Artificial steroid hormone response element generated by dam-methylation.
Using the interaction of steroid hormone receptors with their palindromic response elements as an example, we show here that cloning in dam+ bacterial strains can lead to artifactual results due to methylation of adenine residues at the N-6 position. Substitution of the T by an A in the third position of the half palindromes of the hormone responsive element TGTTCT(1) yields a functional element only when amplification is made in dam+ bacteria. Mutant palindromes methylated at the N-6 position of this adenine exhibit the same affinity for progesterone and glucocorticoid receptors as the consensus response element, whereas their unmethylated counterpart binds with negligible affinity. These observations underline the significance of hydrophobic interactions between receptors and the major groove of the DNA for discrimination among various responsive elements, and point to the importance of using dam- bacterial strains for the correct identification of the nucleotide sequence of cis-acting elements.
Truss
M
M
Institut für Molekularbiologie und Tumorforschung, Marburg, Germany.
Bartsch
J
J
Chalepakis
G
G
Beato
M
M
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D045167:Progesterone Congeners / Q000494:pharmacology; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D011980:Receptors, Progesterone / Q000378:metabolism; D011987:Receptors, Steroid / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D014407:Tumor Cells, Cultured
D045167:Progesterone Congeners; D011965:Receptors, Glucocorticoid; D011980:Receptors, Progesterone; D011987:Receptors, Steroid; D004247:DNA; D008780:Methyltransferases; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)