1997 Fiscal Year Final Research Report Summary
Characterization of DNA sequences of junction regions and development of vectors for high frequency transformation
| Project/Area Number |
08458239
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Developmental biology
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| Research Institution | HIROSHIMA UNIVERSITY |
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Principal Investigator |
MORIKAWA Hiromichi Fac.of Sci., HIROSHIMA UNIVERSITY Professor, 理学部, 教授 (00089129)
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| Co-Investigator(Kenkyū-buntansha) |
GOSIMA Naoki Fac.of Sci., HIROSHIMA UNIVERSITY Associate Professor, 理学部, 助教授 (70215482)
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| Project Period (FY) |
1996 – 1997
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| Keywords | Trangene / Integration / S / MAR / Position Effect / Particle Bombardment / Site-directed / Topoisomerase / Cultured tobacco cells |
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| Research Abstract |
A 12.5-kb DNA fragment containing junction regions between transgene and genomic DNA was cloned from a transgenic tobacco cell line that was obtained by microprojectile bombardment of plasmid pCaMVNEO.Nucleotide sequence analysis of the fragement (DDBJ accession no.84238) showed that it contained a 7.7-kb core sequence (concatemer of a complete pCaMVNEO and a partial pCaMVNEO) and two identical 1.3-kb junction sequences flanking at both 5' and 3' ends of the core sequence with an inverted orientation. The 1.3-kb sequences contained topoisomerase II (Topo II) consensus sequences and AT-rich sequences that are known to be specific to the nuclear scaffold associated regions (SARs). An in vitro binding assay showed that a 507-bp fragment (designated as TJ1) taken from the 1.3-kb sequence had ability to bind to nuclear scaffold preparations of cultured tobacco cells, confirming that the 1.3-kb sequence is an S/MAR.
The yield of geneticin-resistant transformant was increased by 5- to 10-fold
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by the insertion of the 507-bp fragment at the 5' and 3' sides of the expresion cassette for the nptII gene in the plasmid. The uptII enzyme activity per copy of the gene expression per copy was 10-fold higher in the transformants that had TJ1-containing plasmid than those that had TJ1-free wild type plasmid. It is suggested that TJ1 and TJ2 sequences were very useful for transgenic technology in plants.
Three transgenic Arabidopsis lines indicating a single Southern hybridization band with a selectablegene used as probes were analyzed. The junction regions flanked by transgenes were cloned using inverse polymerase chain reaction methods. All but one of the junction regions were AT-rich sequences containingg motifs characterized by an S/MAR,and calculations showed that seven of them should have a propensity towards curvature. An in vitro binding assay to tobacco nuclear matrices proved that all junction regions had the ability to bind to nuclear matrices and showed that the two input DNA did not. Less
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