CHANGES IN SEDIMENTATION PROPERTIES OF HELA CELL DNA AND NUCLEOPROTEIN DURING REPLICATION

1. Short pulses of [3H]thymidine were used to label the newly replicated DNA of HeLa suspension cultures which had been grown for a generation time in [14C]thymidine. DNA was extracted by several methods from whole cells, nuclei and dispersed nucleoprotein, and the relative sizes of the newly replicated and bulk DNA were compared by sedimentation in alkaline and neutral sucrose gradients. 2. The results show that DNA is first synthesized in small pieces (less than 12 S) which are joined within 1 min into intermediate replicon-sized segments (26 S) and in 30-45 min into the final DNA product (> 39 S). 3. Dispersed nucleoprotein containing the newly replicated DNA sediments faster than the bulk nucleoprotein. However, when extracted from nucleoprotein by alkali or 2 M NaCl, the newly replicated DNA sediments more slowly than the bulk DNA. 4. Evidence is presented which suggests that DNA labeled with high specific activity [3H]thymidine can undergo significant radiolytic degradation within 1 week. © 1969.