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

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Calgene Pacific

PO Box 53, Ivanhoe, Victoria 3079, Australia

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Calgene Pacific

PO Box 53, Ivanhoe, Victoria 3079, Australia

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Molecular Surgery, City of Hope National Medical Center

Duarte, CA 91010, USA

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Molecular Surgery, City of Hope National Medical Center

Duarte, CA 91010, USA

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Received:

04 February 1988

Revision received:

28 March 1988

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D.M. Woodcock, P.J. Crowther, W.P. Diver, M. Graham, C. Bateman, D.J. Baker, S.S. Smith, RgIB facilitated cloning of highly methylated eukaryotic DNA: the human L1 transposon, plant DNA, and DNA methylated in vitro with human DNA methyltransferase , Nucleic Acids Research, Volume 16, Issue 10, 25 May 1988, Pages 4465–4482, https://doi.org/10.1093/nar/16.10.4465
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Abstract

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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