of size differences between Alu and L1 elements. Alternatively, it may be the result of a reduced rate of amplification of L1 elements in some of the nonhuman primate genomes in which Alu elements appear to be currently undergoing rapid retrotransposition (e.g., owl monkey). In addition, the likelihood for precise insertion site homoplasy with L1 elements is lower since this type of event would involve two independent L1 insertions at the same genomic location along with two variable truncations of newly integrated L1 elements to about the same length (a reasonably rare event).
The other identified sources of variability in the L1 Ta element preintegration sites in nonhuman primates can be attributed to common mutation processes that generate sequence diversity. A single mutational event during primate evolution, resulting from the process of replication, recombination, gene conversion, or repair, is sufficient to account for the sequence variation observed at 12 L1 Ta loci. Similarly, three loci differ among nonhuman primates due to variable microsatellite sequence lengths caused by slippage during the replication process. These minor variants spanning less than 100 bp account for 50% (15/30) of the sequence variation observed at the L1 Ta preintegration site loci.
The three definitive examples of deletions caused by the L1 insertions represent 8.11% (3/37) of the 30 insertion sites sequenced in this study in combination with seven additional insertion sites sequenced previously (Morrish et al. 2002). If we include the two putative single-base deletion events, the estimated percentage of L1 insertions that cause target site deletions during retrotransposition jumps to 13.5%. Although lower than expected, these figures are in relatively good agreement with previous estimates of 8/37 (21.62%) of retrotransposition events in cultured human cells causing deletions at the target site, considering that some deletion events may become obscured or eliminated at the sequence level over time via negative selection, mutation, recombination, or repair (Gilbert, Lutz-Prigge, and Moran 2002).
Five of the 30 variable preintegration loci analyzed (16.67%) consisted of successive mobile element insertion events that were inherited throughout the primate lineage as an integral part of the preintegration site into which the L1 Ta element subsequently inserted. The sequences of these orthologous loci allow us to trace the succession of mobile element integrations at a single locus throughout primate evolution. These clustered insertions appear to support the notion that regions of previously existing mobile elements such as Alu poly (A) tails and L1 A/T-rich sequence, can serve as potential target sites for subsequent mobile element integration. The L1HS301 locus provides an interesting examination of the alteration of a genomic locus that can occur from multiple mobile element insertions and sequence deletions within the interior of preexisting mobile elements. Sequence analysis may be necessary to confirm the sequence composition of loci containing adjacent mobile elements, particularly when PCR analysis indicates variability in the sizes of preintegration loci across several distantly related species.
In conclusion, about 12% of L1 Ta element insertion sites show sequence size and content variability at their orthologous loci in nonhuman primate genomes. About half of these variants are minor, involving short sequence tracts of less than 100 bp. Target site deletions occur as a result of approximately 10% of the L1 insertion events in the genome, and these deletions range from a single nucleotide to over 25 kb. Insertion site sequence architecture may also be altered in approximately 17% of variable L1 integration sites over the course of evolution due to the successive accumulation of multiple mobile elements within a small genomic region. Parallel independent insertions of mobile elements into orthologous loci in nonhuman primates are an additional source of sequence variation. The rates of parallel insertions vary predictably according to species and mobile element type due to differential amplification rates over the course of primate evolution. These events can potentially introduce homoplasy into retroelement-based phylogenetic and population genetic data that is otherwise homoplasy free. Fortunately, these events are easily discernible as variably truncated product sizes when L1 elements are involved in the parallel insertion events. Although parallel independent Alu insertions are more difficult to detect without sequence analysis, the overall percentage of parallel mobile element insertion events is relatively low, affecting only seven out of 1,336 or 0.52% of loci examined. These occurrences appear to be random and exceedingly rare, even when considering primate species that are separated by evolutionary time periods on the order of 50 to 60 Myr. In addition, no independent mobile element insertion events of any type have been found at orthologous loci in human and chimpanzee species, reaffirming the utility of L1 and Alu element integrations as essentially homoplasy free characters well suited to the study of population genetics and phylogenetic relationships within closely related species.
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Acknowledgements |
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Footnotes |
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