*Departamento de Bioloxía Fundamental, Facultade de Bioloxía, Universidade de Santiago de Compostela, Santiago de Compostela, Spain;
and
Departamento de Bioloxía Celular e Molecular, Facultade de Ciencias, Universidade de A Coruña, A Coruña, Spain
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Abstract |
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Introduction |
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Alu and L1 are the major families of human interspersed repeated DNA, amounting to 10% and 15% of the genome, respectively (Kazazian and Moran 1998
). The evolutionary histories of these two families have been determined by comparative sequence analysis of a number of different inserted copies (Shen, Batzer, and Deininger 1991
; Deininger et al. 1992
; Smit et al. 1995
). The major conclusion of this so-called paleogenomic approach has been that most Alu and L1 elements were produced by only one or a few source genes ("master elements") at any one time during evolution of the family. Accumulation of substitutions in the master elements, or replacement of these masters by others from their own derivatives, can give rise to distinct subfamilies of pseudogene copies defined by shared differences with the consensus of the family. These characteristic (or diagnostic) nucleotide positions can be sequentially ordered, with the youngest subfamilies sharing older differences as well as new diagnostic substitutions. Thus, the evolution of L1 and Alu families has been shown to be driven essentially by a single lineage of master elements ("sequential masters" or "molecular drivers"). The existence of several minor subfamilies besides the main lineage suggests that from time to time, elements distinct from the sequential masters may retain their capability of retrotransposition over a significant period of time.
Another interesting type of repetitive DNA elements consists of retrovirus-like elements (RLEs), or endogenous retroviruses (ERVs), representing about 1% of the human genome (Lowër, Lowër, and Kurth 1996
). Their structure closely resembles that of retroviruses, carrying internal sequences with homology to gag, pol, and, sometimes, env open reading frames (ORFs) flanked by long terminal repeats (LTRs), which contain several transcriptional regulatory sequences. A large number of solitary LTRs that have arisen by homologous recombination between the 5' and the 3' LTRs of full-length elements are scattered throughout the mammalian genomes (Lowër, Lowër, and Kurth 1996
).
RLEs can be regarded as highly specialized intragenomic parasites, with their parasitic capability being the product of natural selection among RLE copies within the genome (Doolittle and Sapienza 1980
; Orgel and Crick 1980
). Evolutionary consequences for the host of carrying these intragenomic parasites have been a matter of debate for years. Apparently, the majority of characterized RLE insertions do not have an adaptative value, so RLEs may be considered insertional mutagens that affect host genes either by direct disruption of their coding sequences or by altering their expression patterns (Leslie, Lee, and Schrader 1991
; Wu et al. 1993
; Mitreiter et al. 1994
). Nevertheless, it has been suggested that the ERV LTRs may be an evolutionary tool for coupling expression of regulatory factors and their receptors (Kapitonov and Jurka 1999
), or they may provide a cellular reservoir of control elements with diverse transcriptional specificities (Keshet, Schiff, and Itin 1991
). Furthermore, regulation of human genes by ERV homologous sequences has been shown or suggested in several cases (Suzuki et al. 1990
; Ting et al. 1992
; Di Cristofano et al. 1995a
; Long et al. 1998
; Kapitonov and Jurka 1999
). Finally, some data suggest that human ERVs may be involved in the prevention of infections with related exogenous retroviruses or act as pathological agents in certain autoimmune disorders (Lowër, Lowër, and Kurth 1996
; Patience, Wilkinson, and Weiss 1997
).
Despite these important biological interactions between ERVs and mammalian genomes, very little is known about evolution of ERV families (Clough et al. 1996
). Although it would seem likely that most copies of an active RLE are able to retrotranspose, a few observations suggest an expansion of mammalian ERVs similar to the master gene model proposed for L1 and Alu families (Lee et al. 1996
; Medstrand and Mager 1998
).
ERV9 elements, first identified by La Mantia et al. (1991)
, constitute one of the families of ERVs mobilized during primate evolution (Di Cristofano et al. 1995b
). This family is represented in the human genome by 3040 members besides at least 4,000 solitary LTRs (La Mantia et al. 1989, 1991
; Lania et al. 1992
). The prototype ERV9 element is about 8 kb long, including the two LTRs of approximately 1.8 kb; their precise size depends on a variable number of two tandemly repeated subelements. One of these subelements is 41 bp long, with about 12 repeats within the U3 region, whereas the other is 72 bp long, with about 4 repeats in the U5 region (La Mantia et al. 1991
; Lania et al. 1992
).
To get a better insight into the evolution of ERV families, we conducted a comparative sequence approach to reconstruct the evolutionary history of the ERV9 family using sequences gathered from GenBank. Our results clearly indicate the existence of several lineages with distinct evolutionary success which retained their capacity of transposition over extended periods of primate evolution.
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Materials and Methods |
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Element subfamilies were established by grouping sequences into different sets according to shared nucleotide deletions and nucleotides observed in the most variable sites of the alignment. Although several of these sites were CpG positions, with little discrimination for subfamilies due to the fast mutation rate of these dinucleotides to TpG or CpA, characteristic differences could be detected occasionally. Further inspection of initial subsets led to the identification of correlated diagnostic positions so that these previous groups could be further split. A nucleotide position was considered diagnostic of a sequence set whenever more than 70% of the sequences grouped into it shared the same nucleotide, which differed from that characterizing at least some other similar groups. Subfamily status was conferred on a sequence set when five or more sequences of the group contained at least three diagnostic positions.
Consensus sequences for each subfamily and for the whole sample ("general" consensus sequence) were obtained by choosing the more frequent nucleotide at each position, with one exception. If a combination of dinucleotides of the three pairs CpG, CpA, and TpG were present at the same doublet position, the CpG dinucleotide was chosen as the consensus dinucleotide unless the T or A nucleotides were present in more than 70% of the sequences. When either of two bases occurred in a site with 50% frequency (ambiguous site), IUPAC-IUB base codes representing multiple bases were used (Cornish-Bowden 1985
).
The accuracy of these consensus sequences was evaluated by comparing observed and expected pairwise divergences between sequences from the same subfamily, considering the consensus sequence the best reconstruction of the source gene (genes) that originated the subfamily. Expected divergences were calculated by the following formula (Smit et al. 1995
):
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To estimate the ages of ERV9 subfamilies, we first calculated the average level of nucleotide substitutions from the consensus (K), excluding CpG dinucleotides and gaps, using Kimura's (1981)
two-parameter model with the usual transition/transversion ratio of 2. This method of estimation of the actual number of nucleotide substitutions, originally derived for two sequences diverged from a common ancestor, is also valid when one of the two sequences is the unchanged source gene (Kapitonov and Jurka 1996
). Assuming 0.16% per million years as the rate of change of pseudogene sequences in primates (Britten 1994
; Kapitonov and Jurka 1996
), the average transposition age of the subfamily (T) has been estimated as T = K/0.0016.
Comparison of the rates of change between CpG and non-CpG positions was done after correction of CpG divergence by dcorr = -ln(1 - dobs), according to Labuda et al. (1991)
. dobs at CpG dinucleotide positions was calculated again as the relative number of mismatches after removing all gaps from the alignment. The programs DNADIST from the PHYLIP package (Felsenstein 1993
) and DnaSP3 (Rozas and Rozas 1999
) were used to calculate some of these values.
The phylogenetic analysis of subfamily consensus sequences using maximum parsimony was performed with the program DNAPARS from the PHYLIP package (Felsenstein 1993
). Since the probability of parallel or convergent evolution is much lower for indels than for point mutations, each indel received a higher weight than point mutations (with one indel being counted as three point mutations, irrespective of their length, as a conservative weight). The bootstrap analysis was performed with 1,000 replications using the programs SEQBOOT, DNAPARS, and CONSENSE from the PHYLIP package (Felsenstein 1993
).
Analysis of mosaicism was performed as described by Robertson, Hahn, and Sharp (1995)
. This method is based on the distribution of phylogenetically informative sites supporting alternative tree topologies among four taxa: the putative mosaic sequence, one representative of each of the two "parental" lineages, and a known outgroup. SimPlot (Ray 1999
) was used to carry out a sliding-window analysis of bootstrap values determined by maximum parsimony.
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Results |
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Although the majority of differences appearing in a master gene are shared by subsequent source genes, some subfamilies show a few private differences (fig. 1 ). This may be indicative that these subfamilies are not the direct ancestors of the younger subfamilies of their lineage.
The subfamily consensus sequence is the best reconstruction of the active sequence that yielded members of the subfamily by retrotransposition. After their insertions, most of these elements are not functionally constrained and accumulate mutations at a neutral rate. Thus, the average divergence of the members of each subfamily from their respective subfamily consensus sequence would reflect the age of the subfamily. This is not true for CpG dinucleotides, whose mutation rate, caused by deamination of the methylated cytosine, is very high (Bird 1980
). Therefore, CpG positions were excluded from the estimation of the average number of nucleotide substitutions. Obviously, these estimates should be considered approximate due to the differences in local mutation rates and the small size of the sequence sample (the average coefficient of variation was 0.28). As it appears in table 2
, the estimated ages of individual subfamilies are in general good agreement with their sequential order within each lineage based on shared ancestry (fig. 2
). A conspicuous exception is subfamily X. Although this subfamily shares five more sequence variants than IX with younger subfamilies of its lineage, it seems to be clearly older than subfamily IX according to the average within-subfamily divergence. This incongruence disappears if sequence AC005332d (2.5 times as divergent as the average) is excluded from the analysis of subfamily X.
Evolution of active elements may be inferred from the comparison between consensus sequences of the oldest and youngest subfamilies (I and XII, respectively). Of the 63 observed nucleotide differences (excluding the two ambiguous sites, 162 and 253, in the consensus sequence of subfamily I), 29 correspond to CpG dinucleotides, which represents 21.6% and 6.3% of CpG and non-CpG sites affected, respectively. After correction of observed divergence for CpG positions, due to its nonlinear substitution rate (see Materials and Methods for details), we obtained a CpG/non-CpG ratio of 3.9. To compare this value with that for pseudogene copies of ERV9, we used subfamily IX, accounting for 55% of the sequences for the lineage. Interestingly, the obtained value (dobs at CpG = 0.19, dcorr = 0.21, CpG/non-CpG = 3.8) is quite similar to that for active elements.
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Discussion |
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The time of insertion of two ERV9 elements included in our study has been analyzed experimentally by different authors. One of them is an element inserted into the ZNF80 locus (GenBank accession number X83497). This insertion has been detected by genomic PCR at the same position in gorillas, chimpanzees, and humans, but it is absent from the orangutan genome (Di Cristofano et al. 1995b
). This result is in good agreement with our classification of this element within subfamily IX, whose estimated age of transposition is 18.7 MYA. The other element (GenBank accession number AF064190) is located in the ß-globin locus of humans and gorillas, the only two primate genomes tested (Long et al. 1998
). This element belongs to subfamily XIV, whose transpositional age was estimated as 15.6 MYA.
The major finding of our study was the existence of several lineages of ERV9 elements simultaneously active over long periods of time. This fact contrasts with previous data on other mammalian RLEs, such as HERV-K and the retrotransposon mys of Peromyscus. Unlike ERV9, the HERV-K and mys subfamilies seem to arise in a sequential order from the same master lineage and expand during different periods of time (Clough et al. 1996
; Lee et al. 1996
; Medstrand and Mager 1998
). Thus, whether the main mode of expansion of RLEs occurs via a master lineage or through several competitive lineages is an open question.
Our results clearly indicate that ERV9 elements have been actively retrotransposing over an extended period of primate evolution. Nevertheless, the propagation of ERV9 was not at all constant through this time. Subfamily IX alone represents 26% of all of the sampled ERV9 sequences, and its period of activity overlaps at least that of subfamily VIII (table 2
); both together account for one third of the total recorded ERV9 insertions. In view of the extended period of ERV9 activity within primate genomes as well as its putative discontinuous transpositional dynamics, it is likely that some ERV9 elements retained its transpositional activity after the split of great apes. Thus, the presence of a functionally transposing subset of ERV9 in the human lineage, as described for HERV-K (Medstrand and Mager 1998
), may be more probable than it is for other types of ERV sequences.
It has been suggested that mosaic evolution (by novel combination of preexisting mutations) may be a likely mechanism during the evolution of repetitive elements, facilitated by the existence of a large pool of genomic copies (Kass, Batzer, and Deininger 1995
; Kido et al. 1995
; Zietkiewicz and Labuda 1996
). Furthermore, RLEs are expected to be especially prone to genetic rearrangements due to the possibility of recombination between two RNA genomes packaged within the same capsid (McDonald 1993
). Our results clearly suggest three cases of mosaic evolution, caused by either recombination or gene conversion. One of them has given rise to lineage D by combination of sequences from lineage B and C (fig. 3
). The relative evolutionary success of this new assembly of sequences is revealed by the two expansions of lineage D during different periods. Another two putative cases of mosaic evolution of ERV9 elements have been detected. One is constituted by the three closely linked characteristic differences (233, 257, and 279) shared by subfamily I and lineage C, and the other is present in subfamily X. This subfamily acquired eight private differences (autopomorphs), including a deletion. Six of these differences are clustered in the interval from position 429 to position 488. Taken together, these findings indicate that mosaic evolution is a likely mechanism in ERV9 evolution. Interestingly, the youngest subfamily of HERV-H LTRs also originated from a rearrangement of preexisting mutations, in this case by recombination between the other two described subfamilies (Mager 1989
).
Finally, it is worth mentioning that the rate of substitutions at CpG dinucleotides during the evolution of active ERV9 sequences is clearly higher than the substitution rate at non-CpG positions. Furthermore, the CpG/non-CpG substitution ratio of source genes is similar to that of pseudogene copies of ERV9. This contrasts with the situation found in the Alu family, whose mutation rate in CpG dinucleotides of source genes is as low as that in non-CpG sites (Deininger and Slagel 1988
; Labuda and Striker 1989
). These data suggest that the active ERV9 sequences for retrotransposition are not protected from cytosine methylation despite the fact that one of the main functions for cytosine methylation within mammalian genomes seems to be suppression of parasitic elements by transcription inhibition (Yoder, Walsh, and Bestor 1997
; but see Bird 1997
for opposite explanations).
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Acknowledgements |
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Footnotes |
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1 Abbreviations: ERV, endogenous retrovirus; LTR, long terminal repeat; RLE, retrovirus-like element.
2 Keywords: ERV9
endogenous retrovirus
mosaic evolution
source gene
master model
3 Address for correspondence and reprints: Javier Costas, Departamento de Bioloxía Fundamental, Facultade de Bioloxía, Universidade de Santiago de Compostela, Campus Sur s/n E-15706 Santiago de Compostela (A Coruña), Spain. E-mail: bfcostas{at}usc.es
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