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DNA of replication foci attached to the nuclear matrix was isolated from Chinese hamster ovary cells and human HeLa cells synchronized at different stages of the G(1) and S phases of the cell cycle. The abundance of sequences from dihydrofolate reductase ori-beta and the beta-globin replicator was determined in matrix-attached DNA. The results show that(More)
To study the organization of DNA replication in mammalian rRNA genes, the sites of initiation of DNA synthesis in rat and human rRNA genes were mapped by two independent techniques. In rat cells the growth of the nascent DNA chains was blocked by Trioxsalen cross-links introduced in vivo. The fraction of "restricted" nascent DNA chains labeled in vivo was(More)
1. The effect of gamma-irradiation (4000rd) on the synthesis of ribosomal (pre-rRNA) and heterogeneous nuclear RNA (pre-mRNA) in normal and in regenerating rat liver was studied by using 40 min labelling with [6(-14)C]orotic acid. 2. Partial hepatectomy caused a sharp transient increase in the specific radioactivity of the endogenous low-molecular-weight(More)
Using a previously developed procedure (Gencheva et al. [1996] J Biol Chem 271:2608-2614), we isolated a DNA fraction consisting of short fragments originating from the regions of initiation of DNA synthesis from exponentially growing Chinese hamster ovary cells. This fraction arbitrarily designated as "collective origin fraction" was labeled in vitro and(More)
HeLa cells were synchronized at late G1, early S, and late S phase of the cell cycle by nocodazole treatment. The cells were permeabilized with Triton X-100, digested with DNAse I, and extracted with 0.2 M ammonium sulfate to remove the digested chromatin. DNA was isolated from the residual chromatin attached to the nuclear matrix, digested with Hind III,(More)
The role of poly(ADP-ribosyl)ation in chromatin replication and the activity of poly(ADP-ribose) synthetase in the newly synthesized and old chromatin was studied. It was found that 3-aminobenzamide, which is an inhibitor of poly(ADP-ribose) synthetase, had no effect on the initiation of DNA synthesis and only a moderate effect on DNA chain elongation.(More)
In the pH interval 10.5-11.8, 70% of the nonhistone proteins normally present in rat liver chromatin were dissociated. The rest remained complexed with DNA even at pH 13. Dodecylsulfate-polyacrylamide gel electrophoresis revealed that the majority of the high-molecular-weight nonhistone proteins together with a few characteristic fractions with molecular(More)
Three types of density gradients - neutral metrizamide, alkaline NaOH-metrizamide and alkaline triethanolamine-metrizamide - were used for studying the distribution of histones between the two DNA strands in alkali-denatured chromatin. It was found possible to avoid both protein redistribution and dissociation by using triethanolamine-metrizamide density(More)
In higher eukaryotic cells, DNA is tandemly arranged into 10(4) replicons that are replicated once per cell cycle during the S phase. To achieve this, DNA is organized into loops attached to the nuclear matrix. Each loop represents one individual replicon with the origin of replication localized within the loop and the ends of the replicon attached to the(More)
Chinese hamster ovary (CHO) cells were synchronized by a thymidine-hydroxyurea block. At different times after release from the block, cells were treated with trioxsalen and long-wavelength ultraviolet light to crosslink DNA in vivo and were labelled with [3H]thymidine for 30 min. This technique permits labelling of only the short nescent DNA fragments(More)