Cloning of the resistant EcoRII recognition site of phage T7 into an EcoRII-sensitive plasmid makes the site susceptible to the restriction enzyme.
The recognition sequence 5'-CC(A/T)GG for EcoRII in the bacteriophage T7 genome is refractory to this restriction endonuclease, despite not bearing the specific (protective) methylation. Following the integration of this site as part of a 219 bp fragment (in which the recognition sequence is flanked by about 100 bp of T7 origin) into the EcoRII-sensitive vector pUC18, the T7 site becomes susceptible to cleavage, too. The same is true of recombinant pBR322 plasmids containing the T7-derived recognition site. The results show that the flanking sequences are not immediately responsible for the refractory behaviour of EcoRII sites and are in agreement with data according to which EcoRII requires the coordinated presence of at least two recognition sites in its DNA substrate.
Krüger
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DH
Institut für Medizinische Virologie, Humboldt-Universität, Berlin.
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Reuter
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Goebel
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D003001:Cloning, Molecular; D004279:DNA, Viral / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D015971:Gene Expression Regulation, Enzymologic; D010957:Plasmids / Q000235:genetics; D015183:Restriction Mapping; D013604:T-Phages / Q000235:genetics*
D004279:DNA, Viral; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific