Cloning, expression and sequence analysis of the SphI restriction-modification system.
SphI, a type II restriction-modification (R-M) system from the bacterium Streptomyces phaeochromogenes, recognizes the sequence 5'-GCATGC. The SphI methyltransferase (MTase)-encoding gene, sphIM, was cloned into Escherichia coli using MTase selection to isolate the clone. However, none of these clones contained the restriction endonuclease (ENase) gene. Repeated attempts to clone the complete ENase gene along with sphIM in one step failed, presumably due to expression of SphI ENase gene, sphIR, in the presence of inadequate expression of sphIM. The complete sphIR was finally cloned using a two-step process. PCR was used to isolate the 3' end of sphIR from a library. The intact sphIR, reconstructed under control of an inducible promoter, was introduced into an E. coli strain containing a plasmid with the NlaIII MTase-encoding gene (nlaIIIM). The nucleotide sequence of the SphI system was determined, analyzed and compared to previously sequenced R-M systems. The sequence was also examined for features which would help explain why sphIR unlike other actinomycete ENase genes seemed to be expressed in E. coli.
Guthrie
E P
EP
New England Biolabs, Inc., Beverly, MA 01915, USA. guthrie@neb.com
Quinton-Jager
T
T
Moran
L S
LS
Slatko
B E
BE
Kucera
R B
RB
Benner
J S
JS
Wilson
G G
GG
Brooks
J E
JE
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D015254:DNA Modification Methylases / Q000235:genetics*; D004269:DNA, Bacterial; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D004926:Escherichia coli; D015870:Gene Expression; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D015254:DNA Modification Methylases; C102921:DNA modification methylase SphI, Streptomyces; C046184:endodeoxyribonuclease PaeI; D015252:Deoxyribonucleases, Type II Site-Specific