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Nucleic Acids Research, 1988, Vol. 16, No. 10 4465-4482
© 1988

Articles

RgIB facilitated cloning of highly methylated eukaryotic DNA: the human L1 transposon, plant DNA, and DNA methylated in vitro with human DNA methyltransferase

D.M. Woodcock*, P.J. Crowther, W.P. Diver, M. Graham1, C. Bateman1, D.J. Baker2 and S.S. Smith2

Molecular Science Group, Peter MacCallum Cancer Institute 481 Little Lonsdale St.Melbourne, Victoria 3000, Australia 1Calgene Pacific PO Box 53, Ivanhoe, Victoria 3079, Australia 2Molecular Surgery, City of Hope National Medical Center Duarte, CA 91010, USA

*To whom correspondence should be addressed

Received February 4, 1988. Revised March 28, 1988. Accepted March 28, 1988.

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 Kpnl 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.


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