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. 2000 Feb 1;28(3):784-90.
doi: 10.1093/nar/28.3.784.

cis and trans factors affecting Mos1 mariner evolution and transposition in vitro, and its potential for functional genomics

L R Tosi  1 , S M Beverley
Affiliations

cis and trans factors affecting Mos1 mariner evolution and transposition in vitro, and its potential for functional genomics

L R Tosi et al. Nucleic Acids Res. .

Abstract

Mos1 and other mariner / Tc1 transposons move horizon-tally during evolution, and when transplanted into heterologous species can transpose in organisms ranging from prokaryotes to protozoans and vertebrates. To further develop the Drosophila Mos1 mariner system as a genetic tool and to probe mechanisms affecting the regulation of transposition activity, we developed an in vitro system for Mos1 transposition using purified transposase and selectable Mos1 derivatives. Transposition frequencies of nearly 10(-3)/target DNA molecule were obtained, and insertions occurred at TA dinucleotides with little other sequence specificity. Mos1 elements containing only the 28 bp terminal inverted repeats were inactive in vitro, while elements containing a few additional internal bases were fully active, establishing the minimal cis -acting requirements for transposition. With increasing transposase the transposition frequency increased to a plateau value, in contrast to the predictions of the protein over-expression inhibition model and to that found recently with a reconstructed Himar1 transposase. This difference between the 'natural' Mos1 and 'reconstructed' Himar1 transposases suggests an evolutionary path for down-regulation of mariner transposition following its introduction into a naïve population. The establishment of the cis and trans requirements for optimal mariner transposition in vitro provides key data for the creation of vectors for in vitro mutagenesis, and will facilitate the development of in vivo systems for mariner transposition.

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Figures

Figure 1
Figure 1
Purification of the Mos1 transposase from E.coli. A Coomassie blue stained 12% SDS–PAGE gel containing samples from the various steps involved in Mos1 transposase purification is shown. Lane 1, bacterial lysate before IPTG induction; lane 2, bacterial lysate after induction; lane 3, washed inclusion bodies; lane 4, purified Mos1 transposase after chromatography and refolding steps.
Figure 2
Figure 2
In vitro mariner transposition assay. (A) Organization of the modified Mos1 element, mosK. The Tn903 kanamycin resistance marker (Kanr) was inserted into the unique SacI (S) site. The gray arrowheads and the black arrow represent the inverted terminal repeats and the transposase ORF, respectively; ClaI (C) and DraI (D) sites are indicated. (B) The donor plasmid, pMD13-mosK, contains a tetracycline resistance marker (Tcr), an R6K replication origin (oriR6K) and mosK. In this example, the target DNA is carried in the vector pEL-HYG (24), which contains a hygromycin resistance marker (Hygr) and a minimal colE1 replication origin (oriColE1).
Figure 3
Figure 3
Effect of different conditions on Mos1 transposase activity. In vitro transposition was performed as described in Materials and Methods, using the donor pMD13-mosK and the target pELHYG-H2. (A) Effect of enzyme concentration and (B) amount of donor plasmid on transposition efficiency. Transposition efficiency values were calculated from the ratio of Kanr + Hygr to Hygr colonies. In (A) the efficiency for the Himar1 transposase is shown as dotted lines; the peak height has been normalized to the maximal peak height seen for Mos1 and corresponds to an efficiency of 6.3 × 10–3 (26). Mos1 transposase protein concentration was determined by the BCA method; if the UV absorbance method was used, the X-axis for Mos1 should be multiplied by a factor of 3 (Materials and Methods).
Figure 4
Figure 4
Randomness of mosK insertion and target specificity in the presence of Mg2+ or Mn2+. (A and B) Insertion of mosK into the 12 kb insert of pELHYG-H2 in reactions containing Mg2+ or Mn2+, respectively. The vertical arrows represent individual insertions; those above or below the map represent insertions with the Kanr marker in the forward or reverse orientation, respectively. (C and D) Target site analysis of 111 and 21 mosK insertions, obtained in the presence of Mg2+ and Mn2+, respectively. The consensus was determined and displayed using the Sequence Logo algorithm (49). The height of each base corresponds to its prevalence at that position.
Figure 5
Figure 5
cis requirements for transposition. Deleted versions of pMD13-mosK were used as transposon donors in a standard in vitro transposition reaction, using the target plasmid pELHYG-H2. The terminal inverted repeats are indicated as arrows; restriction sites are: C, ClaI; S, SacI; D, DraI. The transposon sizes are: mosK, 2.4 kb; mosKΔCD, 1.2 kb; mosKΔCS, 1.7 kb; mosKΔDS, 1.9 kb; mmosK, 1.1 kb.
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