Characteristics of Nuclear‐Ribosomal and DNA‐Like Ribonucleic Acids Differentially Extracted by Hot‐Phenol Fractionation

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Abstract
The purpose of the present study was to define more exactly the conditions of the method of hot‐phenol fractionation of nuclear RNA (Georgiev and associates). The thermal RNA fractions obtained were characterized by agar‐gel electrophoresis, determination of the specific radioactivity and base composition analysis, both of the total and newly synthesized RNA.It was found that the fraction extracted at 0–4 °C (considered as cytoplasmic RNA in the literature) contained some nuclear ribosomal precursor RNAs, expecially 32‐S and 23‐S RNAs.The composition of the different thermal RNA fractions strongly depended on the extraction conditions (time, pH, volume ratiosetc.). Conditions were well‐defined (15‐min extraction at each temperature, pH 6.2) for obtaining, as separate fractions, the nuclear RNA species of ribosomal type (40‐°C RNA,i.e.that obtained at 40 °C) and two classes of DNA‐like RNA (63‐°C and 85‐°C RNA). The 55‐°C RNA fraction was found to be of mixed character (rRNA + DNA‐like RNA) regardless of the extraction conditions.The 85‐°C RNA contained the “giant” RNA molecules, had the highest specific radioactivity and a (G + C)/(A + U) ratio of about 0.80. These findings are in agreement with the view that 85‐°C RNA is the very first product of transcription. 63‐°C RNA was also DNA‐like but different from 85‐°C RNA with respect to electrophoretic profile, specific radioactivity and base composition. A high content of adenylic acid (above 30%) was found in the region 8 to 18 S of 63‐°C RNA. It is suggested that poly(A) segments are attached at the 18‐S stage of the processing of 63‐°C RNA as a precursor to the cytoplasmic mRNA.The results presented demonstrate that under well‐defined conditions the method of the hot‐phenol fractionation of Georgiev may be used as a reproducible procedure for obtaining different types of nuclear RNA without preliminary isolation of nuclei and nucleoli.