Mode of enhancement in ribonucleic acid synthesis directed by chromium (III)-bound deoxyribonucleic acid

By forming a complex with calf thymus DNA, Cr(III), i.e., CrCl3 and Cr(NO3)3, significantly enhanced its template activity for in vitro RNA synthesis as assayed by 3H incorporation from [5-3H]uridine triphosphate (UTP). The extent of the augmentation in RNA synthesis was proportional to the binding ratio of Cr(III) to the template DNA. K2CrO4, on the other hand, neither bound to DNA nor enhanced its template activity. Experiments using rifampicin and heparin suggested that incorrect and nonviable initiation sites for RNA synthesis became functional in Cr(III)-bound DNA. The incorporation of [gamma-32P]adenosine triphosphate (ATP) into RNA synthesized on Cr(III)-bound DNA was 8 to 9 times greater than that on control DNA. This value was much higher than that of the 3H incorporation form [5-3H]UTP, i.e., the incorporation of 32P on Cr(III) bound DNA was 8 to 9 times greater that of 3H and less than twice that on control DNA. These results suggest that Cr(III) possibly induces the abnormal synthesis of RNA of a very low molecular weight, for most if not all the molecules, by binding to the template DNA.     

Control of RNA synthesis by chromatin proteins.

The effect of chromatin proteins on template activity has been studied. Using both E. coli RNA polymerase and calf thymmus polymerase B we have measured the number of initiation sites on chromatin and various histone-DNA complexes. Chromatin can be reconstituted with histone proteins alone and this complex is still a restricted template for RNA synthesis. The removal of histone f1 causes a large increase in the template activity. Chromatin is then treated with Micrococcal nuclease and the DNA fragments protected from nuclease attack ("covered DNA") are isolated. Alternatively, the chromatin is titrated with poly-D-lysine, and by successive treatment with Pronase and nuclease, the DNA regions accessible to polylysine are isolated ("open DNA"). Both fractions were tested for template activity. It was found that RNA polymerase initiation sites are distributed equally in open and covered region DNA.     

In the Simian Virus 40 In Vitro Replication System, Start Site Selection by the Polymerase α-Primase Complex Is Not Significantly Altered by Changes in the Concentration of Ribonucleotides

The simian virus 40 (SV40) in vitro replication system was previously used to demonstrate that the human polymerase (Pol) alpha-primase complex preferentially initiates DNA synthesis at pyrimidine-rich trinucleotide sequences. However, it has been reported that under certain conditions, nucleoside triphosphate (NTP) concentrations play a critical role in determining where eukaryotic primase initiates synthesis. Therefore, we have examined whether increased NTP concentrations alter the template locations at which SV40 replication is initiated. Our studies demonstrate that elevated ribonucleotide concentrations do not significantly alter which template sequences serve as initiation sites. Of considerable interest, the sequences that serve as initiation sites in the SV40 system are similar to those that serve as initiation sites for prokaryotic primases. It is also demonstrated that regardless of the concentration of ribonucleotides present in the reactions, DNA synthesis initiated outside of the core origin. These studies provide additional evidence that the Pol alpha-primase complex can initiate DNA synthesis only after a considerable amount of single-stranded DNA is generated.     

DNA primase-DNA polymerase alpha from simian cells: sequence specificity of initiation sites on simian virus 40 DNA.

Unique single-stranded regions of simian virus 40 DNA, phage M13 virion DNA, and several homopolymers were used as templates for the synthesis of (p)ppRNA-DNA chains by CV-1 cell DNA primase-DNA polymerase alpha. Intact RNA primers, specifically labeled with an RNA capping enzyme, were typically 6 to 8 ribonucleotides long, although their lengths ranged from 1 to 9 bases. The fraction of intact RNA primers 1 to 4 ribonucleotides long was 14 to 73%, depending on the template used. RNA primer length varied among primers initiated at the same nucleotide, as well as with primers initiated at different sites. Thus, the size of an RNA primer depended on template sequence. Initiation sites were identified by mapping 5' ends of nascent RNA-DNA chains on the template sequence, identifying the 5'-terminal ribonucleotide, and partially sequencing one RNA primer. A total of 56 initiation events were identified on simian virus 40 DNA, an average of 1 every 16 bases. Some sites were preferred over others. A consensus sequence for initiation sites consisted of either 3'-dCTTT or 3'-dCCC centered within 7 to 25 pyrimidine-rich residues; the 5' ends of RNA primers were complementary to the dT or dC. High ATP/GTP ratios promoted initiation of RNA primer synthesis at 3'-dCTTT sites, whereas low ATP/GTP ratios promoted initiation at 3'-dCCC sites. Similarly, polydeoxythymidylic acid and polydeoxycytidylic acid were the only effective homopolymer templates. Thus, both template sequence and ribonucleoside triphosphate concentrations determine which initiation sites are used by DNA primase-DNA polymerase alpha. Remarkably, initiation sites selected in vitro were strikingly different from initiation sites selected during simian virus 40 DNA replication in vivo.     

CHANGES IN CHROMATIN TEMPLATE ACTIVITY AND THEIR RELATIONSHIP TO DNA SYNTHESIS IN MOUSE PAROTID GLANDS STIMULATED BY ISOPROTERENOL

Chromatin template activity of mouse parotid glands increases after a single injection of isoproterenol (IPR), a procedure that causes, after a lag period of 20 hr, a marked stimulation of DNA synthesis and cell division in salivary glands of rodents. The increase in chromatin template activity occurs as early as 1 hr and peaks between 6 and 10 hr after IPR, paralleling previously reported changes in the incorporation of uridine-³H into total cellular RNA of mouse parotids. Template activity was measured in vitro in a system in which parotid gland chromatin was incubated with an exogenous RNA polymerase isolated from Escherichia coli. Similar results were obtained when template activity of parotid gland chromatin was assayed using an homologous RNA polymerase from mouse liver. Chromatin template activity in mouse parotids was also studied after the administration of drugs capable of inducing in salivary glands both DNA synthesis and secretion or secretion alone. The results indicate that the increased chromatin template activity occurring 6 hr after IPR is related to the subsequent onset of DNA synthesis. Furthermore, the increased chromatin template activity caused by IPR is inhibited by the previous administration of puromycin, an inhibitor of IPR-stimulated DNA synthesis.     

Template activity of chromatin during stimulation of cellular proliferation in human diploid fibroblasts

1. Contact-inhibited confluent monolayers of WI-38 human diploid fibroblasts can be stimulated to divide by replacing the medium with fresh medium containing 30% foetal calf serum. 2. Of the cells 40–75% are stimulated to divide with a peak DNA synthesis between 15 and 21h and a peak mitotic index between 28 and 30h after stimulation. 3. In the first 12h before the initiation of DNA synthesis there is a biphasic increase in the incorporation of [³H]uridine into RNA of whole cells. 4. This is paralleled by a similar biphasic stimulation of chromatin template activity measured in vitro in a system in which purified cell chromatin is incubated with an exogenous RNA polymerase isolated from Escherichia coli. 5. The changes in chromatin template activity are believed to represent activation of the genome, with more sites available for RNA synthesis, and to account almost entirely for the changes in RNA synthesis occurring in the whole cell.     

Abscisic acid as an inhibitor of RNA synthesis by RNA polymerase in vitro

Abscisic acid interacts with DNA which increases the stabilityof the double strand against heat or alkaline denaturation.Abscisic acid added to RNA polymerase systems isolated fromthe chromatin of coconut nuclei inhibits RNA synthesis by loweringthe template activity of DNA. No interaction with RNA polymeraseor the initiation factor (factor B) is discernible. (Received May 18, 1972; )