Synthesis of Deoxyribonucleic Acid by Isolated Liver Nuclei

Abstract

Abstract The incorporation of 3H-TTP into the DNA of intact nuclei isolated from the livers of normal and partially hepatectomized rats has been studied. The regenerating nuclei incorporate 3H-TTP at about 10 times the rate of normal nuclei. The requirements for a maximal rate of incorporation include the three unlabeled deoxynucleoside triphosphates, ATP, and Mg++. Three pieces of evidence are presented to show that the replication of DNA by the isolated regenerating nuclei is a continuation of the process that was going on in vivo. First, with one exception, a constant relationship exists between the abilities of the nuclei to form DNA in vivo and in vitro after various manipulations of the partially hepatectomized rat. The exception is in the responses of the nuclei after treatment of the rat with cycloheximide. Second, only nuclei that were forming DNA in vivo are able to incorporate 3H-TTP in vitro. Finally, the isolated nuclei use few or no improper sites of initiation of synthesis. Most or all of the DNA formed is added to polydeoxynucleotide strands that were growing in vivo. The possibility is considered that one of the differences between normal and regenerating nuclei is that the nonreplicating nuclei lack sites of initiation for DNA synthesis. Treatment of normal nuclei with pancreatic DNase causes a marked stimulation in 3H-TTP incorporation.