| Budget Amount *help |
¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 1990: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1989: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1988: ¥5,300,000 (Direct Cost: ¥5,300,000)
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| Research Abstract |
1. We have constructed an in vitro system involving Tn3 transposase that catalyzes the formation of DNA deletions. Activity of the reaction depened on Tn3 transposase and IHF (Proc. Japan Acad. Ser. B, 1-4, 1989). However, structure of the in vitro deletion products differed from that of in vivo products in that the deletions did not start from one of the ends of Tn3. Sequence analysis of the in vitro products showed that the deletions had occurred between two short homologous sequences (J. Dept. Liberal Arts Kansai Med. Univ., No.13, in press).
2. We analyzed the effect of the IHF and HU proteins on the deletion formation associated with Tn3 in vivo by using mutants lacking these proteins. Unexpectedly, both IHF and HU were unnecessary for the deletion formation (Gene, 76,359-362,1989 ; J. Kansai Med. Univ. Suppl., 41, 79-86). We also analyzed the effect of the activities of DNA polymerase I on DNA rearrangements associated with Tn3 in vivo and found that DNA polymerizing activity was
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not needed for the transposition of Tn3 into the host chromosome and that the 5^' to 3^' exonuclease activity was unnecessary for the DNA deletion forming activity (J. Dept. Liberal Arts Kansai Med. Univ. No. 12, 4-8, 1989).
3. We found that Tn3 transposase binds preferentially to superhelical DNA molecule (J. Dept. Liberal Arts Kansai Med. Univ. No. 13, in press) and that specific and non-specific DNA binding of the transposed are inhibited independently by different monoclonal antibodies (manuscript in preparation).
4. Plasmids with a unique "staggered head-to-head dimer" structure were obtained, possibly as the results of a reaction associated with the Tn3 transposase (manuscript in preparation).
5. We found an insertion of IS4 into the tnpA gene and sequenced the insertion site (Gene, 96, 129-132, 1990).
6. As for technical contributions, we improved the transformation procedure by PEG (Nucleic Acids Res. 18, 6169) and developed a method for measurement of DNA concentration with high sensitivity by fluorescence (Hitachi Scientific Instrument News, 34, in press). Less
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