Presence of an SAR-like sequence in junction regions between an introduced transgene 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 genomic DNA was cloned from a transgenic tobacco cell line obtained by microprojectile bombardment of plasmid pCaMVNEO. Nucleotide sequence analysis of the 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 two 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 topoisomerase II (Topo II) cleavage sites and adenine and thimine-rich sequences known to be specific to nuclear scaffold-attachment regions (SARs). An in vitro binding assay showed that a 507-bp fragment (designated TJ1) from the 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 the npt II gene increased transformant yields 5- to 10-fold and the NPT II enzyme 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 producing transgenic plants. This is the first report on an SAR-like sequence that is located in the transgene locus and enhances transformation efficiency in eukaryotic cells. The possible role of TJ1-SAR in the molecular evolution of plant genome is discussed.
Shimizu
K
K
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima, 739-8526 Japan.
Takahashi
M
M
Goshima
N
N
Kawakami
S
S
Irifune
K
K
Morikawa
H
H
D020856:AT Rich Sequence; D001483:Base Sequence; D019470:Biolistics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004250:DNA Topoisomerases, Type II; D018744:DNA, Plant / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D019143:Evolution, Molecular; D018628:Gene Dosage; D018745:Genome, Plant; D007613:Kanamycin Resistance / Q000235:genetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000166:cytology / Q000235:genetics*; D011485:Protein Binding; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D014026:Nicotiana / Q000166:cytology / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics*; D019076:Transgenes
D018744:DNA, Plant; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004250:DNA Topoisomerases, Type II