Cloning and expression of the NaeI restriction endonuclease-encoding gene and sequence analysis of the NaeI restriction-modification system.
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.
Taron
C H
CH
New England Biolabs, Beverly, MA 01915, USA.
Van Cott
E M
EM
Wilson
G G
GG
Moran
L S
LS
Slatko
B E
BE
Hornstra
L J
LJ
Benner
J S
JS
Kucera
R B
RB
Guthrie
E P
EP
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
D011994:Recombinant Proteins; C060630:endodeoxyribonuclease NaeI; D015252:Deoxyribonucleases, Type II Site-Specific