208 Hornby

pBR322 ~4~~

plaemld Hi:t/~lb:te d

CUT Hlncll

SlteHIncll(s) ~

Cleaved

Llneerlsed

Plasmld

Two DNA

fragments

Fig. 2. Selective inactivation of partially degenerate restriction sites.

In a second approach, the use of a DNA Mtase to protect a restriction
site involves the methylation of bases that are part of overlapping
Mtase sites. Thus, in the example shown in Fig. 3, BamHI and MspI
sites overlap by two bases in the sequence GGATCCGG. Methylation
of the 5' dC of the MspI site renders the BamHI site refractory to
BamHI endonuclease. Those BamHI sites that are not followed by a
GpG dinucleotide are not methylated by MspI Mtase and are therefore
not protected.

3.3. In Vitro Methylation of Eukaryotic Genes
Methylation of dC bases in mammalian DNA has been implicated
in the control of gene transcription. The availability of methylated and
unmethylated DNA for the experimental investigation of this phenom-
enon is highly desirable. In order to reproduce eukaryotic methylation
patterns in vitro, the DNA must either be synthesized chemically from
a 5-methyldCTP precursor or prepared enzymically using a prepara-
tion of a CpG-specific Mtase. Alternative but somewhat less satisfac-
tory Mtases include HpalI (CCGG-specific) and HhaI (CGCG), which