The transposable element Ac has been the subject of intensive studies and is thought to transpose via a cut-and-paste mechanism. Nevertheless, little is known on how it excises and what are the intermediates of transposition. In order to test the possibility that extrachromosomal circles are formed upon Ac excision, we have used PCR with primers shown in Figure 1, to search for joined ends. The presence of joined ends is indicative of either circle formation or of presence of two adjacent elements in direct orientation (Fig. 1). Nested PCR was performed with primers 2 and 3 in the first round and with primers 1 and 4 in the second round. The templates consisted of genomic DNA from transgenic tobacco plants transformed with constructs pAGS4411 and pAGS4081, which carry Ac and Ds elements respectively (Dooner et al., Plant Cell 3: 473-482, 1991). DNA from a line carrying the bz2::Ds2 allele and an active Ac element was also used as template.

In all Ac-carrying plants, a band of ~520 bp was observed on EtBr-stained gels. This band has the size expected for precise joining of the terminal inverted repeats (TIRs). It was found only in lines carrying an actively transposing Ac or Ds element, but not in the absence of transposition, suggesting that its formation is transposition dependent. The 520 bp band was cloned and individual clones sequenced (Table 1). The amplified sequences corresponded to Ac joined ends with short insertions or deletions in between the TIRs. Note that no molecules were found with perfect joined ends. Short deletions in both TIRs were found in 9 out of 26 sequenced joined ends. These stuctures are probably unable to reintegrate in the genome. Moreover, they cannot correspond to tandem jumps as at least one end should remain intact. Therefore we conclude that these deleted joined ends were amplified from circular molecules which are abortive transposition products formed upon element excision. Another type of molecules, which had one end intact and a deletion in the other end, were found in 5 out of 26 sequences. These molecules could be interpreted either as transposition of Ac in itself near its termini, or as an Ac circle. In the latter case, such a circle would probably be unable to reinsert. Sequences with intact TIRs are of two types: those with insertions resembling the flanking donor site, and those with insertions unrelated to the donor. The latter are probably not caused by tandem jumps but rather by circularization of the ends. The former could in principle be caused by tandem jumps, or alternatively, flanking sequences might be carried by the circularized element as a result of the excision process. We are in the process of determining the origin of the joined ends and of the footprints between the ends. Moreover we are testing whether circular Ds molecules with intact termini can reintegrate into the genome via the transposition pathway.

Table 1. Sequence at the junction of joined Ac ends
 
 

Conceptual head-to-head joining of Ac ends

---- -

CATCCTACTTTCATCCCT G Aa)

TAGGGATGAAAACGGTC

-----

Sequences of the PCR clones

1 2 3 4a) 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- - ---- -

CATCCTACTTTCATCCCT G CATCCTACTTTCATCCCT G CATCCTACTTTCATCCCT G CATCCTACTTTCATCCCT G CATCCTACTTTCATCCCT G CATCCTACTTTCATCCCT G CATCCTACTTTCATCCCT G CATCCTACTTTCATCCCT G CATCCTACTTTCATCCCT G CATCCTACTTTCATCCCT G CATCCTACTTTCATCCCT G CATCCTACTTTCATCCCT G CATCCTACTTTCATCCCT G CATCCTACTTTCATCCCT G CATCCTACTTTCATCCCT G CATCCTACTTTCATCCCT G CATCCTACTTTCATC CATCCTACTTTCATC CATCCTACTTTCATCCCT CATCCTACTTTC C deletion of 41 bp CATCCTACTTTCATCCC CATCCTACTTTCATCCCT G CATCCTACTTTCATCCCT G CATCCTACTTTCATCCCT G

G C AC GC GC CT CC AA TTG CTAA 7 bpb) 21 bp 21 bp 27 bp 64 bp GC TAC T

TAGGGATGAAAACGGTC TAGGGATGAAAACGGTC TAGGGATGAAAACGGTC TAGGGATGAAAACGGTC TAGGGATGAAAACGGTC TAGGGATGAAAACGGTC TAGGGATGAAAACGGTC TAGGGATGAAAACGGTC TAGGGATGAAAACGGTC TAGGGATGAAAACGGTC TAGGGATGAAAACGGTC TAGGGATGAAAACGGTC TAGGGATGAAAACGGTC TAGGGATGAAAACGGTC TAGGGATGAAAACGGTC deletion of 33bp TAGGGATGAAAACGGTC ATGAAAACGGTC GGGATGAAAACGGTC GATGAAAACGGTC ATGAAAACGGTC AACGGTC TAGGGATGAAAACGGTC GGGATGAAAACGGTC deletion of 29 bp AACGGTC

----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- -----

a) The bz2::Ds2 allele of maize, which generated the sequence #4, contains an insertion of Ds element with perfect TIRs.
b) Clones 11 - 15 contain insertions of the indicated size, sequences are not shown.

Figure 1.  (A) Set of PCR primers (arrows) designed to amplify joined Ac ends.  (B) Molecules that could serve as templates for amplification with the primers 1, 2, 3, 4.

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