Biological activity of hemimethylated and single-stranded DNA after direct gene transfer into tobacco protoplasts

AbstractIn this investigation we analyzed the effect of DNA methylation on gene expression in tobacco protoplasts. Methylation of the chimeric GUS gene (CaMV 35s/GUS/NOS) with the HpaII methyltransferase had no effect on transient and on stable GUS gene expression. The chimeric gene was then cloned as single-stranded DNA. In vitro second-strand DNA synthesis was performed with 5-methylcytosine, leading to methylation of every cytosine in one DNA strand. After transfection this DNA was totally inactive. Control experiments revealed that the single-stranded DNA was as active as the double-stranded form