RglB facilitated cloning of highly methylated eukaryotic DNA: the human L1 transposon, plant DNA, and DNA methylated in vitro with human DNA methyltransferase.
In vitro methylation of Bluescribe plasmid DNA (pBS) with human placental DNA methyltransferase to 6% 5-methylcytosine (mC) reduced transformation efficiencies in rglB+ host strains C600 and DS410 by almost 2 orders of magnitude. By contrast, the rglB- derivative of DS410 showed no reduction in transformation efficiency with methylation while the rglB- derivative of C600 was partially tolerant to methylation. Further, we show that the 1.8 kilobase (kb) and 1.2 kb KpnI fragments derived from the human L1 repeat have respectively 18.3% and 2.3% mC in vivo. Using these hyper- and hypo-methylated genomic segments ligated into the pBS plasmid, transformants with the highly methylated 1.8 kb L1 insert were recovered at 17 to 40 fold higher frequency with the rglB- host strains than with the rglB+ hosts. In addition, recombinant phage (lambda 2001) containing inserts of plant genomic DNA with 26.7% mC (from Petunia hybrida) when plated on rglB- hosts gave titres up to 222 times higher than on the rglB+ strains.
Woodcock
D M
DM
Molecular Science Group, Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia.
Crowther
P J
PJ
Diver
W P
WP
Graham
M
M
Bateman
C
C
Baker
D J
DJ
Smith
S S
SS
D002460:Cell Line; D003001:Cloning, Molecular; D003596:Cytosine / Q000378:metabolism; D004247:DNA / Q000235:genetics*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D006801:Humans; D008745:Methylation; D010944:Plants / Q000235:genetics*; D010957:Plasmids; D013154:Spleen / Q000378:metabolism; D014169:Transformation, Bacterial
D004251:DNA Transposable Elements; D003596:Cytosine; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases