Department of Biological Sciences, Duquesne University
Department of Entomology, University of Illinois
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Abstract |
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Introduction |
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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. 1995
; Lohe et al. 1995
; Robertson and Lampe 1995
). 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 1997
; Robertson and Zumpano 1997
) (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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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.
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Materials and Methods |
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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. 1994
) 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 1998
). These are the donor plasmids shown in figure 2
.
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Transposase Purification
Himar1 transposase was purified as described previously (Lampe, Churchill, and Robertson 1996
). 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)
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 1996
). 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 1015 µl of sequencing stop buffer (US Biochemicals). Size standards and footprint reactions were resolved as in Lampe, Churchill, and Robertson (1996)
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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.
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Results |
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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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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 1983
). Indeed, even Mos1 mariner has mismatches between its ITRs which make each end ca. 90% identical to the other (Maruyama, Schoor, and Hartl 1991
). One way to analyze homologous sequences to determine important conserved positions is through sequence logo analysis (Schneider and Stephens 1990
). 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 38 and positions 1418, 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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Discussion |
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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 1999
).
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 1996
; Lohe and Hartl 1996b
; Hoy et al. 1997
). 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 1999
).
Second, Himar1 and Mos1 have been successfully used as genetic tools in a variety of organisms (Lidholm, Lohe, and Hartl 1993
; Lohe and Hartl 1996b
; Gueiros-Filho and Beverley 1997
; Fadool, Hartl, and Dowling 1998
; Sherman et al. 1998
; Rubin et al. 1999
). 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)
and others (Robertson 1993
; Robertson and MacLeod 1993
; Robertson and Lampe 1995
; Hartl et al. 1997
) 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. 1995
; Robertson and Lampe 1995
) 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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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 1995
). 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 1997
). 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 1996a
).
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. 1997
; Witherspoon 1999
). 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. colibased screen (Lampe et al. 1999
).
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Acknowledgements |
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Footnotes |
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1 Abbreviation: ITR, inverted terminal repeat.
2 Keywords: mariner,
transposon
transposase
3 Address for correspondence and reprints: David J. Lampe, Department of Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania 15282. lampe{at}duq.edu
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