NICK TRANSLATION OF ACTIVE GENES IN INTACT NUCLEI

Transcriptionally active genes in chromatin are maintained in a conformation such that they are exceedingly sensitive to attack by the enzyme DNase I. We have exploited this observation to introduce radioactive nucleotides specifically into active regions of the chromosome by performing nick translation reactions on intact nuclei. Nuclei are treated with low concentrations of DNase I such that active genes are nicked rather than solubilized. The subsequent addition of DNA polymerase I results in the incorporation of nucleotides at these nicked sites. This reaction results in the introduction of label into nucleosomal structures. Annealing reactions with the products of nuclear nick translation reactions and total cellular RNA or DNase I-treated DNA indicate that over 85% of the label is incorporated into sequences transcribed in the oviduct. Three enzymatic processes participate in this reaction. We have demonstrated that the discrimination we observe for active regions of chromatin is imparted by the nicking action of DNase I. These studies may now provide the technology necessary to effect the successful isolation of active genes from chromatin. © 1979.