Purification and characterization of LetA and LetD proteins of the F plasmid, proteis which control cell division of Escherichia coli.
| Project/Area Number |
60480463
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | Kyushu University |
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Principal Investigator |
HORIUCHI TADAO Faculty of Pharmaceutical Sciences, Kyushu University, Professor, 薬学部, 教授 (10037567)
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| Co-Investigator(Kenkyū-buntansha) |
SAGAR YASUHIRO Faculty of Pharmaceutical Sciences, Kyushu University, Assistant, 薬学部, 助手 (60162319)
KOGA HIDEO Faculty of Pharmaceutical Sciences, Kyushu University, Assistant, 薬学部, 助手 (20101173)
MIKI TAKEYOSHI Faculty of Pharmaceutical Sciences, Kyushu University, Associate Professor, 薬学部, 助教授 (40037586)
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| Project Period (FY) |
1985 – 1986
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| Project Status |
Completed (Fiscal Year 1986)
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| Budget Amount *help |
¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 1986: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1985: ¥5,700,000 (Direct Cost: ¥5,700,000)
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| Keywords | Cell division / DNA-replication / letA gene / letD gene / protein purification / F plasmid / 大腸菌 |
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| Research Abstract |
Coupling between DNA replication of the F plasmid and cell division of the host E. coli cells is controlled by at least seven host coded-genes including groES (unpublished observation) and two F plasmid-coded genes, letA and letD (Miki, et al., J.Mol.Biol.174 605-625 (1984), Miki et al. ibid. 174 627-646 (1984)). The letD gene product acts to inhibit cell division of the bacteria, whereas the letA gene product acts to suppress the activity of the letD gene product and to induce cell division. We have constructed a plasmid that overproduces the LetA and LetD proteins and have purified them to homogeneity by monitoring its presence after polyacrylamide gel electrophoresis. Both gel filtration on a TSK G3000SW column and ion exchange chromatography on a TSK DEAE-5PW indicate that the LetA and LetD proteins exist in a form of a complex of Mr 64,000. After denaturing the complex by guanidine hydrocloride, we have purified the LetA and LetD proteins separately using reversed phase chromatography. The amino acid sequences of the first seven amino acids and overall compositions of the purified LetA and LetD proteins match those predicted by the nucleotide sequences of the letA and LetD genes, respectively. The amino acid composition of the LetA-LetD complex indicates that the complex is composed of two molecules of LetA proteins and four molecules of LetD proteins. Using nitrocellulose filter binding assay, the LetA-LetD complex was indicated to bind to the mini-F DNA sequence. These results suggest that the LetA and LetD proteins act directly to mini-F DNA sequence in a form of a complex in coupling DNA replication of the F plasmid and cell division of the host bacteria.
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Report
(1 results)
Research Products
(7 results)
