--Min-gang Li and Peter Starlinger

We have used the phenotypic assay system developed in this laboratory (Baker et al., EMBO J. 6:1547, 1987) to study the 5' coding region of transposable element Ac in Nicotiana tabacum. In this system both Ac and Ac derivatives were inserted into the leader sequence of neomycin phosphotransferase gene (NPTII) and introduced into tobacco protoplasts via Agrobacterium tumefaciens. Excision or transposition of Ac allows us to detect Km-resistant calli.

There are several AUGs located 3' to the long untranslated leader of the Ac transcript. AUGs 1, 2 and 10 are located in the longest open reading frame (ORFa) which encodes an 807-amino acid protein initiated from AUG1. AUGs 7, 8 and 9 are completely covered by the ORFa sequence, but are located in a different open reading frame (ORFb) which potentially encodes a 102-amino acid protein and is not detected by Western analysis (Kunze et al., EMBO J: 6:1555, 1987). We altered all AUGs 7, 8 and 9 to AAGs by using oligonucleotide site-directed mutagenesis, to eliminate initiation of ORFb. In addition, we have inserted an amber codon into ORFb that causes the reading frame to stop 38 amino acids earlier, but without altering the amino acid sequence encoded by ORFa. The phenotypic assay showed that in both cases mutant Ac elements can still excise or transpose efficiently. No difference in transposition frequency between mutant Ac elements and wildtype Ac elements can be detected. These results indicate that the protein encoded by ORFb does not affect Ac transposition.

Furthermore, in order to find out whether a truncated protein starting from AUG 10 still has a biological function, we shifted the reading frame initiated at AUGs 1 and 2, deleted the fragment containing AUGs 3 to 6 and destroyed AUGs 7 to 9 by oligonucleotide site-directed mutagenesis, which causes the large protein to be translated only from AUG 10, producing a protein lacking 101 amino acids at the N-terminus. This Ac derivative gives rise to a similar number of Km-resistant calli as does wildtype Ac, indicating that the truncated protein is sufficient for Ac transposition.

Km-resistant calli from all assays mentioned above were regenerated into plants. Mutant constructs of Ac in transgenic tobacco plants were confirmed by Southern analysis of DNA isolated from these plants and/or by direct sequencing of DNA amplified by the polymerase chain reaction. Southern analysis also detected the DNA fragment left after correct excision of Ac by hybridization to either a NPTII probe or to a probe from the 1'-promoter.

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