Structural Changes in Deoxyribonucleic Acid during Early Stages of Lens Regeneration in Triturus

Abstract

Abstract When the lens is removed from the newt Triturus, a new lens will form from epithelial cells of the dorsal iris. DNA was isolated from dorsal irises of animals which had been engaged in lens regeneration for 0, 3, 5, and 7 days. The nitrocellulose affinities of the DNA preparations were: 0-day, 12%; 3-day, 15%; 5-day, 86%; and 7-day, 60%. This suggests that structural alterations occur during the early stages of lens regeneration (and initiation of DNA synthesis). These structural changes can be correlated with the cell cycle. The DNA preparations were characterized by hydroxyapatite chromatography, buoyant density in CsCl and CsSO4(Hg), thermal denaturation, and sedimentation in neutral and alkaline sucrose gradients. The DNA isolated from 0-day regenerates (G0 phase of the cell cycle) behaved as native duplex structures containing few nicks. The DNA from 3-day regenerates (early G1 phase) contained a significant number of nicks. The DNA from 5-day regenerates (late G1 phase) contained gaps or missing runs of nucleotides. The DNA from 7-day regenerates (S phase) existed in two configurations. One possessed a great deal of single-stranded character, and the other was similar, but not identical, to the DNA from 5-day regenerates. These results lead us to suggest that prior to entry into the cell cycle, DNA exists in vivo as chromosomal duplexes which are isolated as structures possessing a maximum charge density. During G1, DNA is activated for replication by a process which at first leads to nicks, then gaps. During replication much of the DNA exists in vivo as an extended structure which is isolated as molecules which possess a great deal of single-stranded character.