Analysis of in vitro transcription of duck reticulocyte chromatin using mercury-substituted ribonucleoside...

We have employed mercury-substituted UTP to study the transcription of duck reticulocyte chromatin in vitro by Escherichia coli RNA polymerase. We find that the use of this method results in large overestimates of the amount of de novo synthesis of globin-specific RNA sequences. The artefact arises because endogenous globin RNA can serve as a template for the RNA polymerase, resulting in the formation of a duplex product in which one strand is the endogenous message, and the other is the mercury-labeled complementary strand. Subsequent purification of the mercury-substituted RNA on thiol-agarose results in copurification of endogenous globin sequences. We document the details of this mechanism and describe methods which will eliminate the artefact.