title: Cloning and expression of the NaeI restriction endonuclease-encoding gene and sequence analysis of the NaeI restriction-modification system.
abstract: NaeI, a type-II restriction-modification (R-M) system from the bacterium Nocardia aerocolonigenes, recognizes the sequence 5'-GCCGGC. The NaeI DNA methyltransferase (MTase)-encoding gene, naeIM, had been cloned previously in Escherichia coli [Van Cott and Wilson, Gene 74 (1988) 55-59]. However, none of these clones expressed detectable levels of the restriction endonuclease (ENase). The absence of the intact ENase-encoding gene (naeIR) within the isolated MTase clones was confirmed by recloning the MTase clones into Streptomyces lividans. The complete NaeI system was finally cloned using E. coli AP1-200 [Piekarowicz et al., Nucleic Acids Res. 19 (1991) 1831-1835] and less stringent MTase-selection conditions. The naeIR gene was expressed first by cloning into S. lividans, and later by cloning under control of a regulated promoter in an E. coli strain preprotected by the heterologous MspI MTase (M.MspI). The DNA sequence of the NaeI R-M system has been determined, analyzed and compared to previously sequenced R-M systems.
authors.affiliation: New England Biolabs, Beverly, MA 01915, USA.
authors.forename: C H
authors.initials: CH
authors.lastname: Taron
authors.forename: E M
authors.initials: EM
authors.lastname: Van Cott
authors.forename: G G
authors.initials: GG
authors.lastname: Wilson
authors.forename: L S
authors.initials: LS
authors.lastname: Moran
authors.forename: B E
authors.initials: BE
authors.lastname: Slatko
authors.forename: L J
authors.initials: LJ
authors.lastname: Hornstra
authors.forename: J S
authors.initials: JS
authors.lastname: Benner
authors.forename: R B
authors.initials: RB
authors.lastname: Kucera
authors.forename: E P
authors.initials: EP
authors.lastname: Guthrie
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D015252:Deoxyribonucleases, Type II Site-Specific / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009615:Nocardia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; C060630:endodeoxyribonuclease NaeI; D015252:Deoxyribonucleases, Type II Site-Specific