The restriction enzyme AlwNI is blocked by overlapping methylation.

Most strains of E.coli contain two sequence specific methylases, Dam and Dem. The adenine residue of the sequence GATC is methylated at the N 6 position by the Dam methylase whereas the Dem methylase recognizes the sequences CC(A/T)GG methylating the internal cytosine at the C 5 position. The commercially available restriction enzyme AlwNl (New England Biolabs) has a recognition sequence CAGN 3 /CTG and is a useful enzyme for creating deletions. During our study of the 5' flanking sequence of the amyloid precursor protein (APP) gene, we cloned a 3.8 kb Bamffl fragment (1) into pUC18 for subsequent deletional analyses. AlwNl digestion of recombinant plasmid which had been transformed into JM105 always resulted in a partial digest where only 4 out of 5 theoretical restriction enzyme sites were cut (Figure 1, lane 2). In addition to the expected fragments of 2179 bp, 213 bp and 190 bp, a band of 3902 bp was also generated. This large fragment contained an internal AlwNl recognition site of the following sequence ggcgC-AGCCCCTGgcaa (1). Since the underlined sequence was a potential dan methylation site, the plasmid was transformed into the dcm~ bacterial strain GM272 (genotype: F~ dam-3 dcm-6 hsdS2l mefol lacYl or Z4 galK2 gcdTll mtl-2 tonte or A31 tsx-l or-78 supEM (r/u-1)) (2). AlwNl digestion of this preparation of plasmid yielded two fragments of 2612 bp and 1290 bp (Figure 1, lane 3) instead of the 3902 bp fragment. The restriction enzyme AlwNl can therefore be blocked by overlapping dem methylation of its recognition site. Figure 1. Digestion of APP recombinant plasmid with/4/wNI. Lane 1: 1 kb ladder (BRL); lane 2: preparation of plasmid from JM105 (dcm +); lane 3: preparation of plasmid from GM272 (dcm~).