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

The capacity of tobacco-leaf extracts to incorporate ATP and GTP into a cold-trichloroacetic-acid insoluble product has been studied. Almost all the four nucleotide-dependent incorporating activity is localized in the 1000 × g fraction of such extracts. The RNA character of the product is indicated by the dependence of the reaction on all four nucleotides (ATP, GTP, UTP, and CTP), by the fact that the product is readily solubilized by RNAase treatment, and the fact that both ATP and GTP are incorporated at comparable rates. The incorporation of [³²P]ATP is enhanced by the addition of an ATP-generating system. The system is inhibited by the presence of DNAase, RNAase, or actinomycin D in the incubation mixtures. The product of incorporation is solubilized by RNAase but not by DNAase. Pretreatment of the 1000 × g fraction with DNAase or actinomycin D destroys the RNA synthesizing capacity. Within the 1000 × g fraction, RNA synthesizing activity was associated with both chloroplasts and nuclei, the former being the major site of activity.

The ribonucleic acid of rat-liver nuclei labeled with [³H]cytidine and harvested from liver homogenates using a phenol-NaCl mixture may be fractionated by successive extractions at temperatures between 20 and 75°, using a phenol-NaCl extraction medium. Successive extractions at any one temperature produce extracts whose ribonucleic acid content decreases markedly. The analysis of the specific activity of extracted nuclear ribonucleic acid indicates two fractions: (1) a relatively low specific-activity fraction obtained at 25, 40 and 50°; and (2) a relatively high specific-activity fraction obtained at 65 and 75°, temperatures at which measurable amounts of deoxyribonucleic acid are also extracted.

It is demonstrated that InCl₃ at appropriate concentration and ionic strength, effectively precipitates all soluble ribonucleic acid as well as all deoxyribonucleic acid as the indic nucleates. The indic nucleates are solubilized in 0.38 N KOH which results in the precipitation of In (OH)₃ in an exchange reaction that is complete in about 2 h at room temperature. In this way the ribonucleic acid may be hydrolysed to constituent nucleotides and the unhydrolysed deoxyribonucleic acid may be recovered by perchloric acid precipitation.

The results of extraction experiments with nuclei harvested from liver of young and old adult rats indicate: (1) the specific activity of ribonucleic acid extracted from “old” liver nuclei is consistently higher than that extracted from “young” nuclei; (2) the amount of ribonucleic acid extracted from “young” liver nuclei is greater than that extracted from “old” liver nuclei; and (3) the amounts of deoxyribonucleic acid extracted from “young” and “old” liver nuclei are approximately equal.

Calf-thymus histone fractions were found to inhibit the biosynthesis of DNA in vitro to a substantial extent. The inhibition could be explained on the basis of DNA-histone interaction. The very lysine-rich histones (Fraction 1) complexed with DNA most readily and inhibited the DNA replication at a histone-DNA ratio of 1:1 and higher. Higher ratios of the moderately lysine-rich histones (Fraction 2b) and of the arginine-rich histones (Fractions 2a and 3) were required. Complete inhibition was observed only at ratios of histone-DNA of 2:1 and higher. Histone stabilizes the helical molecule of DNA towards heat denaturation. Melting temperatures of reconstituted nucleohistones were determined. Most resistant to the heat denaturation were nucleohistones with a high content of lysine whereas the arginine-rich nucleohistones showed melting temperatures close to those of the DNA.