We have developed a method to detect the frequency with which Ac excises from a neomycin phosphotransferase II (NPTII) gene (Baker et al., this issue). This system is now being used to determine which sequences within Ac are required for the excision process. Ac derivatives were constructed in vitro and inserted at the same position within the NPTII gene as described previously for Ac.
These experiments differed slightly from those of Baker et al. in that the T-DNA of the Ti-plasmid also contained a hygromycin resistance gene expressed in tobacco protoplasts, which rendered these resistant to the antibiotic hygromycin. Therefore, in these experiments, hygromycin resistance indicated the T-DNA was successfully transferred to tobacco, and kanamycin resistance indicated that Ac had excised from the NPTII gene.
The Ac derivatives tested so far are shown in Figure 1. Whether or not they are capable of excision is also indicated. These data, together with our knowledge of the only Ac transcript so far detected (Kunze et al., this issue) allow us to make certain predictions of how Ac may be organized.
Four Ac derivatives (in plasmids pKU36, pKU32, pKU33 and pKU31) which contain deletions within the long 5' untranslated region of the Ac transcript are all capable of excision. At least 400 bp of the leader (in pKU31) are unnecessary for transposase expression and for excision. However, the Ac derivative present in pKU30 is incapable of excision. This derivative contains BglII linkers inserted at the EagI site within the leader. It is not clear why the insertion of BglII linkers in pKU30 prevents transposition while the insertion of the same linker at the endpoints of the deletions indicated above does not. It is known that in pKU30 there is more than one BglII linker in tandem as this has created a PstI site, but this is also true of pKU31 and pKU32. We are presently determining the nucleotide sequence of these derivatives to try to answer these questions.
Although deletions within the leader of the Ac transcript did not prevent excision, deletions removing part of the long open reading frame did. This was indicated by the Ac derivatives present in pKU35 and pKU9. In pKU9 the deletion has removed the 5' end of the open reading frame including the first two potential ATG initiation codons within the long open reading frame. This further suggests that translation of the open reading frame initiates at one of these two ATG codons, and not at one farther downstream (see Kunze et al., this volume).
The sequences deleted from the Ac derivatives present in plasmids pKU37, pKU19 and pKU29 are not within the Ac encoded transcript. These three derivatives cannot undergo self-catalyzed excision. The derivative present in pKU37 has lost the terminal sequences of Ac which are probably necessary for recognition of the ends of Ac by the transposase prior to excision. The sequences deleted from pKU19 and pKU29 may be required for recognition of the end of Ac by the transposase, for expression of the transposase (e.g., a promoter sequence) or for both of these. Experiments are underway to distinguish these possibilities. In addition, we are constructing further deletions to characterize more exactly the sequences required at the ends of Ac for recognition by the transposase, and are constructing point mutations within the open reading frame to try to determine which areas of this are required for transposase expression.
George Coupland1, Barbara Baker2, Jeff Schell2 and Peter Starlinger1
1Institut fur Genetik
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