Biochimica et Biophysica Acta (BBA) - Specialized Section on Nucleic Acids and Related Subjects最新文献

The conditions that were optimal for incorporation of [¹⁴C]uridine triphosphate into RNA of nucleoli of normal liver cells were found to be optimal for incorporation of [¹⁴C]uridine triphosphate into nucleoli of thioacetamide-treated rat livers. Maximum incorporation of isotope was found at 15–30 min after the incubation was begun. Thioacetamide produced a marked increase in the rate of uptake of total isotope into RNA and in the specific activity of the RNA despite the increased amount of RNA in the nucleoli obtained from thioacetamide-treated animals. These data suggest that thioacetamide may derepress the genomic segments involved in biosynthesis of nucleolar RNA.

The postulation that RNA synthesis is the molecular site of action of insulin was tested by examining the effect of actinomycin on the response of isolated rat diaphragm muscle to the hormone. Actinomycin markedly suppressed, but did not completely inhibit, nucleic acid synthesis in muscle; the effect of the antibiotic on protein synthesis was not nearly as great. Actinomycin treatment of diaphragm muscle did not prevent the insulin stimulation of protein synthesis, of glucose uptake, or of aminoisobutyric acid accumulation; nor, for that matter, did it prevent the influence of the hormone to increase incorporation of [¹⁴C₈]adenine into nucleic acids. The results do not lend comfort to the theory that an effect of insulin on the synthesis of messenger RNA is fundamental to its action, although it does not rule out that possibility with certainty.

• 1.

  1. [¹⁴C₆]Glucose was injected intravenously into rats and the incorporation of ¹⁴C into the ribose and deoxyribose of nucleic acids was investigated.

• 2.

  2. In the RNA of all tissues studied, namely, thymus, spleen, liver and intestinal mucosa, the incorporation into the purine-bound ribose was higher than that into the pyrimidine-ribose. Further analysis showed that the specific activity of the ribose attached to cytosine was the lowest of the values for the four individual nucleotides; this was especially marked in the thymus.

• 3.

  3. Similarly in the DNA, the specific activities of the deoxyribose moieties bound to the two purine bases were greater than the corresponding values of the pyrimidine-deoxyribose.

• 4.

  4. These findings are in agreement with the results obtained previously from similar investigations in vitro. They are discussed here with reference to the formation of deoxyribotides from their respective ribose analogues.

Two DNA preparations of mammalian origin, viz., the DNA from calf thymus and the DNA from human spleen, were employed in a comparative study of different methods suitable for the investigation of the arrangement of pyrimidine nucleotides in the polymer chain. The conditions under comparison were (a) the degradation of DNA with dilute sulfuric acid; (b) the degradation of DNA with formic acid-diphenylamine; (c) the degradation of apurinic acid with dilute alkali. The released pyrimidine nucleotide runs of length 1 to 6 were determined directly by two-dimensional paper chromatography.

The range and validity of the several procedures described here and in the literature are considered in detail and the bearing of the findings on the general problem of the nucleotide sequence in DNA is discussed.

It was shown that cortisol increases the level of tyrosine-α-ketoglutarate transaminase (l-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) of the isolated rat liver, when perfused with diluted rat blood containing an amino acid mixture; a more striking increase was obtained in the presence of a supplementary amount of tyrosine. This enhancement of activity was blocked by inhibitors both of protein synthesis (chloramphenicol, puromycin, ethionine) and of ribonucleic acid synthesis (actinomycin D and mitomycin C).

Adding [6-¹⁴C]orotate to the perfusing blood, caused a rapid incorporation of radioactivity into nuclear ribonucleic acid to take place; cytoplasmic ribonucleic acid was poorly labelled after 30 min, but its radioactivity rose thereafter. Cortisol stimulated the labelling of both the nuclear and the cytoplasmic ribonucleic acid; moreover it induced the increase of tyrosine transaminase after a lag of about 30 min. In both cases, the effect of the hormone was prevented by actinomycin D, or by mitomycin C.

It was finally found that livers perfused with cortisol have a higher nuclear ribonucleic acid polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) activity than control ones.

The facts that the synthesis of nuclear ribonucleic acids precedes the increase of tyrosine transaminase and also that cortisol stimulates ribonucleic acid polymerase strongly suggest that the action of the hormone can be related to an increase in the synthesis of messenger ribonucleic acid molecules for the synthesis of the enzyme.