The presence of such elements has been extensively examined primarily in insect species (411) although they are found in many phyla including vertebrates, arthropods, ciliates and fungi (see (368) and references therein). The elements have been detected using a specific PCR probe which exploits the presence of a highly conserved Tpase sequence.
The mariner family (369) includes the Tc elements originally detected in C. elegans (128). These are the subject of extensive analysis. Alignment of their Tpases (120) and their target specificity suggest that they are related to the IS630 family of bacterial elements. Tc1/3 and mariner show significant similarities (Fig.). Moreover, the insertion specificity of both Tc1 and Tc3 is strict. Analysis of 204 Tc1 and 166 Tc3 insertions indicated that all occurred into a TA dinucleotide which was duplicated during the insertion event (491), a target choice which is identical to that of IS630.
While Tc1A and Tc3A also carry a DD(34)E motif ( 120)), the mariner subgroup generally carries a DD34D catalytic motif. More extensive database analysis (437a) has uncovered elements with DD37D DD39D and DD37E motifs(see 412a , 227a, 465a, 273a). It has been proposed that elements with these three variant motifs constitute three new variant mariner subgroups.
Both Tc1 and the related Tc3 element carry a single transposase gene, tc1A and tc3A respectively, each interrupted by one intron at a different position. Tc1 is 1610 bp long and carries terminal IR sequences of 54 bp while Tc3 is 2335 bp in length with 462 bp IRs. The reason for this large difference in IR length is unknown. The IR sequences are recognised and bound by their cognate Tpases in vitro (497),(89). This is consistent with the limited conservation of nucleotides between the IRs of each element. Moreover, it has been shown that transposase induction in vivo from a heatshock promoter leads to high levels of transposition of the cognate element (489),(497). Point mutations in the D,D and E residues render the Tc3 enzyme inactive in vivo (490). Like several bacterial Tpases, a sequence-specific DNA binding domain is located at the N-terminal end of both Tpases. It is followed by a region which overlaps the DD(35)E region and binds DNA non-specifically. Interestingly, the N-terminal domain of Tc1A shows similarities and identities over a 40 amino acid stretch with the N-terminal region of the IS30 Tpase which is itself sufficient for binding to IS30 IRs. Also in the case of Tc1A, band shift assays, footprinting and methylation interference studies have demonstrated a binding site between nucleotides 5 and 26 of the IR. Nucleotides 1-4 are not necessary for binding (497). This observation is consistent with the notion that the Tc1 IR sequences are organised into a 2-domain structure similar to that of several bacterial elements ( Fig 1). The binding site can be further subdivided into two distinct regions: one closer to the tip which is contacted by the specific N-terminal transposase domain and the other which is contacted by the non-specific DNA binding domain (496). A leucine zipper motif has also been detected in the Tpases of certain members of this group (223). As in the IS3 family this may represent a multimerisation domain although recent structural studies with a truncated transposase derivative indicated that this region did not assume a coiled-coil motif (126).
Purified Tc1A has been demonstrated to have both endonuclease and phosphoryltransferase activities in vitro. The major endonucleolytic activity detected was at the 5' rather than, as might have been expected based on the activities of other Tpases (496), at the 3' ends of the element. Moreover, cleavage was shown to occur specifically at the phosphodiester bond between the second and third nucleotide within the transposon. Phosphotransferase (strand transfer) activity was also observed in a "disintegration" assay.
Transposon circles have been detected in vivo (128), (413), (386) but their role as possible intermediates in transposition is not entirely clear. Tc1/3 appear to transpose using a cut and paste mechanism. Induction of Tc3A in vivo results in excision of Tc3 as a linear form. One interesting characteristic of these molecules is that while the cleavage at the 3' ends of the element occurs precisely between the transposon and its flanking sequences, 5' cleavage occurs two nucleotides within the transposon in a manner similar to that observed in vitro (490). The result of these cleavages is that two nucleotides of each transposon end remain behind in the donor site leaving a "footprint": TACATA (where the flanking TA dinucleotide is that generated by duplication during the original integration) ( Fig). It has been noted by Plasterk (368) that a related "footprint" observed for Mos1 (TACCATA) suggests that excision of these elements occurs with a 3 nucleotide stagger.
Recently systems which support Tc1 and mariner transposition in vitro have been described (497), (273). In both cases it has been demonstrated that the transposase is the only protein required for transposition. For Tc1, double-strand cleavage was observed not only at each end of an element carried on a supercoiled plasmid donor molecule but also on molecules carrying a single end. Excision, but not cleavage of a single end, appeared somewhat reduced when the donor plasmid was linearised suggesting that supercoiling stimulates the two ended-reactions. The cleaved ends were shown to carry a 2 base 3' overhang as shown in Fig). Intermolecular transposition was demonstrated using a genetic assay and the resulting products showed a similar target specificity as those analysed in vivo. For mariner (Himar1 from the hornfly, Haematobia irritans), the purified transposase was demonstrated to generate a footprint in the terminal IR and to promote double strand cleavage similar to that found with Tc1 but with a 3 base 3' overhang (273).
Mahillon J. and
Chandler M.
(1998)
Microbiology
and Molecular Biology
Reviews.
62 : 725-774
Chandler, M. and Mahillon, J.(2002) Insertion Sequences Revisited
Mobile DNA II Edited by N.L., Craig et al.
ASM
Press 305-366
with permission of American Society of Microbiology the 10-26-01.
Last modification : December 19 2001