Effect of prostaglandins on protein, RNA, DNA and collagen synthesis in experimental wounds

The effect of exogenous prostaglandins E₁, E₂ and F₂ (PGE₁, PGE₂ and PGF₂) on ³H-leucine, ³H-uridine, ³H-thymidine and ³H-proline incorporation in experimental cutaneous wounds has been studied in rats.

Prostaglandins E₁ and E₂ markedly stimulate the incorporation of these tritiated precursors, into protein, RNA, DNA and collagen synthesis, whereas F₂ inhibits it. All tested prostaglandins exhibit their maximum effect within the first hours following administration. Most active is PGE₁. These observations indicate that application of prostaglandins significantly stimulate incorporation with protein, RNA, DNA and collagen synthesis in the skin of wounded rats and thus, may play a role in epidermal cell growth and division as well as in scar-forming tissue.     

Effect of prostaglandins on protein, RNA, DNA and collagen synthesis in experimental wounds.

The effect of exogenous prostaglandins E1, E2 and F2alpha (PGE1, PGE2 and PGF2alpha) on 3H-leucine, 3H-uridine, 3H-thymidine and 3H-proline incorporation in experimental cutaneous wounds has been studied in rats. Prostaglandins E1 and E2 markedly stimulate the incorporation of these tritiated precursors, into protein, RNA, DNA and collagen synthesis, whereas F2 inhibits it. All tested prostaglandins exhibit their maximum effect within the first hours following administration. Most active is PGE1. These observations indicate that application of prostaglandins significantly stimulate incorporation with protein, RNA, DNA and collagen synthesis in the skin of wounded rats and thus, may play a role in epidermal cell growth and division as well as in scar-forming tissue.     

Initiation, elongation and pausing of in vitro DNA synthesis catalyzed by immunopurified yeast DNA primase: DNA polymerase complex.

Yeast DNA primase and DNA polymerase I can be purified by immunoaffinity chromatography as a multipeptide complex which can then be resolved into its functional components and further reassembled in vitro. Isolated DNA primase synthesizes oligonucleotides of a preferred length of 9-10 nucleotides and multiples thereof on a poly(dT) template. In vitro reconstitution of the DNA primase:DNA polymerase complex allows the synthesis of long DNA chains covalently linked to RNA initiators shorter than those synthesized by DNA primase alone. The SS (single-stranded) circular DNA of phage M13mp9 can also be replicated by the DNA primase:DNA polymerase complex. Priming by DNA primase occurs at multiple sites and the initiators are utilized by the DNA polymerase moiety of the complex, so that almost all the SS template is converted into duplex form. The rate of DNA synthesis catalyzed by isolated yeast DNA polymerase I on the M13mp9 template is not constant and is characterized by distinct pausing sites, which partly correlate with secondary structures on the template DNA. Thus, replication of M13mp9 SS DNA with the native primase:polymerase complex gives rise to a series of DNA chains with significantly uniform termini specified by the primase start sites and the polymerase stop sites.     

Mechanism of initiation of in vitro DNA synthesis by the immunopurified complex between yeast DNA polymerase I and DNA primase

The immunopurified yeast DNA-polymerase-I--DNA-primase complex synthesizes oligo(rA) and oligo(rG) molecules that are used as primer for replication of poly(dT) and poly(dC). Neither initiation nor DNA synthesis is observed with poly(dA) and poly(dI). Nitrocellulose-filter binding shows that the enzyme complex binds to deoxypyrimidine polymers, but not to deoxypurine polymers. Although the yeast complex initiates DNA synthesis on deoxypyrimidine homopolymers, it prefers to elongate pre-existing primer molecules rather than to initiate de novo DNA replication. The size of the oligo(rA) and oligo(rG) primer molecules has been determined by urea/polyacrylamide gel electrophoresis: longer oligoribonucleotides are synthesized when their utilization is prevented by omitting dNTP. An oligodeoxythymidylate template with a chain length as short as five residues can support oligo(rA) synthesis catalyzed by the yeast DNA-polymerase--DNA-primase complex and the size of the oligoribonucleotide products synthesized with oligodeoxythymidylate of differing chain length has also been determined. The mechanistic properties of the DNA-polymerase--DNA-primase complexes, purified from different eukaryotic organisms, appear to be very similar. The possible biological implication of the studies on the mechanism and specificity of initiation of DNA synthesis in a well-defined model template system has been discussed.     

DNA primase isolated from the yeast DNA primase-DNA polymerase complex. Immunoaffinity purification and analysis of RNA primer synthesis

We have utilized immunoaffinity chromatography as a means of efficiently isolating a stable yeast DNA primase from the DNA primase-DNA polymerase complex, allowing identification of the polypeptides associated with this DNA primase activity and comparison of its enzymatic properties with those of the larger protein complex. A mouse monoclonal antibody specifically recognizing the DNA polymerase subunit was used to purify the complex. Stable DNA primase was subsequently separated from the complex in high yield. The highly purified protein fraction which bound to the DNA polymerase antibody column consisted of polypeptides with apparent molecular masses of 180, 86, 70, 58, 49, and 47 kDa. DNA primase activity eluted with a fraction containing only the 58-, 49-, and 47-kDa polypeptides. Partial chemical cleavage analysis of these three proteins demonstrated that the 49- and 47-kDa polypeptides are structurally related while the 58-kDa protein is unrelated to the other two. A DNA primase inhibitory monoclonal antibody was able to inhibit the activity of the purified DNA primase as well as the activity of the enzyme in the larger complex. In immunoprecipitation experiments, all three polypeptides were found in the immune complex. Thus, these three polypeptides are sufficient for DNA primase activity. In reactions using ribonucleotide substrates and natural as well as synthetic DNA templates, the purified DNA primase exhibited the same precise synthesis of unit length oligomers as did the larger protein complex and was able to extend these RNA oligomers by one additional unit length. An examination of the effects of deoxynucleotides on these DNA primase-catalyzed reactions revealed that the yeast DNA primase is an RNA-polymerizing enzyme and lacks significant DNA-polymerizing activity under the conditions tested.     

Effects of 6-thioguanine-containing RNA on in vitro protein synthesis

6-Thioguanine was administered to rats 12 hr after partial hepatectomy at a dose of 40 mg/kg of body weight; 6 hr later, polyadenylic acid-containing RNA was isolated and was used to measure initiation of protein synthesis $\text{in vitro}$ in a wheat germ system. $\text{In vitro}$ initiation was found to be 2.3-fold greater when 6-thioguanine-containing RNA was employed, than when polyadenylic acid-containing RNA isolated from untreated animals was used. The homopolymer, poly(TG), did not promote peptide synthesis in the wheat germ $\text{in vitro}$ system employed.     

In vitro DNA synthesis in a concentrated yeast lysate

Summary A system is described in which DNA synthesis can be monitored in a yeast lysate. The observed synthesis has many of the properties of in vivo DNA replication. It is dependent upon replication growing points that were active in vivo. The in vitro synthesis proceeds via low molecular weight intermediates, but these do not mature into larger DNA. There is a specific requirement for rATP. Mitochondrial DNA is also synthesised in this system.     

RNA bandages for photoregulating in vitro protein synthesis

'RNA bandages' are composed of two 6-12-mer 2'-OMe RNA strands complementary to a mRNA target that are joined by a photocleavable linker. These tandem oligonucleotides typically exhibit much higher affinity for the mRNA than the individual strands. An RNA bandage with binding arms of different lengths and a 4-base gap blocked translation in vitro of GFP mRNA; subsequent near-UV irradiation restored translation. This provides a general method of photomodulating hybridization for a variety of oligonucleotide-based technologies.     

Effect of ischaemia on protein synthesis in vitro

Changes in the incorporation of 14C-amino acids into proteins in vitro were followed under conditions of ischemia induced by abdominal aorta ligature and subsequent recirculation in dogs. Cell saps isolated from L-S spinal cord, spinal ganglia, the sciatic nerve and medulla oblongata were added to the incorporation mixture composed of ribosomes and an enzymatic system from intact brains. Cytosols isolated from ischemic animals affected the rate of in vitro protein synthesis moderately, while repeated ischemia caused a profound decrease in the incorporation of amino acids into proteins. Cytosols from L-S spinal cord and especially from spinal ganglia after three days of recirculation substantially enhanced incorporation thus indicating a massive response of these tissues to ischemic injury. Cell saps from the medulla oblongata increased amino acid incorporation into proteins in vitro in all experimental groups.     

Effect of 2 MHz ultrasound on DNA, RNA and protein synthesis in Pisum sativum root meristem cells.

The amounts of DNA, RNA and protein synthesis were determined in Pisum sativum root meristem cells at various times after a 1 min exposure to 1 MHz ultrasound at a power density of 30 W/cm2. Immediate depressions in all three macromolecular syntheses occurred after sonication, followed by an apparent recovery several hours later. These events appear to correlate in time with the subsequent reduction and recovery in mitotic index in Pisum sativum root meristem cells exposed to 2 MHz ultrasound.     

Differential effect of neomycin on DNA dependent -DNA and RNA synthesis in vitro

Neomycin inhibits $\text{in vitro}$ DNA dependent DNA and RNA synthesis catalyzed by DNA polymerase I and RNA polymerase from $\text{E. coli}$. The effect of the antibiotic is more pronounced towards DNA synthesis. The inhibition of DNA synthesis is competitive with template DNA, does not reverse with excess deoxynucleoside triphosphate, Mg²⁺ or enzyme $\text{E. coli}$ DNA polymerase I. Neomycin does not reduce the number of potential 3′ -OH end or primer. It seems to shorten the size of the newly formed polynucleotide.