| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1988: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1987: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
The structure of a 1.5-kb DNA sequence that is necessary and sufficient for the DNA replication of an 8.2-kb cryptic plasmid, pFTB14, isolated from a strain of Bacillus amyloliquefaciens has been characterized. The 1.5-kb DNA sequence contains an open reading frame, rep, stretching for 1017 bp, a promoter region for rep expression, and a possible replication origin. Genetic analysis by deletion and insertion mutations suggested that the rep product is trans-active and essential for plasmid automonous replication. The predicted rep protein is a basic protein, as are the RepC protein of pT181, RepB of pUB101 and protein A of pC194 which are found in staphylococci and used as Bacillus vectors. The redicted rep protein has highly similar amino acid sequences with protein A of pC194 and RepB of pUB101 through the protein molecule, but not with RepC of pT181 or protein RepH encoded by and initiating the replication of pC194.
In order to obtain rep protein, it was overproduced in Escherichia coli by the promoter system of lambda phage, and purified partially through DEAE-Toyopearl chromatography. The molecular weight of the rep protein was estimated to be 39,600 dal as same as that expected from DNA sequences. By the result of gel-shift assay analysis the protein was considered as DNA binding protein because of the binding activity on the restricted DNA sequence of 1.5-kb region, which is similar to the DNA regions for the replication origin of pC194 and pUB101.
The above results suggested the rep protein regulates DNA replication of PFTB14 through binding with the origin of DNA replication.
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