Presence of an SAR-like sequence in junction regions between an introducedtransgene and genomic DNA of cultured tobacco cells: its effect on transformation frequency

A 12.5-kb DNA fragment with junction regions between the transgene and geno mic DNA was cloned from a transgenic tobacco cell line obtained by micropro jectile bombardment of plasmid pCaMVNEO. Nucleotide sequence analysis of th e fragment (DDBJ accession no. D84238) showed that it carried a 7.7-kb core sequence (concatemer of a complete pCaMVNEO and a partial pCaMVNEO) and tw o identical 1.3-kb junction sequences that flanked both the 5' and 3' ends of the core sequence and had inverted orientations. These sequences had top oisomerase II (Topo II) cleavage sites and adenine and thimine-rich sequenc es known to be specific to nuclear scaffold-attachment regions (SARs). An i n vitro binding assay showed that a 507-bp fragment (designated TJ1) from t he 1.3-kb sequence had the ability to bind to nuclear scaffold preparations of cultured tobacco cells, confirmation that the 1.3-kb sequence is an SAR . Insertion of TJ1 at the 5' and 3' sides of the expression cassette for th e npt II gene increased transformant yields 5- to 10-fold and the NPT II en zyme activity per copy of the gene 5-fold. TJ1 enhances the integration or expression of the transgene, or both. Clearly, TJ1 is very useful for produ cing transgenic plants. This is the first report on an SAR-like sequence th at is located in the transgene locus and enhances transformation efficiency in eukaryotic cells. The possible role of TJ1- SAR in the molecular evolut ion of plant genome is discussed.