Purification, Properties and Determinations of Recognition Sequence and Cleavage Site of Restriction Endonuclease from "Agrobacterium gelatinovorum" IAM 12617, a Marine Bacterium (AgeI)(Microbiology & Fermentation Industry)

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概要

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• A new restriction endonuclease, designated as AgeI, was purified from cell-free extracts of a marine bacterium, "Agrobacterium gelatinovorum" IAM 12617 by streptomycin treatment, ammonium sulfate fractionation, combined column chromatographies on heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and FPLC on Mono Q (HR 5/5) and Superose 12 (HR 10/30). The purified enzyme was homogeneous on SDS-polyacrylamide gel disc electrophoresis and free from other phosphatase and exonuclease activities on ligation-recutting test. The relative molecular mass of the enzyme was 24,000 daltons by SDS-polyacrylamide gel disc electrophoresis. The gel filtration using Superose 12 (HR 10/30) gave the same calculation (23,000 daltons). These data indicated that the enzyme is a monomer. The isoelectric point of the enzyme was 6.5. The purified enzyme cleaved λ and Ad2 DNAs at 10 or more and 5 sites, respectively. However, the purified enzyme did not cleave SV40, φX174 RF I, M13mpl8 RF I or pBR322 DNAs. pBR328 DNA was cleaved at 1 site by the purified enzyme. The purified enzyme worked best at 37℃ and pH 7.5 in a reaction mixture (50 μl) containing 1.0 μg λDNA, 10mM Tris-HCl, 7mM 2-mercaptoethanol, 7mM MgCl_2 and 50mM NaCl. The purified enzyme did not require monovalent cations necessarily for the enzyme reaction. The enzyme recognized the palindromic hexanucleotide DNA sequence 5'-ACCGGT-3' and cut between A and C, producing a 5'-cohesive tetranucleotide extension.

• 社団法人日本農芸化学会の論文
• 1990-07-23

著者

• Suzuki T

  Tokyo Univ. Fisheries Tokyo Jpn

• YAMADA Yuzo

  Laboratory of Applied Microbiology, Department of Applied Biological Chemistry, Faculty of Agricultu

• Suzuki Toshihiko

  Laboratory Of Applied Microbiology Department Of Agricultural Chemistry Shizuoka University

• YAMASATO Kazue

  Institute of Applied Microbiology, The University of Tokyo

• Yamada Y

  Laboratory Of Applied Microbiology Department Of Agricultural Chemistry Shizuoka University

• Suzuki T

  Laboratory Of Applied Microbiology Department Of Agricultural Chemistry Shizuoka University

• YAMASATO Kazuhide

  Institute of Applied Microbiology, The University of Tokyo

• MIZUNO Hirofumi

  Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Shizuoka University

• AKAGAWA Masayo

  Institute of Applied Microbiology, The University of Tokyo

• Akagawa Masayo

  Institute Of Applied Microbiology The University Of Tokyo:(present Office)department Of Chemistry Un

• Mizuno H

  Shizuoka Univ. Shizuoka Jpn

• Mizuno Hirofumi

  Laboratory Of Applied Microbiology Department Of Agricultural Chemistry Shizuoka University

• Yamasato Kazue

  Institute Of Applied Microbiology The University Of Tokyo

• Yamada Yuzo

  Laboratory Of Applied Microbioligy Department Of Agricultural Chemistry Shizuoka University

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