Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis最新文献

Cellular DNA from a synchronized population of cultured mouse L5178Y cells was labeled with [³H]thymidine at three times during the S stage: early, middle and late S phases. The deoxyribonuclease I digestability of DNA from these three periods in the subsequent cell cycle indicated that the early S phase replicating DNA had higher enzymatic susceptibility at G1 and early S phases than at middle and late S phases, while the middle and late S phase replicating DNA demonstrated a low susceptibility throughout the cell cycle. This suggests that most, if not all, of transcriptionally active DNA is replicated very early in the S phase.

Gamma-irradiation or introduction of hydrocortisone bring about degradation of nuclear DNA in rat thymocytes. The chromatin degradation products were extracted from purified nuclei by 0.7 mM EDTA. The quantity of low molecular weight chromatin fragments formed 6 h after irradiation increases up to the doses of 3 Gy, then remains constant up to 30 Gy and decreases at doses 100 to 300 Gy. Whatever the irradiation dose, DNA degradation starts after a 2-h lag, reaches a maximum by the 6th hour and remains constant between the 6th and 10th hours. The quantity of chromatin fragments formed coincides with the number of cells with pycnotic nuclei. The chromatin fragments present nucleosomes and their oligomers with a normal histone content and an intact structure, as judged from how they are split by DNAase I. The number of intranucleosomal breaks in DNA is negligible. DNA fragmentation is not accompanied by degradation of histones and nonhistone proteins of chromatin. Hence, DNAase I and proteases are not involved in degradation of chromatin. The ratio between mononucleosomes and oligomeres of different lengths does not depend on the dose and the time after irradiation. The quantity of DNA degraded is determined by the number of dying cells in which all DNA is fragmented rather than the degree of chromatin degradation over the whole thymocyte population. Hydrocortisone-induced degradation of chromatin in rat thymocytes occurs similarly. A possible role of chromatin degradation in cell death is discussed.

Antiserum was prepared in rabbits against ultraviolet-irradiated DNA. Using a ¹²⁵I-labeled protein A binding assay it was shown to be specific for ultraviolet-irradiated DNA, binding increasing as a function of logarithmic increase in dose. This antiserum was then used to develop a radioimmunoassay in which the competition between labeled UV-DNA and unlabeled sample DNA for antibody binding sites is monitored. Using this system the specificity of the assay could be changed depending on the nature of the labeled probe. The inability of poly(dA-dT) · poly(dA-dT), as compared with poly(dA) · poly(dT), to act as a competitive inhibitor established that the primary lesion recognized by the antiserum is the thymine dimer. This antigenic response did, however, depend on the presence of at least one nucleotide adjacent to the dimer. The sensitivity of the assay was optimized by using ³²P-labeled plasmid DNA as competitive probe and is capable of detecting photodamage in cellular DNA at doses as low as 2.5 J · m⁻².

Poly(C) and heparin at low concentrations (1 μg/ml) prevent the RNA synthesis termination protein ϱ from functioning during the biosynthesis of RNA from bacteriophage T7 DNA catalyzed by Escherichia coli RNA polymerase. Both of these polyanions inhibit the binding of ϱ to isolated T7 RNA. Heparin also inhibits ϱATPase when isolated RNA transcripts are used as cofactors. It is concluded that the polyanions inhibit termination by binding to the site on ϱ that is normally used for the initial interaction with a nascent RNA transcript in the ϱ-mediated release of RNA. Since one of the inhibitors, poly(C), is itself a potent activator for ϱATPase, it is also concluded that the ATP hydrolysis step that is required for ϱ termination has to be coupled to an action of ϱ on the RNA molecule to be released from the transcription complex.

Three ataxia telangiectasia homozygotes, one heterozygote and normal fibroblast strains were compared as to the capacity of their cellular extracts to enhance the priming activity of gamma-irradiated colicin E1 DNA for purified DNA polymerase (EC 2.7.7.7) of Escherichia coli. It was found that homozygotes had substantially lower activity than normal strains, while no difference was detected between the heterozygote and normal strains. In vitro complementation of the activity occurred between extracts of certain strains of homozygotes, allocating them to two complementation groups.

Hepatoma tissue culture cells, grown in the presence of d-galactosamine and 6-azauridine, demonstrate a strong reduction of the intracellular UTP pool that can be replenished by formation of UTP from uridine and FUTP from 5-fluorouridine within 2 h. Concomitantly with the UTP deficiency, a decrease of dexamethasone-induced tyrosine aminotransferase activity occurs. 5-Fluorouridine, as compared to uridine, is even more efficient in restoring the activity of tyrosine aminotransferase. Treatment of the cells with d-galactosamine alone results in a minor lowering of UTP that is not followed by the inhibition of the enzyme induction. However, the administration of d-galactosamine, simultaneously or at any time up to 5 h before or after dexamethasone, leads to a 1.5- to 2-fold higher induction (superinduction) which appears 24 h later.

Ribosomal 5 S RNA synthesis in Chinese hamster V79 cells treated with an inhibitory antibiotic of protein synthesis, cycloheximide, was quantitated by hybridization of RNA preparations with plasmid ColE1-pSC101 DNA carrying Xenopus 5 S DNA. In V79 cells, cycloheximide produced a notable decrease in the production of the higher molecular weight ribosomal RNA (rRNA) fraction, whereas the accumulation of both 5 S RNA and polyadenylate-containing messenger RNA was much less affected. When the cellular protein synthesis was inhibited by over 85% of the control, accumulation of 5 S RNA and of rRNA was respectively 40–50% and about 10% of the control. The size distribution analysis of RNA species revealed that 5 S RNAs obtained from V79 cells after treatment with 0.1 μg/ml or 2.0 μg/ml cycloheximide were indistinguishable from control. Inhibition of precursor uptake into 5 S RNA by cycloheximide was apparently phase-specific during the cell cycle and was 44–67% and 78–85% of the control for the S and the M phase, respectively. The responses of RNA polymerase III activities to cycloheximide in isolated nuclei from each phase of the cell cycle correlated very well with those of precursor uptake into 5 S RNA observed in vivo, while total solubilized RNA polymerase activities showed no inhibition by the drug at any phase of the cell cycle. Cytosine arabinoside, a specific inhibitor of DNA synthesis, did not cause any decrease in the cellular level of 5 S RNA.

The conformational changes of yeast-tRNA^Phe induced by various intercalators have been studied using limited digestions with nucleases S₁ and T₁. The results show an increased sensitivity to T₁ in the D-loop, suggesting a weakening of the D-loop-T-loop interaction. Furthermore, the results are best explained by a non-intercalative binding of the dyes, probably in the D-loop-T-loop cavity.

Recombinant DNA molecules were constructed from the plasmid pIL203 and the EcoRI-fragment of N3 plasmid containing EcoRII endonuclease and methylase genes and also a gene for resistance to sulfanilamide. The pIL203 plasmid, used as a vector, consisted of the BamHI-EcoRI-fragment of the plasmid pBR322 conferring resistance to ampicillin and the BamHI-EcoRI-fragment of lambda phage containing promoters, a thermosensitive mutation in the cI gene and a suppressible amber mutation in the cro gene. Ampicillin-sulfanilamide-resistant clones were selected and tested for their restriction and modification phenotype. The recombinant plasmid DNA, isolated from Ap^RSu^R-resistant clones, which restricted and modified phage λ imm21 with EcoRII specificity, had the EcoRI-fragment with EcoRII genes in a single orientation. The recombinant plasmid pSK323 was transferred into E. coli strains with su⁻, su1, su2 or su3 phenotypes. The synthesis of products of EcoRII genes by these strains grown at 37°C is increased by 10–50-fold.