Methylation of intact chromosomes by bacterial methylases in agarose plugs suitable for pulsed-field electrophoresis. Methylation of intact chromosomes in agarose by methylases.
Conditions were determined for the methylation of intact yeast chromosomes by EcoRI, HhaI, and MspI bacterial methylases using an endonuclease protection assay while the chromosomes were embedded in agarose plugs suitable for transverse-field electrophoresis. Parameters were also established for the methylation of human chromosomes by EcoRI methylase. Methylation of embedded chromosomes by EcoRI methylase required prewashes with EDTA. EcoRI, HhaI, and MspI methylases showed optimal activity when nonacetylated bovine serum albumin, high levels of S-adenosylmethionine, and high levels of methylase were used. The use of bacterial methylases for methylation of embedded chromosomes will allow investigators to normalize variations in cellular DNA methylation prior to restriction and create new and rare endonuclease recognition sites which will facilitate the detection of chromosomal alterations and deletions.
Wilson
W W
WW
Department of Pediatrics, University of California, San Diego, La Jolla 92093.
Hoffman
R M
RM
D015825:Chromosomes, Fungal / Q000378:metabolism*; D002877:Chromosomes, Human / Q000378:metabolism*; D015254:DNA Modification Methylases / Q000378:metabolism*; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D004492:Edetic Acid; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008745:Methylation; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism
D004492:Edetic Acid; D015254:DNA Modification Methylases; C020098:DNA modification methylase EcoRI; C037664:DNA modification methylase HhaI; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)