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

The nascent ribosomal RNA which has not yet been incorporated into mature ribosomal particles has been reported to sediment as 17 S and 22–23 S as compared with 16 S and 23 S for RNA from mature ribosomes. In this study, we have investigated whether the transformation of sedimentation property from nascent to mature state takes place gradually during ribosome formation or occurs at the later step of ribosome maturation.

Ribosomal RNA's were labelled either with [³H]adenosine or [¹⁴C]adenine at different stages of ribosome maturation, representing pulse-labelled RNA, RNA obtained in the presence of chloramphenicol or 5-fluorouracil, and RNA of mature ribosomes. Comparison of their sedimentation velocities in sucrose density gradient indicated that transformation from 17 S to 16 S occurs at the later step of ribosome maturation. No clear difference of sedimentation velocity was detected between nascent and mature 23-S ribosomal RNA in this study.

• 1.

  1. The nature of the inhibition of DNA nucleotidyltransferase (EC 2.7.7.7) from KB cells (DNA polymerase) by poly ethenesulfonic acid (PES) and other polyanionic materials were examined. The inhibition was found to be competitive with the DNA used to prime the enzyme. The K₁ was found to be 1.72 ± 0.36 μg PES/ml for the 12 900 mol. wt. material in the range of zero to 10 μg inhibitor/ml. The inhibitory action of PES was strongly dependent on the molecular weight of the sample used in the range of 5000 to 13 000 and further increases in molecular weight did not seem to affect efficacy. Other polyanionic polymers, such as heparin, carboxymethyl cellulose, alginic acid, chitosan sulfate, RNA and chondroitin sulfate also inhibited this enzyme to varying degrees.

• 2.

  2. Spermine, which by itself inhibits the DNA polymerase, reversed the inhibition caused by PES. This is explained by the competition between DNA and PES for salt formation (and precipitation) with spermine.

• 3.

  3. The inhibition of pancreatic RNAase (EC 2.7.7.16) by PES exhibited a similar dependence on the molecular weight of the polymer as did the inhibition of DNA polymerase.

• 4.

  4. Addition of PES to DNAase I (EC 3.1.4.5) assays resulted in a bimodal, concentration-dependent curve. At intermediate concentrations the compound stimulated DNA hydrolysis, while at higher concentrations it inhibited.

Fractionation of native DNA from mammalian and bacterial sources by countercurrent distribution has been found to depend on the degree of partial separation of the two strands. This is indicated by thermal denaturation of countercurrent distribution fractions of DNA and by countercurrent distribution of DNA reacted with [¹⁴C]formaldehyde. Countercurrent distribution of DNA double-labelled with [¹⁴C]thymidine and [³H]deoxycytidine, and measurements of T_m and buoyant density in CsCl of countercurrent distribution fractions of DNA indicate that base composition does not play an important part in the separation. Countercurrent distribution fractions of DNA did not show significant differences in s values. Countercurrent distribution in the solvent system used may exaggerate preexistent partial denaturation but it is considered that the patterns obtained do reflect the amount of denaturation which is present in the DNA in the cell.

Soluble RNA and a serine specific transfer RNA fraction were digested by highly purified T₁-RNAase (ribonucleate 3′-guanylohydrolase, EC 3.1.4.8) and phosphomonoesterase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1). Conditions for the separation of the oligonucleotides on DEAE-cellulose columns were worked out with digests of soluble RNA (Fig. 1–3). The splitting products of the serine-transfer-RNA fraction were separated (Fig. 4) and further purified by paper electrophoresis. Their nucleotide composition — including the odd nucleotides — was determined by conventional methods and the sequences partially elucidated. 5 Di-, 6 tri-, 7 tetra-, 2 penta- and 1 hexanucleotide of the two serine-transfer-RNA chains were identified (Table I) and quantitatively determined (Table II). One longer oligonucleotide was partially identified. Some oligonucleotides, which are present in small amounts, were due to impurities in the serine-transfer-RNA sample.