We have developed a soluble enzyme system that replicates exogenously added plasmid DNA (Adv) bearing the replication origin of the bacteriophage A chromosome. The system contains pure phage A 0 and P replication proteins and a partially purified mixture of Escherichia coli replication proteins [the enzyme system of Fuller, R. S., Kaguni, J. M. & Kornberg, A. (1981) Proc. NatL Acad. Sci USA 78,[7370][7371][7372][7373][7374]. The features of Adv replication in this system closely resemble the known characteristics of phage A DNA replication in viva. The system (i) depends completely on exogenously supplied DNA, (ii) specifically replicates supercoiled plasmid DNA that contains a A replication origin, (iii) depends on both the A 0 protein and the A P protein, (iv) depends on RNA polymerase, (v) depends on host replication proteins (e.g., primase, dnaB protein, and several others that function in the priming of DNA synthesis in E. coli) as judged by antibody inhibitions, and (vi) replicates as much as 32% of added Adv plasmid DNA through a single complete round to generate catenated daughter molecules. Furthermore, replication of Adv DNA in vitro requires DNA gyrase and an ATP-regenerating system. It is notable that addition of A 0 and P proteins to the mixture of E. coli replication proteins inhibits replication of plasmids bearing the origin of the E. coli chromosome. Exploitation of this enzyme system should allow a detailed investigation of the biochemical mechanisms involved in bacteriophage A DNA replication and its regulation.An essential feature of the life cycle of both prokaryotic and eukaryotic cells is a strict regulation of the initiation of a new round of chromosome replication. This replication proceeds bidirectionally from unique chromosomal origins (1). Because of the complexity of the process, the biochemical mechanisms involved in the initiation of bidirectional DNA replication and its regulation have not been elucidated for any organism.The chromosome of coliphage A is ideally suited for model studies on the initiation and regulation of duplex DNA replication. The results of more than 25 years of intensive research on the genetics and physiology of phage A gene regulation and DNA replication can be brought to bear on this problem (2). Replication is initiated on supercoiled A chromosomes (3) and proceeds bidirectionally from a single fixed origin (4). At early times after infection, A DNA molecules are replicated in a circular mode via 6 structure intermediates (3-5). This early replication generates catenated circular monomers, nicked circles (a circular DNA molecule containing an interruption in one or both DNA strands), and covalently closed circles as products (3). Both viral and host proteins participate in A DNA replication. Genetic studies indicate that the products of A genes 0 and P (6, 7) and the Escherichia coli dnaB, dnaJ, dnaK, primase, and DNA polymerase III holoenzyme replication proteins (unpublished data; refs. 8 and 9) are all essential for viral DNA replication. Final...
In the present study, we have shown that insulin-like growth factor-I (IGF-I) was released by primary cultures of rat osteoblast-like (ROB) cells into the conditioned medium (CM). Dexamethasone (DEX) caused a dose-dependent inhibition of the IGF-I. At 10(-8) M, DEX reduced IGF-I level to 70% of the control value (P less than 0.05); at 10(-7) M DEX, the IGF-I level was further reduced to 60% of the control (P less than 0.01). The active vitamin D metabolite 1,25-dihydroxycholecalciferol [1,25(OH)2D3] slightly increased the IGF-I level, but the increase was not statistically significant. However, in combined treatments of 10(-7) M DEX and 10(-8) M of 1,25(OH)2D3, the inhibition of DEX was partially antagonized by the presence of 1,25(OH)2D3. Studies with metabolically radiolabeled IGF-I by immunoprecipitation indicated the changes of IGF-I in the CM reflected synthesis of the protein by the cells. The alteration of IGF-I level may mediate some of the actions of these steroid hormones on bone cells.
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