The purification from rat liver of a nuclease hydrolysing ribonucleic acid and deoxyribonucleic acid

1. The purification of a nuclease from rat-liver mitochondria is described. The mitochondria are rendered soluble by treatment with Triton X-100 and, after fractionation with ammonium sulphate and acetone, the active fraction is further purified by chromatography on DEAE-cellulose and Sephadex G-75 to give a purification of over 700-fold. 2. The purified enzyme was only very slightly contaminated with deoxyribonuclease II, phosphodiesterase and phosphomonoesterase. The individual activities of these enzymes did not exceed 0.1% of the activity of the liver nuclease. 3. The purified enzyme attacked RNA more rapidly than denatured DNA and hydrolysed native DNA more slowly than denatured DNA. 4. There is some evidence to suggest that the nucleolytic activity of the purified preparation towards native DNA, denatured DNA and RNA is associated with a single protein. 5. The enzyme is relatively labile but is stabilized in the presence of 20% (w/v) glycerol or 10mm-2-mercaptoethanol.     

Ribonuclease-sensitivity of covalently closed rat liver mitochondrial deoxyribonucleic acid

Preparations of covalently closed mitochondrial DNA of rat liver contain 10-30% of molecules that are converted into relaxed circular molecules after treatment with ribonuclease. Control experiments, with covalently closed bacteriophage PM2 DNA, indicate that ribonuclease-sensitivity cannot be induced either by depurination or by incubation with reducing agents.     

Binding of diethylstilboestrol to deoxyribonucleic acid by rat liver microsomal fractions in vitro and in mouse foetal cells in culture.

Diethylstillboestrol, a synthetic and carcinogenic hormone, binds to DNA as a result of incubation with a liver microsomal preparation in vitro and on incubation with primary mouse foetal cells in culture. Enzymic digestion of DNA samples thus prepared gives several covalent deoxyribonucleoside-diethylstilboestrol products from the microsomal system. One of these is produced in small but significant yield in the tissue-culture system.     

The influence of alcohol-water solvents on the conformation of deoxyribonucleic acid

The influence of alcohol–water solvents on the conformation of native DNA was studied by the methods of flow birefrigence and viscometry. Conformational transitions of DNA were observed at low alcohol concentrations corresponding to the destruction of the water spatial structure. A change in the secondary structure of the DNA molecule was observed at high ethanol concentrations and is discussed in the paper.     

Localization of the ribonucleotide sites in rat liver mitochondrial deoxyribonucleic acid.

Supercoiled rat liver mitochondrial DNA is relaxed by treatment with ribonucleases A, T1 or H. All the supercoiled mitochondrial DNA is sensitive to ribonuclease H and ribonuclease A, but only 35% of the supercoiled population is sensitive to ribonuclease T1. Removal of the ribonucleotides with calf thymus ribonuclease H, followed by denaturation of the mitochondrial DNA and analysis of the single-strand fragment lengths in the electron microscope, showed that the ribonucleotides were randomly located on both strands of the DNA. Endonuclease-S1 digestion of mitochondrial DNA after removal of the ribonucleotides reveals that no unique fragments are produced and ribonucleotides are randomly distributed with respect to one another. The average number of ribonucleotide sites per molecule was estimated to be between 8 and 13. Two possible mechanisms for the origin of ribonucleotide sites are discussed.     

Transfer ribonucleic acid methylase activity in adult and foetal rat liver.

Foetal rat liver extracts were found to have higher tRNA methylene activities than corresponding extracts of adult liver. When the specific activities were expressed per mg of liver or per mg of protein, the foetal tRNA methylating enzymes were respectively 2.5 and 6 times higher than those of adult livers. The presence of an inhibitor in adult liver can be excluded, since the same recoveries of total tRNA methylase activity were obtained after partial purification of both adult and foetal liver extracts: yields were close to 100%. The apparent Km's for the substrates in the methylating reactions were the same when tRNA methylases from either adult or foetal liver were used: values were 0.2 muM for Escherichia coli tRNA and 2.1 muM for S-adenosyl-L-methionine. After T1-T2 ribonuclease digestion of an in vitro methylated tRNA, similar methyl nucleotide patterns were observed in foetal and adult enzymatic extracts. It is concluded that the same tRNA methylase pool is present in adult and foetal liver. In addition, it is hypothesized that the different reaction rates exhibited by these enzymes might be due to the tRNA functional requirements rather than to the presence of a tRNA methylase inhibitor.     

The morphological picture of implanted foetal rat brain tissue

The authors studied the morphological picture of implanted foetal brain tissue. Macroscopically, they found that the grafts used for morphological study had taken in every case and that the size of the graft had doubled or trebled. The surface of the implant grew above the level of the recipient's cortex and numerous branching blood vessels were observed in the graft-cortex contact area. In the light microscope, organization of the cells in circular clusters or strips was found in the implant. Large numbers of blood vessels of varying calibre were present in the vicinity of large cell concentrations. Distinct differences between the cytoarchitectonics of the normal tissue of the recipient and the graft tissue were seen. Cells with a normal appearance or an apolar form, whose nucleus contained irregularly scattered chromatin and mosaic-like nucleoli, were observed at ultrastructural level. The nuclear membrane was thrown into multiple folds which invaginated deep into the nuclear matter. The Golgi complex covered a large area. Axodendritic synapses indistinguishable from the control were found in the neuropil. No changes were observed in the walls of the capillaries. The pericapillary zone was characterized by the presence of light astrocyte processes.     

Inhibition of polyamine accumulation and deoxyribonucleic acid synthesis in regenerating rat liver.

Repeated injections of 1,3-diaminopropane into rats after partial hepatectomy caused a repression-type inhibiton of liver ornithine decarboxylase (EC 4.1.1.17) and totally prevented the marked increases in liver putrescine and spermidine concentrations that normally occur in response to partial hepatectomy. The inhibition of polyamine synthesis by diaminopropane was accompanied by a profound decrease (about 80%) in the synthesis of DNA in the regenerating rat liver without any changes in the synthesis of RNA and total liver protein.     

Evidence for ethylation of rat liver deoxyribonucleic acid after administration of ethionine

1. Administration of a large dose (500mg/kg body wt.) of (3)H-labelled l-ethionine to rats resulted in the incorporation of a small amount of radioactivity into the liver DNA. Considerable evidence that this radioactivity was not due to contamination of the isolated DNA with labelled protein, RNA, S-adenosyl-l-ethionine or l-ethionine was obtained. 2. After acidic hydrolysis of the DNA isolated from the livers of rats treated with labelled l-ethionine, virtually all of the radioactivity present in the DNA was found in a fraction with similar chromatographic properties to 7-ethylguanine. 3. Treatment of rats with comparable doses of l-methionine did not lead to the formation of 7-methylguanine in the liver DNA. 4. These results are discussed in relation to the induction of liver tumours by ethionine.