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

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Abstract
Hg-substituted UTP was used to study the transcription of duck reticulocyte chromatin in vitro by Escherichia coli RNA polymerase. The use of this method resulted 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 duplex product in which 1 strand is the endogenous message, and the other is the Hg-labeled complementary strand. Subsequent purification of the Hg-substituted RNA on thiol-agarose results in copurification of endogenous globin sequences. The details of this mechanism were documented and the methods which will eliminate the artefact were described.