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Factors Affecting Transposition of the Himar1 mariner Transposon in Vitro
David J. Lampea, Theresa E. Granta, and Hugh M. Robertsonaa Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
Corresponding author: David J. Lampe, Department of Entomology, University of Illinois at Urbana-Champaign, 505 S. Goodwin, Urbana, IL 61801, d-lampe{at}uiuc.edu (E-mail).
Communicating editor: M. J. SIMMONS
| ABSTRACT |
|---|
Mariner family transposable elements are widespread in animals, but their regulation is poorly understood, partly because only two are known to be functional. These are particular copies of the Dmmar1 element from Drosophila mauritiana, for example, Mos1, and the consensus sequence of the Himar1 element from the horn fly, Haematobia irritans. An in vitro transposition system was refined to investigate several parameters that influence the transposition of Himar1. Transposition products accumulated linearly over a period of 6 hr. Transposition frequency increased with temperature and was dependent on Mg2+ concentration. Transposition frequency peaked over a narrow range of transposase concentration. The decline at higher concentrations, a phenomenon observed in vivo with Mos1, supports the suggestion that mariners may be regulated in part by "overproduction inhibition." Transposition frequency decreased exponentially with increasing transposon size and was affected by the sequence of the flanking DNA of the donor site. A noticeable bias in target site usage suggests a preference for insertion into bent or bendable DNA sequences rather than any specific nucleotide sequences beyond the TA target site.
HIMAR1 is an irritans subfamily member of the mariner family of class II, DNA-mediated, or short-inverted, terminal repeat-type transposable elements, and it is one of only two known active mariner elements. It was isolated from the horn fly, Haematobia irritans. The active copy is a reconstructed consensus sequence based on a series of genomic clones, each of which differ from the consensus at several positions (![]()
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Activity of mariners in diverse hosts can be attributed to their very simple requirements for transposition. Himar1 can complete transposition in vitro using only its purified transposase (![]()
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The ubiquity of mariners and their apparent ease of transfer between species does not mean that the activity of these elements is unregulated. Indeed, their regulation is complex and most likely has both inherent and stochastic components (![]()
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| MATERIALS AND METHODS |
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Recombinant DNA:
Donor constructs carrying different size Himar1 transposons used in the in vitro transposition reaction (outlined in Figure 1A) were created by adding various DNA fragments to the HpaI site of pMarKan (Figure 1B; ![]()
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A transposon smaller than pMarKan was constructed by first creating a "minimariner" consisting of the first and last 100 bp of Himar1 using a PCR-ligation-PCR technique exactly as described by ![]()
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Donor constructs containing different flanking DNAs were created from in vitro transposition products. MMKan (the Himar1 transposon portion of the plasmid pMMKan) insertions into target TA dinucleotides at positions 2611 and 2622 of the target plasmid were cleaved with XhoI, and the products were separated on a 0.5% 1x TAE agarose gel. Insertions at these positions were known to be in the Ampr gene, so the monomer of the target containing the insertion should be Kanr (kanamycin resistant) but ampicillin sensitive (Amps). The 4.3-kb band containing one target monomer and the MMKan insertion was isolated, diluted to <10 ng/µl with TE, and 1 µl was used in a 10-µl ligation to recircularize the plasmid. The ligation products were transformed into bacteria, and the cells were plated on LB-kan plates. Subsequent plating onto LB-amp plates confirmed that the bacteria were Amps. Because these insertions were in the Ampr gene of the plasmid, no further manipulations were necessary to derive new donor constructs. These donors are pMM26 (insert at 2611) and pMM32 (insert at 2622).
In vitro transposition reactions:
In vitro transposition reactions were performed as described in ![]()
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Conditions were varied from the descriptions above as follows:
- Time: The time at which the transposition reactions were stopped was varied between 30 and 360 min.
- Temperature: The temperature at which the reactions were performed was varied between 15° and 40°.
- MgCl2 concentration: The MgCl2 concentration was varied between 0 and 15 mM.
- Transposase concentration: Transposase concentration was varied between 0.6 and 600 nM.
- Transposon size: The size of the donor construct was varied between 1.3 and 7.3 kb, as described above. Each donor was used in an equimolar concentration to that of the target DNA.
- Donor flanking DNA: The effect of flanking DNA was tested by using the donors pMM26 and pMM32 (see above).
Transposition frequencies were previously reported as the number of Kanr-Ampr colonies/(number of Ampr colonies x 10-3) (![]()
Himar1 insertion sites into the target plasmid were ascertained by sequencing outward from the transposon across the Himar1/target junction and comparing the flanking DNA sequence to that of the known sequence of the target. The products were picked randomly from plates of transposition products produced under the standard conditions. Products from reactions performed on different days were pooled. We attempted to ensure that the colonies picked were independent by allowing the growth of bacteria transformed with transposition products to grow no more than 30 min. The number of the insertion site position is that of the T nucleotide in the TA dinucleotide target sequence using the forward orientation of the pBSKS+ sequence from GenBank (accession number X52331).
A "sequence logo" for the aligned sites of insertion was generated over the internet by the WebLogo program (![]()
| RESULTS |
|---|
Results obtained by varying reaction conditions are presented in raw and transformed formats in Table 1 and graphically in Figure 2 Figure 3 Figure 4 Figure 5. We previously reported an assay to detect the transposition of the Himar1 transposon in vitro (![]()
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Transposase was required for transposition and was most active (>50% maximal activity) between 2.5 and 35 nM transposase, peaking at ~10 nM (Figure 2D). This result is similar to that found for purified Tn10 transposase, which is also inhibited at high transposase concentrations (![]()
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Transposon size dramatically affected the frequency of transposition (Figure 3). Smaller elements transposed much more frequently than larger ones. We fitted an exponential curve to these data based on the behavior of other transposons (![]()
Random transposition products (N = 65) were selected and sequenced to determine the site and orientation of Himar1 insertion into the target plasmid. The sequence, position, orientation, and frequency of use of each insertion site are shown in Figure 4A. A graphical representation of the data in the form of a sequence logo (![]()
The effect of flanking DNA sequence on transposition frequency was tested by creating two new donor constructs. These were derived from transposition products in which the Himar1 transposon was inserted into the Ampr gene of the target plasmid, rendering the transposon-containing monomer Kanr but Amps (see MATERIALS AND METHODS). pMM26, derived from the insertion at site 2611, showed a 2.4-fold increase in transposition frequency, whereas pMM32, derived from the insertion at site 2622, was only slightly more active (1.36-fold higher) than the original pMMKan construct (Figure 5).
| DISCUSSION |
|---|
Mariner family transposable elements are extremely widespread in animals, occurring in several phyla (![]()
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Transposition of Himar1 in vitro is markedly affected by the concentration of the transposase protein, and it is most active over a narrow range of transposase concentration, peaking at ~10 nM. Significantly, these data mirror what occurs in vivo for Mos1. Mos1 can mobilize a nonautonomous mariner element (wpeach) from the white locus in D. mauritiana, leading to a mosaic eye phenotype (![]()
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The important question is whether the property of reduced transposition at elevated transposase concentration is regulatory and under selection, or if it is merely coincidental. All transposable elements have some kind of restrictions on activity, and it would be strange if mariners lacked these. Isolation of mutant mariners that eliminated concentration-dependent inhibition would help resolve this problem. We would predict that such mutants would be hyperactive at high transposase concentrations because more transposase would be available to participate in a transposition reaction rather than being bound up in a nonactive form, whether in inactive multimers or some unstructured aggregation.
Increasing transposon size decreases transposition frequency:
The transposition frequency of Himar1 decreased exponentially with increasing transposon size. This effect has been demonstrated with other transposable elements, but the degree to which it occurs depends on the element and, at least in bacteria, on whether the donor site is on a chromosome or a plasmid. For example, the bacterial transposon Tn10 shows a decrease in transposition frequency of 40% per kb when mobilized from the bacterial chromosome but only 16% when mobilized from a plasmid (![]()
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DNA bending may underlie target site choice and flanking DNA effects at the donor site:
Himar1 showed a pronounced preference for insertion at some sites in the target plasmid used in the in vitro assay (Figure 4A). Sequence logo analysis of these sites shows that there is very little underlying sequence similarity in these sites (Figure 4B). These data are consistent with a lack of insertion site specificity (besides TA) found in vivo (![]()
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Fortuitously, the DNA bending propensity of the exact target region most preferred by Himar1 for insertion was experimentally measured elsewhere by ![]()
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Similar arguments can explain the effects of flanking DNA sequence at the donor site on overall transposition frequency. Several transposases, including the related Tc3 transposase, bend the DNA at the transposon termini upon binding (![]()
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Practical implications:
The ability of mariners to function in diverse hosts makes them attractive candidates for development into generalized animal transformation vectors. The success of elements used in this fashion, however, has been mixed. The Mos1 element has been used to transform D. melanogaster at low frequency, although the construct integrated was >13 kb in length (![]()
In vitro and in vivo studies are revealing the likely foci for the control of mariner activity. One feature of mariner transposases that may make them difficult to use as genetic tools and that may be a property under selection is the inhibitory effect of transposase at elevated concentrations. One of the most common ways to increase the activity of a heterologous genetic system is to increase its level of expression, a strategy clearly counterproductive for mariners. Suitable screens for mutant transposases lacking inherent control mechanisms should reveal forms of mariners more suitable for use as genetic tools; such strategies have worked for the bacterial transposon Tn5 (![]()
| FOOTNOTES |
|---|
1 Because of the great number of mariner family sequences published to date, we follow the formalized naming system for mariner family elements suggested in ![]()
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| ACKNOWLEDGMENTS |
|---|
We thank K. WALDEN for constructing the minimariner; M. BENEDICT, P. GEYER, and G. GLOOR for the gift of plasmids; and S. BERLOCHER, I. BOUSSY, M. CHURCHILL, P. JAMBECK, D. O'BROCHTA, M. SIMMONS, and an anonymous reviewer for helpful comments. This work was supported by Public Health Service grant AI33586-01.
Manuscript received September 10, 1997; Accepted for publication January 15, 1998.
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3). Error bars are SEM.



0.05).