Department of Ecology and Evolutionary Biology, Princeton University
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Abstract |
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Introduction |
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Although the association between mobile elements and microsatellite sequences has previously been documented, the actual molecular mechanisms underlying this relationship are not well understood. The influence of mobile element activity on the genesis of microsatellite repeats is best known from Alu and other mammalian short interspersed elements (SINEs; Alexander, Rohrer, and Beattie 1995
; Arcot et al. 1995
; Nadir et al. 1996
; Gallagher et al. 1999
). In these mobile elements, it is thought that retrotranscripts undergo 3' polyadenylation prior to their incorporation into the genome. As a result, polyA microsatellites have a strong association with the 3' ends of these SINEs, a feature which may serve to guide their retroposition in the genome (Nadir et al. 1996
). While this mechanism is a convincing explanation for the association between SINE elements and their 3' polyA microsatellites, it cannot be invoked to address the origins of microsatellites that are associated with other regions of retroposons. For instance, Ramsay et al. (1999)
found that microsatellites can also be associated with both 5' and internal regions of some retroposons, which is unexplained by the above mechanism.
In our study, we describe microsatellites that arise at internal regions of the mini-me retroposon. We have identified two loci, or proto-microsatellites, where microsatellites have repeatedly evolved. Because these elements are extremely abundant in the genome, we were able to observe the outcome of numerous microsatellite genesis events, all of which were generated from the same proto-microsatellites. Based on these observations, we characterize two modes of microsatellite genesis that can be applied to the creation of microsatellite loci anywhere in the genome.
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Materials and Methods |
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DNA Isolation, Cloning, and Sequencing
Microsatellite libraries were created for D. dunni and D. nigrodunni by T. Diaz, R. W. O'Neill, and J. Kenney. Details of the microsatellite library construction will be presented in a separate manuscript; therefore, we present only a summary of the procedure here. Plasmid libraries for each species were created using DNA isolated from 200 starved, mixed-sex flies. Genomic DNA was digested using the restriction enzymes Sau3AI and RsaI and then ligated into the pBluescript II KS (+/-) cloning vector (Stratagene). The plasmid libraries were then probed using di-, tri-, and tetranucleotide repeat sequences labeled with [33P]-dATP. We sequenced 85 clones that hybridized strongly with the microsatellite probes along a single strand, either manually with an AmpliCycle sequencing kit (Perkin-Elmer) or using an ABIPRISM 377 automated DNA sequencer (Perkin Elmer) maintained by the Princeton University synthesizing/sequencing facility.
Sequence Analysis and mini-me Isolation
We analyzed individually the sequences of 37 clones containing microsatellites from D. nigrodunni and 38 clones from D. dunni. Using the program BioEdit, version 4.7.4 (Hall 1999
), we manually aligned the flanking regions of cloned microsatellites. Based on the alignment, we identified a short, highly conserved sequence adjacent to numerous microsatellites. Between November 1999 and March 2000, we performed BLAST searches using this short conserved region as a query string against the GenBank database (Altschul et al. 1997
). Our analysis initially focused on three separate sequence regions: intron 1 of the RPII215 gene from the species Drosophila madeirensis, Drosophila subobscura, and Drosophila guanche (accession numbers Y18877, Y18876, and Y18878); the distal breakpoint of chromosomal inversion 2j in Drosophila buzzatii (accession numbers AF162799 and AF162797); and the DINE-1 element of Drosophila melanogaster (accession number U66884).
Sequences from the regions listed above were manually aligned using the highly conserved sequence region as a point of reference. Despite an overall lack of conserved nucleotide identity, numerous features were shared between these sequences. We identified a set of these conserved features (described below; fig. 1 ) and used these to create a composite pattern defining the mini-me class of Dipteran genomic elements.
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To confirm that the sequences isolated in this manner represented a discrete class of genomic elements (and not a random collection of sequences that had arbitrarily converged on our pattern-based definition), we performed a phylogenetic analysis on one section of the mini-me element. This section was approximately 120 bp in length and spanned from the putative 5' terminus of mini-me to the end of the conserved region (see fig. 1
). Sequences were aligned using the program CLUSTAL W, version 1.4 (Thompson, Higgins, and Gibson 1994
), and then rechecked manually. The alignment incorporated 28 copies of mini-me elements, isolated from 14 different species (accession numbers AF182164, D89934, M30316, M89990, U49102, AF098329, AF162799, X12536, L13721, AF012415, AF043638, X55391, Y18876, Y18877, AF043637, U66884, AF025540, X01918, AC003925, X62679, and AC005720). The elements used were chosen to maximize phylogenetic representation broadly across the Dipteran family. An unrooted neighbor-joining tree was constructed using PAUP* (Swofford 1998
). Maximum likelihood was used to calculate genetic distances based on the HKY substitution model (Hasegawa, Kishino, and Yano 1985
), allowing variable substitution rates, with model parameters estimated during the tree search.
Genomic Distribution of mini-me Elements in D. melanogaster
In order to determine the density and distribution of mini-me elements in D. melanogaster, the entire genome (Celera, Drosophila Genome Project, version 1) was searched globally using a mini-me element isolated immediately downstream of the hermaphrodite gene (accession number AF025540). Additionally, chromosome 4 was analyzed separately in order to verify the results obtained in the global genome analysis. For chromosome 4, each positive hit recorded from a local BLAST search was manually verified (in order to check for false positives), and the number of mini-me elements counted multiple times was tallied (in order to quantify overcounting). A BLAST E-value cutoff of 1.0 was used for each of these analyses, and the data were not filtered in any way.
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Results |
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Conserved at both termini of mini-me elements are perfect inverted repeats that range in size from 10 to 20 bp (table 1 ). At the 5' end of the mini-me elements, the inverted repeat is partially duplicated, forming a complementary palindrome with the apparent ability to form a hairpin secondary structure. The 3' inverted repeat, which is located in a slightly preterminal position, cannot form this hairpin structure. Also conserved in mini-me elements is a core 33-bp motif that begins approximately 110 bp from the 5' end of the element. This core region has high sequence identity (approximately 80%) across all copies of the element, indicating a possible functional role in transposition of the elements. From the (GTCY)n microsatellite to the 3' preterminal inverted repeat, sequence identity is totally lost in between-species comparisons of the mini-me elements. Subsequent analysis has shown that even between copies of mini-me elements within a single species, the length and sequence identity in this region can be extremely variable. Despite these variations, the 3' end of the mini-me elements is clearly defined by the presence of the previously mentioned preterminal inverted repeat.
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Overall sequence identity is poorly conserved outside of the 33-bp region. This is evidenced in table 1 , which shows the sequences of the inverted repeats from mini-me elements. Both the length and the sequence of the repeats is variable between taxa, although their presence at the termini of the elements was universal. The region between the 5' inverted repeat and the 33-bp core region could be aligned between mini-me isolates from different species (described below). However, beyond this region, sequence alignments between species became completely arbitrary. Across mini-me elements from all species, we could find no apparent coding regions or RNA polymerase III promoters that might enhance the mobility of these sequences.
Phylogenetic Analysis of mini-me Elements
Because our definition of mini-me elements is based on conserved sequence patterns but not conserved sequence identity, we performed a phylogenetic analysis of the mini-me region between the 5' terminus and the 33-bp core region. This analysis revealed a tight clustering of mini-me elements in complete concordance with the recognized taxonomic grouping of their host species (Throckmorton 1975
; fig. 3
). Mini-me elements from closely related taxa are clearly more similar to each other than to elements from more distantly related taxa. The phylogeny of the mini-me elements reveals clades at several taxonomic levels, including within each major family group, within Drosophila subgenera, and within individual species or closely related species groups. Thus, we believe that mini-me elements have been transmitted vertically between species since very early in the Dipteran radiation. Through this process, the nucleotide sequences of mini-me elements evolved to become nearly unrecognizable between lineages but retained basic sequence features and structural characteristics throughout the radiation.
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Analysis of Microsatellites Generated Within mini-me Elements
Microsatellites are regularly associated with internal regions of mini-me elements. Two sites in particular tend to be composed of microsatellite repeats. The first, the tetranucleotide repeat region which initially signaled to us the presence of mini-me elements, lies immediately downstream of the 33-bp core region (see fig. 1
). Microsatellites that are derived from this locus tend to be based on only two different 4-bp repeat motifs, (GTCT)n and (GTCC)n. While perfect repeats as long as (GTCT)13 have been observed at this locus, longer complex microsatellites (based on mixtures of both 4-bp motifs) are also common. The longest observed allele generated at this locus contains mixed (GTCT)n and (GTCC)n motifs and has expanded to a total size of (GTCY)37, which is among the largest microsatellite alleles ever reported from any Drosophila species. While microsatellites are common at this site, they are not universal among mini-me elements. Of the 47 mini-me elements analyzed, 23 (49%) had at least one perfect tandem repeat and had an average of 5.1 perfectly repeated (GTCT)n or (GTCC)n tandem motifs. The remaining 51% did not contain any tandemly repetitive DNA but did contain a 12-bp sequence composed of only the bases, G, T, and C. The presence or absence of microsatellites at this site, as well as the lengths of microsatellites that did occur, was variable between mini-me copies both within and between separate Dipteran species.
The second microsatellite within mini-me elements produces simpler and shorter (TA)n repeats. Unlike the previous proto-microsatellite locus, tandem repeats were almost always present in this region, which lies immediately upstream of the 33-bp core sequence. In most copies of mini-me elements, including those which evidence suggests have been recently mobile, this region consists of a (TA)4 repeat. However, variability in size at this locus seems quite common, with continuous variation observed between (TA)1 and (TA)6. Again, the length variation at this microsatellite exists between mini-me loci both within and between species.
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Discussion |
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Mini-me elements are unusual in that they do not appear to code for any transposition enzymes such as transposase or reverse transcriptase. Consequently, these elements probably do not contain the necessary genetic machinery to initiate their own transposition. We also have not observed them to excise themselves from the genome once incorporating at a locus, indicating that they are probably a retro-element of some sort. However, they do not contain the usual hallmarks of the SINE family of elements, which are among the most common retro-elements in the eukaryotic genome (Makaowski 1995
). Most notably, mini-me elements lack an internal RNA polymerase III promoter or other type of tRNA-derived sequence. Because mini-me elements are not structurally similar to any other known type of mobile sequences, we classify them as a new group of non-LTR retroposons.
Mini-me Elements Are Highly Abundant in the Host Genome
If D. melanogaster can be considered typical, mini-me elements are extraordinarily abundant in the Drosophila genome. With a copy number of >3,000 and a minimal observed size of 500 bp, these elements represent at least 1.2% of the total euchromatic genome of D. melanogaster. Despite their high copy number, their presence in the genome has not been widely detected. This is true even in highly studied portions of the genome, such as the recently annotated 2.9-Mb Adh region (Ashburner et al. 1999
), in which we detected at least 11 copies of mini-me elements. Although, as stated earlier, individual copies of mini-me elements have previously been isolated from a number of species, including D. buzzatii and D. melanogaster, and also from various species in the D. obscura and D. funebris species groups (Marfany and Gonzàlez-Duarte 1992
; Steinemann and Steinemann 1993
; Hagemann et al. 1998
; Amador and Juan 1999
), their prevalence in the host genome and the fact that these copies all belong to the same class of genomic elements has not previously been reported.
Microsatellite Genesis in mini-me Elements
We observed that two types of microsatellites are regularly associated with mini-me elements, (TA)n and (GTCY)n repeats. These microsatellites are variable in length among copies of mini-me elements both within and between host taxa. Since our data indicate that mini-me elements are more closely related to each other within species than between species, we believe that this microsatellite variation has arisen de novo in each host lineage. Because microsatellite genesis repeatedly occurs at two specific loci within mini-me elements, we consider these areas "proto-microsatellites." While we do not contend that mini-me elements are responsible for all, or even most, Dipteran microsatellites, our observations of the patterns of molecular evolution that characterize mini-me proto-microsatellites have revealed two major modes of microsatellite genesis that may be generalized across the entire eukaryotic genome.
The first mode of microsatellite genesis is illustrated by the (TA)n repeat and is relatively simple. At this locus, microsatellite variation arises due to the addition or subtraction of repeat motifs from preexisting tandemly repeated DNA. In this case, a (TA)4 repeat is well conserved across most mini-me retroposon copies. Because the bases T and A are overrepresented in the genome (each comprising approximately 28%), it is possible that this is simply a chance feature of the mini-me element that has remained in all Dipteran lineages. Alternatively, the conservation of this locus might be explained by its being a necessary sequence feature for mini-me retroposition. Regardless, mini-me copies that become fixed in the genome contain this (TA)4 sequence, and this repeat invariantly accumulates mutations along with the rest of the element (see fig. 4A
). Because this locus is composed of tandemly repetitive DNA, slippage mutations may be common, which would tend to produce variation in the number of repeated motifs. Slippage mutations are the mechanism commonly accepted to cause variation at most microsatellite loci (Levinson and Gutman 1987
). Here, we simply apply this mechanism to tandem repeats that are inserted into the genome through retroposition. Microsatellites produced at this locus tend to be invariant with respect to repeat motif, indicating that slippage mutations begin to accumulate before site substitutions break up the (TA)4 proto-microsatellite.
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These data show that some DNA regions with high cryptic simplicity can be converted directly into microsatellite DNA due to the biased pattern of substitution mutations. Interestingly, the reverse of this process has been postulated by Hancock (1999)
, who hypothesizes that microsatellites can be converted into cryptically simple DNA due to substitution mutations that corrupt long stretches of tandemly repetitive sequence. Taken together with our observations from mini-me elements, it appears that some repetitive DNA regions could undergo a cyclic form of molecular evolution mediated by both slippage and substitution mutations, where cryptically simple DNA is converted into microsatellites and vice versa.
Microsatellite Genesis Within mini-me Elements Is Variable Across Taxa
Despite the conservation of the proto-microsatellite sequences, microsatellites are not equally generated within mini-me elements across taxa. This is especially apparent at the (GTCY)n microsatellite, which has expanded to exceptional lengths in numerous mini-me elements. We were able to isolate a total of five mini-me copies from D. virilis, which had an average length of over 11.4 (GTCY)n repeats at the locus, with a range of 0 to 37 repeats. Our elements from the cardini group showed a similar pattern. The five elements we examined had an average of 11.6 repeats (with a range of 4 to 21). Mini-me elements from D. melanogaster, however, have comparatively shorter repeat regions at this locus. The average length of the 10 mini-me copies we used in the phylogenetic analysis was 2.2 tandem (GTCY)n repeats, with a range of 0 to 3. It has been noted that D. melanogaster has unusually short microsatellite loci relative to other taxa (Schug et al. 1998
; Bachtrog et al. 1999
). Indeed much longer microsatellites have been isolated from even closely related taxa, such as D. virilis (Tautz and Renz 1984b
), although no systematic survey has been conducted across the Drosophila genus. Recent studies have also shown that D. melanogaster has an unusually high rate of DNA loss relative to other species (Petrov and Hartl 1997
; Petrov et al. 2000
). If these findings are peculiar to D. melanogaster and not the entire Drosophila genus, they might explain the variation in microsatellite length observed. In the case of mini-me elements, an increased rate of DNA loss might lead to faster degradation of the proto-microsatellite region (lowering the opportunities for DNA slippage) or remove repetitive DNA once slippage has caused it to accumulate.
Contribution of mini-me Elements to Overall Microsatellite Density
Mini-me elements are surely not the source of all microsatellites in the Dipteran genome. However, because these elements occur at a relatively high density in the genome and do regularly decay into microsatellite repeats, they can have a significant effect on the overall microsatellite density for some repeat types. This is definitely the case for the genomewide density of (GTCY)n microsatellites. We performed a BLAST search to survey the number of perfect (GTCC)6 and (GTCT)
6 repeats among Dipterans. Of the 41 microsatellites identified, we found an approximately 2:1 ratio of those located inside of mini-me elements to those not associated with mini-me elements. Furthermore, we found that these types of microsatellites were two to three times as common in the Dipteran genome as arbitrary tetranucleotide repeats with similar base compositions. Because tetranucleotide repeats of any given base composition are relatively rare among Drosophila (Schug et al. 1998
), and presumably other Dipterans, it is easy to envision how elements as common as mini-mes could have a dramatic effect on the relative frequency of any given repeat motif. This is not the case with (TA)n dinucleotide repeats, which are extremely common in the genome (Schug et al. 1998
). We could detect no significant effect of mini-me elements on the overall distribution or frequency of this type of microsatellite.
General Implications For Microsatellite Genesis in Eukaryotes
Mini-me retroposons can clearly be implicated in the widespread genesis of microsatellite DNA in the Dipteran genome. Presently, we do not have examples from non-Dipteran taxa, but mini-me elements may represent a general mechanism of microsatellite genesis common to all organisms that harbor mobile retro-elements. Any such elements that contain preexisting simple or cryptic repeat regions may have the propensity to decay into microsatellite DNA, as we observe in the Dipteran genome. This mechanism offers a broad explanation for the association between mobile elements and microsatellites (e.g., Ramsay et al. 1999
). Unlike the mechanism linking the 3' terminus of Alu elements with polyA microsatellites (Nadir et al. 1996
), mini-me elements illustrate how microsatellites of any motif can be generated at both internal and terminal regions of retroposons.
The two modes of microsatellite genesis that we observe in mini-me elements are broadly applicable to contexts beyond mobile elements in all other eukaryotes. Preexisting tandem duplications or cryptically simple DNAs that are released from selection pressure (as might occur when retroposons integrate into the genome or when genes are retro-transcribed or duplicated; Messier, Li, and Stewart 1996
) may tend to become microsatellites through the mutational processes that we observe in mini-me elements. We contend that this mechanism may be a very widespread source of microsatellite DNA across all taxa.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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1 Keywords: microsatellite genesis
mobile elements
retroposons
Diptera
Drosophila
2 Address for correspondence and reprints: Jason Wilder, Department of Ecology and Evolutionary Biology, Guyot Hall/Washington Road, Princeton University, Princeton, New Jersey 08544. E-mail: jawilder{at}princeton.edu
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