* Department of Biological Sciences, Biological Computation and Visualization Center, Louisiana State University
Department of Anatomy, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
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: primates deletion insertion polymorphism gene conversion
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Introduction |
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Phylogenetic studies of Alu elements suggest that only a small number of Alu elements termed "master" or source genes are retropositionally competent (Deininger et al. 1992). Over time, the gradual accumulation of new mutations within these "master" or source genes created a hierarchy of Alu subfamilies (Deininger et al. 1992). Diagnostic mutation sites can be used to classify each individual element according to subfamily and to stratify Alu subfamily members based upon age from the oldest (designated J) to intermediate (S) and youngest (Y) (Batzer et al. 1996b). Some young Alu subfamilies have amplified so recently that they are virtually absent from the genomes of nonhuman primates (Batzer and Deininger 2002). As a result of the recent integration of some Alu elements into the human genome, individual humans may be polymorphic for the presence/absence of the "young" Alu elements at particular genomic loci (Batzer and Deininger 1991; Perna et al. 1992; Batzer et al. 1994). Since the likelihood of two Alu elements independently inserting into the same exact location of the genome is extremely small, and because there are no known biological mechanisms for the specific excision of Alu elements from the genome, Alu insertions can be considered identical by descent or homoplasy free characters for the study of human population genetics (Roy-Engel et al. 2002).
SINE insertion site homoplasy may occur across distantly related taxa as a function of evolutionary time and variable retroposition rates within various species and can limit the application of SINEs to deep evolutionary questions (Hillis 1999; Cantrell et al. 2001; Roy-Engel et al. 2002). Fortunately, the application of SINE elements to the study of human population genetics is thought to be homoplasy-free as a result of the short evolutionary time frame involved and the current relatively low rate of Alu retroposition within the human genome (Roy-Engel et al. 2002).
We have previously characterized a large number of recently integrated Alu elements found in the human genome that fall in four distinct lineages, termed Ya, Yb, Yc, and Yd based upon their diagnostic mutations (Carroll et al. 2001; Roy-Engel et al. 2001; Xing et al. 2003). Here, we have analyzed 283 members of two newly identified Alu subfamilies termed Yg6 and Yi6 (Jurka 2000; Jurka et al. 2002). We have identified several elements that have been subjected to gene conversion, some that have been involved in lineage-specific deletions, and several new Alu insertion polymorphisms that will be useful tools for the study of the human population genetics. This large data set allows us to begin to estimate the impact of these evolutionary processes on the architecture of primate genomes.
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Materials and Methods |
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PCR Amplification
PCR amplification of 244 individual Alu Yg6 and Alu Yi6 subfamily members was carried out in 25 µl reactions containing 20 to 100 ng of template DNA, 40 pM of each oligonucleotide primer (shown in tables 1 and 2 of Supplementary Material online), 200 µM dNTPs, in 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl (pH 8.4), and Taq DNA polymerase (1.25 units). Each sample was subjected to the following amplification for 32 cycles: an initial denaturation of 150 s at 94°C, 1 min denaturation at 94°C, 1 min at the annealing temperature (specific for each locus), and extension at 72°C for one min. After the cycles, a final extension was performed at 72°C for 10 min. For analysis, 20 µl of each sample was fractionated on a 2% agarose gel with 0.05 µg/ml ethidium bromide. PCR products were directly visualized using UV fluorescence. Phylogenetic analysis of all the ascertained Alu elements was determined by PCR amplification of nonhuman primate DNA samples. The human genomic diversity associated with each Alu element was determined by the amplification of 20 individuals from each of four populations (African American, Asian, European, and South American).
Sequence Analysis
DNA sequencing was performed on gel-purified PCR products that had been cloned using the TOPO TA cloning vector (Invitrogen) and chain termination sequencing (Sanger, Nicklen, and Coulson 1977) on an Applied Biosystems 3100 automated DNA sequencer. The sequence of the nonhuman primate Yi6AH41, Yg6AH42, Yg6AH79, Yi6AH79, Yi6AH55, Yi6AH121, Yi6AH36, Yi6AH46, Yi6AH87, Yg6AH77, and Yg6AH134 ortholog loci have been assigned GenBank accession numbers AY190763 to AY190817 and AY219790 to AY219800. Sequence alignments for all of the Yg6 and Yi6 subfamily members were performed using MegAlign software (DNAStar version 3.1.7 for Windows 3.2). The ages of the Alu Yg6 and Yi6 subfamilies were calculated as previously described (Batzer et al. 1990; Batzer et al. 1995; Carroll et al. 2001; Roy-Engel et al. 2001). Multiple sequence alignments that contain all of the members of the Yg6 and Yi6 subfamilies can be found on the journal's Web site as Supplementary Material and on our Web site (http://batzerlab.lsu.edu) under publications.
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Results |
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To estimate the average ages for the Yg6 and Yi6 Alu subfamilies, we compared the individual Alu repeats with their respective subfamily consensus sequences and calculated the mutation density (from all the subfamily members) and used a neutral rate of evolution to estimate the average subfamily ages as previously described in detail (Carroll et al. 2001). For this analysis we divided the nucleotide substitutions within the elements in each family into those that occurred at CpG dinucleotides and those that occurred at non-CpG nucleotides. The distinction between types of mutations is made because the CpG dinucleotides mutate at a rate that is about 10 times faster than non-CpG positions (Labuda and Striker 1989; Batzer et al. 1990) as a result of the deamination of 5-methylcytosine (Bird 1980). In addition, all insertions, deletions, and 5' truncations were excluded from our calculations. We also excluded Alu elements that did not have all the subfamily-specific diagnostic mutations from the analysis because these Alu elements are largely products of gene conversion events involving older preexisting Alu elements and their inclusion in the analysis would artificially inflate the subfamily age estimates. A total of 130 non-CpG and 161 CpG mutations occurred within the 134 Alu Yg6 subfamily members used in this analysis. For the 96 Alu Yi6 subfamily members analyzed, a total of 183 non-CpG and 149 CpG mutations were observed. Using a neutral rate of evolution for primate intervening DNA sequences of 0.15% per Myr (Miyamoto, Slightom, and Goodman 1987) and the non-CpG mutation density (number of non-CpG mutations divided by the total number of non-CpG bases in the analyzed sequences) of 0.42 % (130/30,820) within the 134 Yg6 Alu elements yields an estimated age of 2.81 Myr for the Yg6 subfamily members. Using only non-CpG mutations in the 96 AluYi6 sequences yields a mutation density of 0.81% (183/22,656) and age estimate of 5.39 Myr old for the Yi6 subfamily.
We can also estimate the ages of each Alu subfamily using CpG-based mutations. The only difference in the estimate is to multiply the CpG mutation density by a mutation rate that is approximately 10 times the non-CpG rate, as previously described (Labuda and Striker 1989; Batzer et al. 1990). In this case, we calculate an average CpG mutation density for the Yg6 subfamily (161 mutations/ 6,700 total CpG bases) of 2.40% and an average CpG mutation density for the Yi6 subfamily (149 mutations/ 4,416 total CpG bases) of 3.49%. Using a neutral rate of evolution for CpG-based sequences of 1.5%/Myr yields average age estimates of 1.65 and 2.30 Myr old for the Yg6 and Yi6 Alu subfamilies, respectively. If we assume a linear rate of expansion for these Alu subfamilies, then the oldest elements would be approximately two times the average ages with an initial expansion of these Alu subfamilies 3.3 to 4.6 MYA. Thus, both estimates are consistent with the initiation of the expansion of the Yg6 and Yi6 Alu subfamilies that is roughly coincident with the divergence of humans and African apes, which is thought to have occurred 4 to 6 MYA. The average age estimates for mobile elements based upon CpG mutation density are typically more accurate than the non-CpGbased estimates because they are less likely to be influenced by sequencing errors as a result of the smaller number of total bases that are sequenced and utilized to generate the CpG-based age estimates (Roy-Engel et al. 2002).
The Yi6 subfamily gave rise to three new derivative Alu subfamilies termed Yi6.1 (21 members), Yi6.2 (57 members), and Yi6.3 (16 members) that have the six diagnostic mutations of Yi6 subfamily in addition to new subfamily-specific mutations (fig. 1). The estimated age of these newly identified Alu subfamilies are 5.02, 2.56, and 2.26 Myr using the non-CpG mutation density, and 2.39, 2.04, and 1.46 Myr based upon the CpG mutation density, respectively.
One hallmark of the integration of an Alu repeat into the genome is the generation of target site duplications flanking newly integrated elements. Of the 283 elements examined, we were able to identify clear target site duplications for 270 elements. The direct repeats of the individual elements range in size from 9 to 21 nt. These types of direct repeats are fairly typical of recently integrated Alu family members (Batzer et al. 1990; Jurka 1997).
We also predicted the endonuclease cleavage sites for the 270 Alu insertions that had clear target site duplications. A complete list of endonuclease cleavage sites is shown in table 1. All but four of the predicted endonuclease sites matched cleavage sites previously reported (Feng et al. 1996; Jurka 1997; Cost and Boeke 1998). The four previously undefined sites may be attributed to a nonstringent or "relaxed" human endonuclease with less specificity (Kajikawa and Okada 2002). Alternatively, these four Alu insertions may be the products of endonuclease independent insertion as part of double stranded DNA break repair similar to that previously reported for LINE elements (Morrish et al. 2002).
The appearance of 5' truncations within a number of the Alu elements (24 elements, which is about 8.5% of the total) presumably occurred as a result of incomplete reverse transcription or improper integration into the genome rather than by postintegration instability. All of the Yi6 and Yg6 Alu family members analyzed have oligo-dArich tails, except one element Yg6AH116 that has both a 5' and 3' truncation, of 5 to 50 nt in length. The 3' oligo-dArich tails of many of the elements have accumulated random mutations, beginning the process of the formation of simple sequence repeats of varied complexity. The oligo-dArich tails and middle A-rich regions of Alu elements have previously been shown to serve as nuclei for the genesis of simple sequence repeats (Arcot et al. 1995).
Phylogenetic Origin
To determine the phylogenetic time of origin of each Alu subfamily member (Yi6 and Yg6) in the primate lineage, we amplified a series of human and nonhuman primate DNA samples using the polymerase chain reaction (PCR) and the oligonucleotide primers shown in tables 1 and 2 of Supplementary Material online. Most of the 160 Yg6 Alu family members were absent from nonhuman primate genomes. However, four Alu elements (Yg6AH42, Yg6AH77, Yg6AH79, and Yg6AH134) had PCR amplification patterns that were unanticipated (PCR products about the size of an Alu-filled site in the nonhuman primate genomes), suggesting that these elements had retroposed much earlier in primate evolution than we suspected (fig. 2). In the Yi6 subfamily, 123 elements were assayed and only seven loci had larger PCR products in humans and Old World monkeys (Yi6AH41), owl monkey (Yi6AH79), great apes (Yi6AH36, Yi6AH46, and Yi6AH87), or all the nonhuman primates tested (Yi6AH55 and Yi6AH121), suggesting either the selective loss of the Alu repeat in some nonhuman primates, parallel independent insertion of Alu elements in multiple primate genomes, or that the insertion of some of the Alu elements predated the radiation of humans and nonhuman primates. Interestingly, the Alu YiAH36 element was present in the human and chimpanzee genomes and absent from the genomes of gorillas and other more evolutionarily distant primates. The results of the PCR-based phylogenetic analysis of orthologous loci are shown in table 2. Detailed sequence analysis of all of these unusual Alu elements indicated that three types of events had occurred: (1) gene conversions of older preexisting Alu elements by an element belonging to a different Alu subfamily, (2) parallel independent insertion of different Alu elements in very close, but not identical, genomic locations, or (3) Alu-mediated deletions of the human genomic sequence during retroposition (as outlined below).
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Alu RetropositionMediated Genomic Deletions
We have also identified two deletions of part of the human genome associated with an Alu retroposition. These deletions were identified in loci Yg6AH42 and Yg6AH77. In the case of Yg6AH42, the deletion was also associated with a gene conversion and involved 68 bp of the 3' flanking region (fig. 3). For Alu Yg6AH77, the Alu element replaced about 300 bp of the genomic sequence that was identified in the nonhuman primate genomes. Based on our data, we estimate the frequency of Alu retroposition mediated deletions of approximately 0.82% (2/244).
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Human Genomic Diversity
To determine the human genomic variation associated with each of the Yg6 and Yi6 Alu subfamily members, each element was subjected to PCR amplification using a panel of human DNA samples as templates. The panel was composed of 20 individuals of European origin, 20 African Americans, 20 Asians and 20 South Americans for a total of 80 individuals (160 chromosomes). Using this approach, 125 Alu Yg6 and 94 AluYi6 subfamily members were monomorphic for the presence of the Alu element, suggesting that these elements integrated in the genome before the radiation of humans. A total of eight Yg6 and Yi6 Alu family members were inserted in other previously unidentified repeated sequences and were not amenable to PCR analysis as a result of paralogous amplification. An additional 31 elements were located in other repetitive regions of the genome that were identified computationally and discarded from further analysis. The remaining elements were polymorphic for the presence of an Alu repeat within the genomes of the test panel individuals (summarized in table 3). Autosomal loci that were polymorphic for the presence/absence of individual Alu insertions were subsequently classified as high, low, or intermediate frequency insertion polymorphisms (tables 4 and 5) with sex-linked polymorphisms shown in table 6. The unbiased heterozygosity values for these Alu insertion polymorphisms were variable and approached the theoretical maximum of 50% in several cases. This suggests that many of these Alu insertion polymorphisms will make excellent markers for the study of human population genetics. Approximately 10.7% (15/140) of the Yg6 and 9.6 % (10/104) of the Yi6 Alu family members were polymorphic for insertion presence/absence within diverse human genomes. In addition, we identified three X chromosome Alu elements that were polymorphic (table 6).
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Discussion |
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The estimated average ages of 1.65 and 2.30 Myr for the Alu Yg6 and Yi6 subfamilies are consistent with their relatively recent origin in primate genomes. Assuming a linear rate of amplification the oldest members of the Alu Yg6 and Yi6 subfamilies would be twice the average age of each group or 3.3 and 4.6 Myr, respectively. Therefore, the estimated ages for the Alu Yg6 and Alu Yi6 subfamilies are in good agreement with what would be expected for groups of Alu repeats that are largely restricted to the human genome and absent from nonhuman primate genomes, since human and nonhuman primates are thought to have diverged from each other 4 to 6 MYA (Miyamoto, Slightom, and Goodman 1987; Stewart and Disotell 1998).
Several members of the Alu Yg6 and Yi6 subfamilies were polymorphic for insertion presence/absence in diverse human genomes. Alu insertion polymorphisms have proved useful in a number of studies of human population genetics (Perna et al. 1992; Batzer et al. 1994; Hammer 1994; Batzer et al. 1996a; Stoneking et al. 1997; Novick et al. 1998; Comas et al. 2000; Jorde et al. 2000; Bamshad et al. 2001; Nasidze et al. 2001; Watkins et al. 2001; Battilana et al. 2002; Romualdi et al. 2002; Bamshad et al. 2003). Individual Alu insertion polymorphisms are useful tools for the study of human population genetics since the Alu alleles are generally thought to be reliable, homoplasy-free characters (Roy-Engel et al. 2002) with a known ancestral state (Perna et al. 1992; Batzer et al. 1994). In addition, there is no known mechanism for the site-specific deletion of Alu insertions from the genome (Perna et al. 1992; Batzer et al. 1994). Therefore, detailed studies of the human variation associated with the newly identified Alu insertion polymorphisms reported here should prove useful for human population genetics and forensic genomics.
Our data have several implications for Alu insertion and postintegration sequence evolution. First, they support the "master" or limited amplification model (Deininger et al. 1992). This model posits that most Alu copies present in the human genome arose from a few active copies and that different subfamilies were active at different evolutionary periods. Therefore, Alu subfamilies that are active after the radiation of two species should generate new copies at specific loci that are not shared between primate species. In our analysis, only three elements from the AluYi6 subfamily were recovered in pygmy chimpanzee and common chimpanzee genomes, with two of these three elements also present in the gorilla genome. These data are also in good agreement with our age estimates for these Alu subfamilies. The rest of our "PCR positives" from nonhuman primate genomes were either gene conversion events (with or without a deletion), the products of parallel, independent Alu insertions or Alu retoposition associated genomic deletions. Secondly, these data suggest that newly integrated Alu elements are stable integrations within primate genomes and that they are identical by descent. In our study, only two out of 283 loci analyzed contained parallel independent Alu insertions. The rate of parallel Alu insertion events is extremely low when considering the number of loci analyzed and the full length of the evolutionary tree of 6,730 million insert years. Within great apes, we have assayed hundred of sites with a combined total of over 4,730 Myr of site evolution without detecting any parallel Alu insertion events. This represents having sampled across 315 genomic sites analyzed with an average of 15 Myr of evolution per site. Based on this number, if we assume humans diverged from one another as far back as 1 MYA, we would expect to see less than one parallel insertion event per locus in a diverse population of over 4,730 individuals. This estimate is somewhat larger than that published previously (Roy-Engel et al. 2002), however the probability of detecting parallel independent Alu insertions in the human population is still extremely low. Therefore, we conclude that Alu insertion polymorphisms are largely homoplasy-free characters for the study of human evolution.
Gene conversion between Alu repeats has been reported previously (Maeda et al. 1988; Kass, Batzer, and Deininger 1995; Roy-Engel et al. 2002). Here, we have identified and characterized three forward gene conversion events after screening 283 independent Alu-containing loci within the human genome. Based on an examination of low copy number transgenes in the mouse, it has been suggested that the germline recombination machinery in mammals has been evolved to prevent high levels of ectopic recombination between repetitive sequences (Cooper, Schimenti, and Schimenti 1998). It is quite possible that the high copy number of Alu elements allows for pairing between the homologous regions of different Alu elements initiating the start of gene conversion before cellular control systems can terminate the process resulting in the production of small gene conversion tracts.
Genomic deletions created upon LINE-1 retrotransposition using cell culture assays have been recently identified (Gilbert, Lutz-Prigge, and Moran 2002). The rate of LINE element deletion was estimated indirectly in the human genome to be about 3% (Kazazian and Goodier 2002; Myers et al. 2002). However, the precise molecular mechanism of the LINE-mediated genomic deletions is still unclear. Recently, an Alu-mediated deletion that resulted in the inactivation of the human CMP-N-acetylneuraminic acid hydroxylase gene has been identified (Hayakawa et al. 2001). The deletion of the human CMP-N-acetylneuraminic acid hydroxylase gene involved about 478 bp, including a 92-bp exon along with the replacement of an Alu Sq element in nonhuman primates with an AluY element in the human lineage (Hayakawa et al. 2001). Here we report two new examples of Alu retropositionmediated deletions that may have been performed by a mechanism similar to that of the LINE elementmediated genomic deletions since Alu and L1 elements utilize a common mobilization pathway (Boeke 1997; Batzer and Deininger 2002; Kajikawa and Okada 2002).
In the first case, Alu Yg6AH42, the deletion appears to have occurred during the process of gene conversion similar to the lineage-specific Alu deletion reported previously (Hayakawa et al. 2001). In the second case, Alu Yg6AH77, two scenarios for the deletion can be envisioned. In the first scenario, the deletion would have occurred before the Alu insertion as a result of double-stranded DNA break repair since this element has no direct repeats (a hallmark of LINE elementmediated, endonuclease-independent, double-stranded DNA break repair) (Morrish et al. 2002). In the second scenario, the deletion would have occurred during the integration of the Alu element in the genome, possibly during TPRT, suggesting a new role for Alu elements in creating human genomic diversity.
Here, we have estimated the frequency of Alu retroposition associated genomic deletions of approximately 0.82%. New Alu integrations have been estimated to occur in vivo at a frequency of one new event in every 10 to 200 births (Deininger and Batzer 1999). If sizable deletions accompany one in every 100 new Alu retroposition events in vivo, the impact on genomic evolution could be substantial. This is not a trivial number of deletions when extrapolated to the copy number of Alu elements in the human genome, which is over 1,000,000 (Batzer and Deininger 2002). About 8,000 Alu elements may have been involved in retropositionmediated deletion events within primate genomes. If each of these deletion events removes 150 bp of genomic sequence, this would mean that Alu retroposition may have been responsible for the deletion of over 1.2 Mb of the primate genomic sequence. If the Alu-associated deletions have involved larger sequences similar to those recently reported for LINE elements (Gilbert, Lutz-Prigge, and Moran 2002), then the impact of these events may be 12 to 120 Mb of lineage-specific deletions. In either case, these types of events represent a novel mechanism of lineage-specific deletion within the primate order. Detailed studies of the orthologous regions of primate genomes deleted in this manner may prove instructive for understanding the genetic basis of the difference between humans and nonhuman primates.
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Acknowledgements |
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Footnotes |
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