Loss of Transposase-DNA Interaction May Underlie the Divergence of mariner Family Transposable Elements and the Ability of More than One mariner to Occupy the Same Genome

David J. Lampe, Kimberley K. O. Walden and Hugh M. Robertson

Department of Biological Sciences, Duquesne University
Department of Entomology, University of Illinois


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Mariners are a large family of eukaryotic DNA-mediated transposable elements that move via a cut-and-paste mechanism. Several features of the evolutionary history of mariners are unusual. First, they appear to undergo horizontal transfer commonly between species on an evolutionary timescale. They can do this because they are able to transpose using only their own self-encoded transposase and not host-specific factors. One consequence of this phenomenon is that more than one kind of mariner can be present in the same genome. We hypothesized that two mariners occupying the same genome would not interact. We tested the limits of mariner interactions using an in vitro transposition system, purified mariner transposases, and DNAse I footprinting. Only mariner elements that were very closely related to each other (ca. 84% identity) cross-mobilized, and then inefficiently. Because of the dramatic suppression of transposition between closely related elements, we propose that to isolate elements functionally, only minor changes might be necessary between elements, in both inverted terminal repeat and amino acid sequence. We further propose a mechanism to explain mariner diversification based on this phenomenon.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Mariner elements are short inverted terminal repeat (ITR)–type transposons that compose a large family of related transposons that are widely distributed among metazoans. To date, more than 13 subfamilies of these elements have been described, typically sharing ca. 25% amino acid identity between subfamilies and from 25% to nearly 100% amino acid identity within a subfamily (Robertson 1993Citation ; Robertson and MacLeod 1993Citation ).

The phylogenetic history of mariner family elements is dominated by extensive horizontal transfer between species, some separated by great phylogenetic distances (e.g., Garcia-Fernandez et al. 1995Citation ; Lohe et al. 1995Citation ; Robertson and Lampe 1995Citation ). When species are examined for the presence of mariners, multiple different kinds are commonly found occupying the same genome (fig. 1 ). For example, the human genome contains two distinct mariner family elements. Hsmar1 (Homo sapiens mariner 1) belongs to the cecropia subfamily, while Hsmar2 belongs to the irritans subfamily (Robertson and Martos 1997Citation ; Robertson and Zumpano 1997Citation ) (fig. 1 ). These two elements share about 37% amino acid identity, and each is present in many hundreds of copies. They presumably result from two independent invasions of the human genome lineage. Some species harbor even more kinds of mariners. The nematode Caenorhabditis elegans, for example, contains nine distinct kinds of mariner elements from three different subfamilies.



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Fig. 1.—A phylogenetic tree of mariners based on aligned transposase sequences. This is a phylogenetic tree based on transposase sequences using the data set from Robertson and Asplund (1996)Citation , with exceptions noted below. The alignment was produced using Clustal X (Jeanmougin et al. 1998Citation ). The elements used in this study, highlighted in bold, are Himar1 (Haematobia irritans mariner 1), Mpmar1 (Mantispa pulchella mariner 1), Hsmar2 (Homo sapiens mariner 2), Ammar1 (Apis mellifera mariner 1), Mos1 (Mosaic factor 1, a particular copy of Drosophila mauritiana mariner 1), and Csmar1 (Carpelimus species mariner 1). The sequences used in this tree were derived from several sources. C.capitata.mar1 (Gomulski et al. 1997Citation ) (GenBank accession number U40493) and H.bacteriophora.mar1 (Grenier et al. 1999Citation ) (GenBank accession number U68392) were obtained from GenBank. C.elegans.mar3a, C.elegans.mar4, C.elegans.mar5, C.elegans.mar6, C.elegans.mar8, C.elegans.mar9, C.briggsae.mar1, and C.briggsae.mar2 were created by us as consensus sequences by aligning multiple sequences from the Caenorhabditis elegans or C. briggsae sequencing projects. The alignment for this tree can be found at http://www.home.duq.edu/~lampe/MBE/AAalign.htm. The tree was produced with the program PAUP*, version 4.0b4a (Swofford 2000), using the neighbor-joining feature and the heuristic search option. It was rooted with the representative set of elements from the Tc1 family. Bootstrap support values (1,000 neighbor-joining runs) exceeding 75% are shown above the branches

 
The presence of distinct mariner family elements in the same genome, each with multiple copies, suggests that they do not interact. If interactions occurred, then elements would be subject to each other's regulatory systems, and it is difficult to imagine how a new element could invade a genome already occupied by another mariner. Regardless of the mechanism that leads to the presence of distinct mariners, some interesting mechanistic questions are raised. Can divergent elements interact through sequence-specific DNA binding? If so, can divergent elements cleave DNA at the ITRs? Finally, how much divergence is necessary before elements fail to interact? The latter question is particularly interesting, as it may underlie the diversification of mariners over evolutionary time.

To investigate whether mariner family elements were capable of interacting, we analyzed the ability of purified Himar1 and Mos1 mariner transposases (the only known active mariners) to mobilize Kanr-marked elements representing different mariner subfamilies in an in vitro reaction. These reactions were followed by DNAse I footprinting and DNA cleavage reactions to determine the nature of the interactions, or lack thereof. We conclude that a relatively small amount of divergence in ITR sequence is necessary to substantially reduce the interactions between elements, and we propose two scenarios in which this divergence may drive mariner evolution.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Recombinant DNA
Mini-mariner elements representing irritans, mellifera, cecropia, and mauritiana subfamilies (see fig. 1 ) were constructed by selecting genomic clones that contained ITR sequences closest in DNA sequence to the subfamily consensus. The Himar1 mini-mariner was previously constructed (Lampe, Grant, and Robertson 1998Citation ). Mantispa pulchella4.1 (a genomic clone containing a mariner insertion isolated from the neuropteroid insect Mantispa pulchella) was used to create the most closely related mini-mariner to Himar1 (Robertson and Lampe 1995Citation ). A third representative of the irritans subfamily was constructed from the Hsmar2 group using human cDNA clone 199288 and human cDNA clone 27609 for the 5' and 3' ITR ends, respectively, because they represent the consensus sequence (Robertson and Martos 1997Citation ). Genomic libraries were constructed and screened for the honeybee Apis mellifera and for a beetle, Carpelimus sp. (Staphylinidae), essentially as described (Robertson and Lampe 1995Citation ), using specific probes obtained by PCR (Robertson 1993Citation ; Robertson and MacLeod 1993Citation ). For the mellifera subfamily, genomic clones A.mellifera.2 and A.mellifera.19 were used for the 5' and 3' ends. Genomic clones Staphylinid.18.3 and Staphylinid.18.1 served as the starting material for the cecropia subfamily 5' and 3' ITRs (Csmar1). Mos1 (a gift of D. Hartl) was used as the template for a mauritiana subfamily representative.

Genomic copies of mariners from the four subfamilies were amplified by PCR using primers designed to the ITR regions of each genomic clone. The PCR primers contained an ATA at the 5' end to facilitate TA cloning into a t-tailed pcDNAII vector and to provide a duplicated target site context in which all mariners were found. The appropriate single ITR from each clone was then amplified during PCR using Pfu DNA polymerase (Stratagene) with a pcDNAII vector primer and a custom internal mariner primer. The custom internal mariner primers, designed to the 5' end of the ITR, contained a SmaI site at their 5' ends. PCR products corresponding to the 5'- and 3'-end ITRs of each subfamily of mini-mariner were kinased and ligated together. They were then used in a PCR using Pfu polymerase with vector primers to amplify the ligated ITR ends. The products were digested with XhoI and HindIII and cloned into an XhoI/HindIII cut pcDNAII vector. Individual clones were manually sequenced with pcDNAII vector primers to verify the sequence. The mini-mariner constructs were next digested with SmaI, and a 1.5-kb fragment of pK19 (Schafer et al. 1994Citation ) digested with BspHI and BglII containing a kanamycin-resistance (KanR) gene was cloned into the SmaI site, separating the two ITRs. Finally, a portion of the AmpR gene was removed as described previously, making the clones KanR only (Lampe, Grant, and Robertson 1998Citation ). These are the donor plasmids shown in figure 2 .



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Fig. 2.—The in vitro transposition assay. An in vitro transposition assay was used to test the ability of purified transposases to mobilize a KanR-marked transposon from a donor plasmid into a target plasmid (after Lampe, Churchill, and Robertson 1996Citation ). The donor and target plasmids have the same origin of replication and thus are incompatible in the same bacterial cell. The only way to recover KanR/AmpR in the same cell is through the formation of a transposition product, i.e., a plasmid containing both markers. Donor plasmids were identical to each other except for the sequence of the ITRs (see table 1 ). The target plasmid is a stable tetramer of a pUC plasmid, which ensures that all transposition events can be recovered, including transpositions into an AmpR gene or an origin of replication

 
For footprint and cleavage analysis, single ITR's were amplified by PCR using Pfu polymerase and the subfamily mini-mariner constructs as templates. The vector primer T7 and a custom internal mariner primer, designed with a SalI site at its 5' end, generated products that contained the first 61 bp of each 5'-end ITR. These products were digested with XbaI and SalI and then cloned into similarly cut pKAN19. Individual clones were sequenced for verification.

Transposase Purification
Himar1 transposase was purified as described previously (Lampe, Churchill, and Robertson 1996Citation ). Mos1 mariner transposases were a gift of D. Finnegan and S. Beverley.

In Vitro Transposition Assays
In vitro transposition assays were carried out according to Lampe, Churchill, and Robertson (1996)Citation with the following modifications (fig. 2 ). Donor plasmids from the four different subfamilies were combined in equimolar amounts with the pBSKS+ target plasmid and Himar1 transposase and allowed to react for 2 h at 28°C. The target plasmid was a tetramer of a pUC plasmid, and so all transposition products formed by insertion into it, even those into the AmpR gene or origin of replication, could be recovered. Two concentrations of Himar1 transposase (0.5 nM and 2.5 nM) were tested with each mariner subfamily donor. After reaction cleanup and electroporation into competent cells, 200 µl of the cell culture was plated on LB-kanamycin-ampicillin plates, and 100 µl of a 10-3 dilution was plated on LB-amp plates. Several colonies containing transposition products were miniprepped and analyzed by restriction digestion with BamHI. Mpmar1 (Mantispa pulchella mariner 1) transposition products were further verified by digesting with BamHI and NsiI to discriminate potentially contaminating Himar1 products that contained NsiI. Genuine Mpmar1 transposition products were sequenced manually with Oncor's Fidelity sequencing kit and 35S dATP to determine insertion site sequences. Mos1 in vitro transposition assays were carried out as described by Tosi and Beverley (2000) using 125 nM purified Mos1 transposase (a gift of S. Beverley).

Footprinting and Cleavage Assays
Footprint fragments containing one ITR sequence were radiolabeled on the top strand by first cutting ~3 µg of plasmid DNA with 15 U of SalI in Promega restriction enzyme buffer D in a total volume of 20 µl for 1 h at 37°C and 20 min at 65°C. The buffer was exchanged to water by spinning the sample through a Sephadex G-50 column for 2 min. To the elute we added 5 µl BRL React 2 buffer, 1 µl 1.5 mM dGTP, 1 µl 1.5 mM dTTP, 4 µl 3,000 mCi/mmol 32P dATP, 4 µl 3,000 mCi/mmol 32P dCTP, 5 U Klenow, and water to bring the volume to 50 µl. The reactions were left at room temperature for 25 min and were then chased with 1 µl of 10 mM dNTPs for 5 min. The reactions were heated to 65°C for 20 min to destroy the polymerase. The DNA was again digested with 15 U of XbaI for 1 h at 37°C and 20 min at 65°C. The radiolabeled footprint fragments were isolated as described previously (Lampe, Churchill, and Robertson 1996Citation ). The DNA was resuspended in 50 µl of TE (10 mM Tris-Cl, pH 8.0, 1 mM EDTA), and 1 µl was counted in a scintillation counter. Each sample was diluted to 25,000 cpm/µl.

DNase I footprinting reactions were carried out as described previously using purified Himar1 transposase and purified Mos1 transposase. The reactions were incubated at room temperature for 30 min, after which 0.75 U of DNase I was added to each tube. After precisely 2 min, the reactions were stopped with 70 µl of stop buffer (64.5 µl 100% ethanol, 0.5 µl tRNA [1 mg/ml], 5 µl saturated ammonium acetate) and chilled in a dry ice/ethanol bath for 15 min. The DNA was centrifuged for 30 min, and the pellets were washed once with 70% ethanol. The pellets were resuspended in 10–15 µl of sequencing stop buffer (US Biochemicals). Size standards and footprint reactions were resolved as in Lampe, Churchill, and Robertson (1996)Citation .

Cleavage assays were set up in the same way as the footprint reactions and incubated at 30°C for 2.5 h, omitting the DNase I step. The reactions were terminated and processed as per the footprint reactions.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Choice of mariner Elements
We chose to examine the interactions of two purified mariner family transposases with the ITRs from six different mariner family elements. The elements examined in this study are listed in table 1 , along with the sequences of the ITRs for each and the percentage of identity of the ITRs to those of Himar1 and Mos1 mariner, elements whose transposases were used in this study. The tree shown in figure 1 graphically represents the relationships of the elements to each other. We examined a mixture of elements from the same subfamily and from different subfamilies. Himar1, Mpmar1, and Hsmar2 are all members of the irritans subfamily (fig. 1 ). Mpmar1 was the element that was most closely related (82% amino acid identity) to Himar1, which showed any significant divergence in its ITRs, while Hsmar2, although in the subfamily, was very divergent (39% identity with Himar1). Each of the others, Csmar1 (cecropia subfamily), Ammar1 (mellifera subfamily), and Mos1 (mauritiana subfamily), was an element from another subfamily. We reasoned that a mixture of members within a subfamily and from other divergent subfamilies would allow us to probe the limits of site-specific DNA binding within the mariner family of transposons. Finally, it was important for us to limit ourselves to naturally occurring elements because these are presumably selected for activity in nature.


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Table 1 Mariner Inverted Terminal Repeat (ITR) Sequences Used in this Study and % Identities of ITR Sequences Between Himar1, Mos1, and Other Elements

 
In Vitro Transposition
In vitro transposition was performed using combinations of both purified transposases at various concentrations and all six elements as donors (fig. 2 ). As table 2 shows, Himar1 transposase was able to mobilize its cognate element and, to a much lesser degree, the Mpmar1 element, whose ITR is 83% identical to that of Himar1. The degree to which Himar1 transposase was able to interact with Mpmar1 ITRs was strongly dependent on the transposase concentration. At 2.5 nM Himar1 transposase, Mpmar1 was mobilized about 26-fold less frequently than was Himar1. At 0.5 nM purified Himar1 transposase, this value plummeted such that Mpmar1 was mobilized 419-fold less frequently than was Himar1. These results indicate a much lower affinity of Himar1 transposase for Mpmar1 ITR DNA than for the cognate Himar1 ITR sequence. Insertions of Mpmar1 were sequenced to determine the site of insertion and were similar to those of Himar1 (data not shown).


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Table 2 Results of in vitro Transposition Assays Using Purified Transposases and the Various Marked Mini-mariner Transposons

 
Hsmar2, which is also an irritans subfamily element, was not mobilized at all by Himar1 transposase. No member of any other subfamily was mobilized by Himar1 transposase. Mos1 transposase was able to mobilize only its cognate element, with no evidence of mobilization of any other element. We note, however, that the level of activity shown by Mos1 transposase to its own ITRs was lower than that of Himar1; thus, the limit of detection for interactions was reduced in the Mos1 comparisons.

Interaction of Purified Transposases with ITR DNAs from Various mariner Family Elements
We next asked what is responsible for the failure of the purified transposases to mobilize other elements. In principle, failure to transpose could be caused by either lack of binding to ITR DNA or a failure to cleave the DNA after binding. Figure 3 shows the results of DNAse I footprinting and cleavage analysis using purified Himar1 and Mos1 transposases with radiolabeled ITR DNA from each of the elements used in this study. In each case in which a transposase was able to mobilize an element in the in vitro transposition assay, the transposase was able to bind and cleave the ITR DNA of the element. The footprint of Himar1 on Mpmar1 ITR DNA above required approximately five times the amount of transposase necessary to bind the cognate Himar1 ITR DNA, and still the footprinting and cleavage was not as intense as that produced on the cognate ITR. The amount of transposase used here was the maximum amount that could be practically accommodated in the assay. These results indicate a decreased affinity of Himar1 transposase for Mpmar1 DNA, which correlates with the decreased ability to mobilize the Mpmar1 element.



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Fig. 3.—DNase I footprinting of mariner family ITRs by purified transposases. Results of a DNase I footprinting study using six different mariner subfamily inverted terminal repeat (ITR) sequences and two different purified transposases. The name of the element ITR is listed above each panel. Footprinting analyses are shown on the left half of each panel. Cleavage assays are shown on the right half of each panel. — = no transposase; H = Himar1 transposase used; M = Mos1 transposase used. Thick black bars indicate the extent of the ITR. The 5' end is at the top

 
In every case in which the transposase was unable to mobilize the element in the in vitro assay, there was no indication of an interaction of the transposase with the ITR of the element. We therefore conclude that the failure of mariner family elements to interact with each other lies at the level of site-specific DNA binding of the transposase to the ITR.

Are All Nucleotide Positions in the Inverted Repeats Important?
We were surprised at the degree to which transposition was suppressed between Himar1 and Mpmar1, given that their ITRs are 83% identical. Such a degree of difference is often seen between the two ITRs in the same transposon in other systems. For example, the bacterial insertion sequence IS50 has inverted terminal repeats (the "outer end" and "inner end") that differ at 7 out of 19 bp (i.e., 63% identical) (Sasakawa, Carle, and Berg 1983Citation ). Indeed, even Mos1 mariner has mismatches between its ITRs which make each end ca. 90% identical to the other (Maruyama, Schoor, and Hartl 1991Citation ). One way to analyze homologous sequences to determine important conserved positions is through sequence logo analysis (Schneider and Stephens 1990Citation ). We generated a sequence logo of the ITRs used in this study and two other mariners from two other subfamilies (C.elegans.mar1 and B.mori.mar1; see fig. 1 ), which are representative of mariner element diversity as a whole (fig. 4 ). The sequence logo strongly suggests that certain positions in mariner ITRs are more conserved than others. Two regions of the ITRs, positions 3–8 and positions 14–18, appear to be most conserved. Conservation of these positions suggests that the transposase might be making base-specific contacts within these regions of the ITR. Indeed, the most conserved positions in each region (positions 5 and 15) are one helical turn away from each other, suggesting that transposase might be making contact with the DNA on one face of the ITR in two locations of the major groove. Conservation at a subset of ITR positions also suggests that not all of the five differences between Mpmar1 and Himar1 ITRs may be equally important. Positions 7, 10, and 11 in the sequence logo are not strongly conserved between mariners, while positions 6 and 17 are moderately conserved. Thus, only positions 6 and 17 might be contributing most of the functional divergence between the two elements. This implies that relatively few changes in the inverted terminal repeat may have dramatic effects on the ability of transposase to interact with the ITR, which may strongly affect how these elements diverge from one another.



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Fig. 4.—Sequence logo of mariner family transposon ITRs. A sequence logo of ITR sequences from the six mariner family elements examined in this study. The sequence logo was generated using the WebLogo server (http://www.bio.cam.ac.uk/seqlogo/) and computed using makelogo (Schneider and Stephens 1990Citation ). A sequence logo is a graphical way to represent aligned sequence data that is easier to interpret than a strict-consensus sequence. At each position, the nucleotides are stacked on top of one another, with the most frequent nucleotide on top and the height of each nucleotide proportional to its frequency. A logo displays the frequencies of bases at each position, along with the degree of sequence conservation, measured in bits of information. The vertical scale is in bits, with a maximum of two bits possible at each position. A bit is a unit used in information theory and is the amount of information necessary to decide between two equally probable choices. The alignment used to produce this sequence logo can be found at http://www.home.duq.edu/~lampe/MBE/ITRalign.htm. More information on sequence logos can be found at http://www.bio.cam.ac.uk/seqlogo

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Lack of Site-Specific DNA Binding Underlies the Ability of More than One mariner to Occupy a Single Genome
Phylogenetic analysis of mariner family transposable elements reveals several striking features about their evolutionary history. First, a phylogeny based on mariner sequences usually shows large discrepancies with the phylogenies of the host species in which they are found (Robertson 1993Citation ; Robertson and MacLeod 1993Citation ; Robertson and Lampe 1995Citation ). Although long-branch attraction is sometimes invoked to explain incogruencies in phylogenetic trees, the most likely explanation for this phenomenon is horizontal transfer between host species (Maruyama and Hartl 1991Citation ; Lohe et al. 1995Citation ; Robertson and Lampe 1995Citation ). We do not understand how this happens, but we do know that transposition is possible using only the mariner-encoded transposase protein which infers a lack of host-specific factors in transposition and which would greatly facilitate the process of horizontal transfer (Lampe, Churchill, and Robertson 1996Citation ; Tosi and Beverley 2000Citation ).

Two other features of mariner element phylogenies are the sheer diversity of the elements and the fact that any given host genome may harbor more than one kind of mariner element. In some cases, at least nine distinct kinds of mariners have been detected in the same genome (see fig. 1 ). Our analysis here demonstrated that divergent mariner elements do not interact and, indeed, the elements need not be very diverged to lose a substantial degree of interaction. This has important evolutionary and practical implications. Any given mariner element is likely to see a new genome as virgin territory upon a horizontal transfer, even if that genome is already occupied by other mariner elements and their regulatory systems, assuming the invading element is sufficiently divergent. This phenomenon probably underlies the fact that many genomes contain more than one kind of mariner element. Similar conclusions have been reached using genetic crosses combining different elements in flies, although this study did not compare elements on as fine a scale as the work herein (De Aguiar and Hartl 1999Citation ).

Practical Implications
Noninteraction of divergent elements has practical implications as well. First, mariner elements have been advanced as genetic tools for germ line transformation of a variety of organisms, some intended for eventual release into the wild (Lampe, Churchill, and Robertson 1996Citation ; Lohe and Hartl 1996bCitation ; Hoy et al. 1997Citation ). The stability of mariner-based transgenes in these organisms has been questioned given the apparent ubiquity of mariners. We feel that there is very little likelihood of any transgene instability given the fact that most mariners we know about are sufficiently divergent so as to not interact with either Mos1 or Himar1, the elements most likely to deliver transgenes. This is in contrast to the situation found between the related hAT family elements, Hermes and hobo, which do cross-react (Sundararajan, Atkinson, and O'Brochta 1999Citation ).

Second, Himar1 and Mos1 have been successfully used as genetic tools in a variety of organisms (Lidholm, Lohe, and Hartl 1993Citation ; Lohe and Hartl 1996bCitation ; Gueiros-Filho and Beverley 1997Citation ; Fadool, Hartl, and Dowling 1998Citation ; Sherman et al. 1998Citation ; Rubin et al. 1999Citation ). It is now clear that these elements could be used together in the same genome to create organisms containing more than one transgene without fear of cross-mobilization.

Speculation: Does Divergence of Transposase-DNA Interactions Drive mariner Family Diversity?
Despite the great diversity of mariner elements, we do not yet understand how this diversity is generated. A model for the life cycle of mariners based on that of Hartl et al. (1997)Citation and others (Robertson 1993Citation ; Robertson and MacLeod 1993Citation ; Robertson and Lampe 1995Citation ; Hartl et al. 1997Citation ) is shown in figure 5 . A key feature of this model is horizontal transfer, and hereinafter we will refer to this model as the horizontal transfer model. The portion of the model outlined in solid arrows indicates the probable fate of the vast majority of mariners in nature, namely, vertical inactivation and stochastic loss from the species (Lohe et al. 1995Citation ; Robertson and Lampe 1995Citation ) and the possible further horizontal transfer of the identical element. Nevertheless, our data suggest that other fates are available to certain diverged mariner copies.



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Fig. 5.—The evolutionary fate of mariner elements. A model for the evolutionary fate of a mariner element based on the life cycle of mariner elements proposed by Hartl et al. (1997)Citation is shown. Components of the original Hartl et al. (1997)Citation figure are shown in light type and solid lines. Components proposed herein are shown in bold print and broken lines. Path 1 emphasizes repeated horizontal transfer of slightly divergent elements and is essentially a drift model of mariner diversification accompanied by selection for activity at the time of horizontal transfer and subsequent spread in the newly colonized genome. Path 2 emphasizes within-species diversification and emphasizes selection for escape from parental element regulatory systems and the apparent ease with which slight differences in ITRs can suppress transposition

 
In all cases, elements diverge from the original invading copy by the accumulation of point mutations (Lohe et al. 1995Citation ; Robertson and Lampe 1995Citation ). Many of these mutations inactivate particular copies and lead to the eventual vertical inactivation of the element in the species as proposed by the horizontal-transfer model. Like any mutation, however, some will be neutral and some will change the function of a cis sequence or the transposase-coding sequence. This change in function could be either a loss or a gain of function. Elements carrying neutral or functionally different (yet still active) mutations can proceed down two other paths besides the most common one emphasized by the horizontal-transfer model. The first path is horizontal transfer of a divergent copy to another species. Phylogenetic evidence indicates that this almost certainly involves the invasion of a single active copy of a mariner into a new genome. Horizontal transfer and establishment of the element in a new lineage implies that the transferred element must be functional (Silva and Kidwell 2000). There is no guarantee, however, that the mutated copy will have the same degree of activity as the parental element. Mutations isolated in mariner in D. melanogaster and in Himar1 in an Escherichia coli screen show that both inactivating and hyperactive mutations can be isolated (Lohe, De Aguiar, and Hartl 1997Citation ; Lampe et al. 1999Citation ). After horizontal transfer, the number of copies an element can make is probably directly dependent on its intrinsic activity. Thus, any mutation that can increase an element's activity will be of benefit to the element, provided that increased activity does not grossly affect the fitness of the host. If, for example, an amino acid mutation is present in a transferred copy that lowered, but did not eliminate, its activity, one class of mutations that might increase the activity of this enfeebled element would be a cis-acting suppressor mutation in the ITR. A side effect of such a suppressor mutation may be reduced interaction with the parental element. If this process were to be repeated sequentially, the descendants of the original element could be expected to diverge to the extent that they could no longer interact with the parent if the two were ever to reestablish contact. This is essentially diversification by repeated founder effects, with selection at the time of horizontal transfer for some degree of activity.

The second alternative path emphasizes divergence primarily within a single species. This path consists of a new (i.e., noninteracting) element arising within the same host species as the parental element. If this model were true, slight coevolutionary changes in ITR and transposase sequences would be sufficient to allow the new element to preferentially mobilize itself, which is not normally the case. A transposase gene transcribed and translated from one particular copy of an element normally has no guarantee that it will mobilize the same copy from which it was transcribed.

We realize that coevolutionary changes occurring in the same element would be rare. Nevertheless, at least three factors mitigate this rarity. First, elements have many chances to undergo this phenomenon, since they can occupy many genomes essentially simultaneously (Robertson and Lampe 1995Citation ). Second, recent evidence on Tc1 transposition in C. elegans indicates that gap repair mechanisms that repair donor sites after transposition sometimes produce chimeric elements due to template switching (S. Fisher, E. Wienholds, and R. Plasterk, personal communication). Thus, the host can perform gene shuffling of the transposable elements, which may mean that the actual diversity of changes in elements is much greater than that derived from simple point mutations. This would dramatically increase the chances of coevolutionary changes occurring in the same element. Future studies on the interaction of mariner transposases and their cognate ITRs will shed much light on the degree to which slight changes in the ITRs can disrupt transposase binding and whether, or if, these changes can be compensated for by slight mutations in the transposase.

Finally, there may be a selective advantage to any particular copy of an element that can undergo the process of within-species divergence. The ultimate fate of mariners in a species is inactivation through point mutation, emergent regulatory processes, or stochastic loss (Hartl, Lohe, and Lozovskaya 1997Citation ). Any element that can escape the regulatory systems of the parental element can found its own lineage and start down the mariner life cycle anew (Lohe and Hartl 1996aCitation ).

To date, sequences collected from a variety of species by PCR and data mining from genome projects overwhelmingly support the diversification of mariners by the first path. Nevertheless, the data presented herein demonstrate that a relatively slight degree of divergence between ITRs and transposase sequences is sufficient to dramatically decrease the amount of interaction between two elements, so in a minority of cases, path 2 conceivably might be utilized. Evidence gathered primarily from elements in protist genomes suggests that diversification can occur within a genome and not depend solely on a benefit to the host (Witherspoon et al. 1997Citation ; Witherspoon 1999Citation ). Divergence by path 2 could be tested in two ways. First, a careful analysis of element lineages within a single species should show closely related elements coexisting and certain fixed amino acid sequences correlating with certain fixed ITR changes. Second, this process could be tested with Himar1 in the laboratory by introducing mutations into the ITR that lower transposition frequency and then screening for transposase mutants that suppress these changes in an E. coli–based screen (Lampe et al. 1999Citation ).


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
We thank D. Hartl for the gift of Mos1 DNA; D. Finnegan and S. Beverly for providing purified Mos1 transposase; A. Dawson for help in interpreting the Mos1 cleavage data; W. Engels, G. Herrick, and D. Witherspoon for helpful discussions; S. Fisher, E. Wienholds, and R. Plasterk for permission to cite unpublished data; and two anonymous reviewers for their comments. This work was supported by PHS grant 2RO1AI33586-04 to H.M.R. and D.J.L. D.J.L. and K.K.O.W. contributed equally to this work.


    Footnotes
 
Thomas H. Eickbush, Reviewing Editor

1 Abbreviation: ITR, inverted terminal repeat. Back

2 Keywords: mariner, transposon transposase Back

3 Address for correspondence and reprints: David J. Lampe, Department of Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania 15282. lampe{at}duq.edu Back


    literature cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 

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Accepted for publication February 5, 2001.