Worm Breeder's Gazette 14(4): 18 (October 1, 1996)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The NetherlandsOne of the key-features of Tc1 transposition is that this transposon always integrates into a TA dinucleotide. Not all TA dinucleotides are equally used, however. Two consensus sequences for integration of Tc1 have been obtained by analysis of respectively 12 and 16 Tc1 insertions (Mori et al. (1988), Proc.Natl.Acad.Sci.USA 85:861-864, Eide and Anderson (1988), Mol.Cell.Biology 8:737-746). These consensus sequences are based on flanking sequences of independent Tc1 alleles of mainly unc-54 and unc-22. Recently a consensus sequence based on 344 endogenous Tc1 insertions was derived (Korswagen et al. (1996), Proc.Natl.Acad.Sci.USA in press). A fourth consensus sequence was derived by van Luenen and Plasterk (van Luenen and Plasterk (1994), Nucl.Acids.Res. 22:262- 269), after analysing 204 de novo Tc1 insertions into the gpa-2 gene. These insertions were detected using PCR after overexpression of the Tc1A transposase. These four consensus sequences are all similar. The consensus sequence derived by Korswagen et al. is depicted in table 1.
In vitro studies using gpa-2 as a target gene showed that the distribution of Tc1 elements in this DNA fragment was virtually the same as in vivo; so chromatin structure does not determine the observed preferences. What then determines the hotness of a given TA dinucleotide?
To analyse the role of the basepairs flanking the TA dinucleotide, we subcloned a 24 bp fragment, containing the hottest TA dinucleotide of the gpa-2 gene plus two adjacent cold TA sites, into the lacZ polylinker of pUC19. This DNA was then used as target DNA for transposition in vitro. Among 65 independent Tc1 integrations into the lacZ region, 33 were found in the gpa-2 hot-site. The two other TA dinucleotides were never used. When the hot gpa-2 fragment is shortened to 13 bp (with 5 and 6 bp on each site of the TA) again more than 50% of the inserts were found in this gpa-2 hot-site. Although the entire lacZ region counts 27 TA dinucleotides, still 17 out of 32 insertions are found in this particular site. This indicates that whatever makes the site preferred is contained within these 5/6 bp flanks. The simplest mechanistic explanation is that the transposase requires recognition of a TA sequence, and prefers recognition of some additional nucleotides before integration occurs. We are now further shortening and mutating the flanks of the hot TA dinucleotide in order to get a more detailed picture of the target site recognition by the Tc1A protein.
Sequence Consensus CAYATATRTG gpa-2 hot site GGTGTATGTC
Table 1. (Y=T or C, R=A or G) The consensus sequence derived by Korswagen et al. (1996) is depicted. Especially the GT dinucleotide at the position +2 and +3 is highly significant. This dinucleotide is also found in the gpa-2 hot-site.