Optimized genomic sequencing as a tool for the study of cytosine methylation in the regulatory region of the chicken vitellogenin II gene.
Some critical parameters of genomic sequencing are described. As an example we show a 200-nucleotide (nt) sequence of the estradiol-regulated avian vitellogenin gene II upstream region containing four CpG nt pairs. The two CpG's at positions -612 and -618 are in the sequence binding estradiol-receptor complex. While all four CpG's are methylated in erythrocytes, they are hypomethylated in the DNA of estradiol-responsive organs of egg-laying hens. A simple electroblot DNA transfer system which gives no distortion of DNA bands and quantitative transfer of denatured DNA from the gel to the filter membrane is described. Using Gene Screen membranes, a maximal hybridization signal was obtained when 30-50% of the input DNA was stably bound to the filters. Hybridization background signal and exposure time could be largely reduced by using highly purified fractionated DNA. Using a 90-120 nt long homogeneous single-stranded DNA probe of high specific activity it was possible to read a genomic sequence of up to 200 nt. The resolution was further improved by reducing the extent of chemical modifications of the DNA during the Maxam-Gilbert sequencing reactions.
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D003596:Cytosine; D004247:DNA / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D005796:Genes; D005809:Genes, Regulator; D006358:Hot Temperature; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008745:Methylation; D009693:Nucleic Acid Hybridization; D014466:Ultraviolet Rays; D014819:Vitellogenins / Q000235:genetics*
D014819:Vitellogenins; D003596:Cytosine; D004247:DNA