EVIDENCE FOR GENE SILENCING BY ENDOGENOUS DNA METHYLATION

Transformed cells can spontaneously silence genes by de novo methylati on, and it is generally assumed that this is due to DNA methyltransfer ase activity. We have tested the alternative hypothesis that gene sile ncing could be due to the uptake of 5-methyl-dCMP into DNA, via the di - and triphosphonucleotides. 5-Methyl-dCMP would be present in cells f rom the ongoing repair of DNA, We have isolated a strain of Chinese ha mster ovary (CHO) cells, designated HAM(-), which spontaneously silenc es two tested genes at a very high frequency, We have shown that this strain incorporates 5-[H-3]methyldeoxycytidine into 5-methylcytosine a nd thymine in DNA, It also has low 5-methyl-dCMP deaminase activity. A nother HAM(+) strain has high deaminase activity and a very low freque ncy of gene silencing. The starting strain, CHO K1, has a phenotype in termediate between HAM(-) and HAM(+).