| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1993: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
We are studying about the function of the replication protein of plasmid Rtsl which is not able to replicate at higher temperature (42 C). A mutant, RepAz279, which showed inability of Rtsl ori-activation and incompatibility with mini-Rtsl was previously isolated. DNA binding activity of RepAz279 was tested with gel shift assay using fragments containing (1) Rtsl-ori and incII, (2) Rtsl-ori, incII,and operator-promoter, (3) incI,and (4) operator-promoter. As results, the mutant protein RepAz279 did not show any significant differencies from wild-type RepA protein in their patterns of band shifting. Additionally, RepAz279 was observed to bend the Rtsl-ori fragment as same as RepA wild-type. RepAzs279 protein showed autorepressor activity as same with wild type by studying with promoter-lac fusion.
It was concluded that amino acid change of 279th (Arg to Gly) did not have any significant effect on DNA-binding activity of RepAz279
To study the role of C-terminal of RepA protein became to be important, since the amino acid change of RepAz279 is closer to its C-terminus. To do that, some of hybrid proteins which were composed of N-terminus peptide from Rtsl RepA and C-terminus peptide from P1 RepA.Rtsl RepA and P1 RepA share high homology in its amino acid sequence. To study those hybrid protein brought us interesting observations. Hybrid RepA protein containing C-terminal 25 amino acids from P1 RepA had autorepressor activity but that containing 80 amino acids from P1 RepA did not. It is reported for another RepA protein that dimer form has the autorepressor activity. It is possible that C-terminus of RepA protein has important role for its dimerization. Therefore amino acid change near C-terminus in RepAz279 may affect the interaction between RepA proteins.
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