Nature of the deoxyribonucleic acid made by isolated liver nuclei

Abstract

To better study the nature of the process of deoxyribonucleotide incorporation by isolated nuclei from regenerating rat liver, improvements have been made in the assay mixture. The changes include the addition of a Ca 2+ binder, ethylene glycol-bis-(2- aminoethyl ether)-N,N′- tetraacetate (EGTA), a high molecular weight compound such as dextran, and a greater concentration of buffer. The EGTA and increased buffer depress the hydrolysis of DNA during incubation of the nuclei at 37 °C. Under the new conditions, the initial rate of DNA synthesis is raised by about 3-fold. Additional evidence is given to show that the formation of DNA in vitro is by the elongation of the chains that were growing in vivo. The growing points of both strands of DNA appear to be advanced by the isolated nuclei. The approximate rates of advancement in vivo and in vitro were 1400 and 200 deoxynucleotides per min per strand, respectively.