M.FnuDII methyltransferase-mediated protection of pMTL85141 against CpaAI endonuclease. A. Time course digestion of pMTL85141 using crude protoplast extracts possessing CpaAI restriction activity, resolved on a 2% agarose gel. Digestion reactions contained 1.0 μg pMTL85141 and 25% protoplast extract in a total volume of 20 μl 1× CpaAI custom buffer. For comparison, pMTL85141 is shown undigested and digested with BstUI, a commercial isoschizomer of CpaAI. Expected digestion products are 1785, 581, 270, 252, and 75 bp. B. M.FnuDII-mediated protection of pMTL85141 from CpaAI digestion (left panel). Protoplast extract digestions contained 1.0 μg pMTL85141 or pMTL85141+pFnuDIIMKn, the vector harboring the M.FnuDII methyltransferase gene, and 25% protoplast extract in a total volume of 20 μl 1× CpaAI custom buffer. pMTL85141 preparation in the presence of plasmid pFnuDIIMKn afforded protection of pMTL85141 from both BstUI and CpaAI restriction, as no digestion products could be detected. Methylation treatment resulted in the presence of high-molecular weight bands. Linearization of the high molecular weight bands by NcoI digestion (right panel) confirmed the presence of pMTL85141 (2,963 bp) and the methylating plasmid, pFnuDIIMKn (6,449 bp), at the correct sizes of the individual linearized vectors.