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

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 3 2 4 and 23-S RNAs.The composition of the different thermal RNA fractions strongly depended on the extraction conditions (time, pH, volume ratios etc.). Conditions were well-defhed (15-min extraction a t each temperature, pH 6.2) for obtaining, as separate fractions, the nuclear RNA species of ribosomal type (40-"C RNA, i.e. that obtained a t 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 specifh 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 fist product of transcription. 63-"C RNA was also DNA-like but different from 85-"C RNA with respect to electrophoretic prose, 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.The nuclei of eukaryotic cells contain a complex set of ribonucleic acids [1-201. The study of their physical-chemical properties, turnover and functions is hampered by difficulties encountered in the complete extraction and good separation of the different types of nuclear RNAs in an undegraded state.One of the approaches followed is the isolation of sufficiently pure nuclei, their fractionation into nucleoli, nucleoplasm, chromatin etc. and extraction of RNA from these nuclear components [ll, [19][20][21][22].Abbreviations. rRNA, ribosomal RNA; dRNA, RNA with DNA-like base composition; nrRNA, nuclear RNA of ribosomal type; ndRNA, nuclear DNA-like RNA; mRNA, messenger RNA.Enzyme. DNAase, DNAase I, deoxyribonucleate oligonucleotidohydrolase (EC 3.1.4.5).Definition. An Azao unit is the quantity of material contained in 1 ml of a solution which haa an absorbance of 1 at 260 nm when measured i n a 1-cm path-length cell.However, these procedures often lead to degradation [23] or loss of some nuclear RNAs [24]. On the other hand, isolation of pure intact nuclei from many cell types (lymphocytes, tumour cells, endocrine organs etc.)...

Effect of removal of f1‐histone on the conformation of nuclear chromatin and on the transcription process

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Indoleamine 2,3-Dioxygenase Serves as a Marker of Poor Prognosis in Gene Expression Profiles of Serous Ovarian Cancer Cells

“…Finally, the absorbance at 450 nm was measured, and the antiproliferating activity of each drug was calculated using the formula: (1 À T / C) Â 100 (%), where T and C represent the mean difference in absorbance at 450 nm of the cells treated with drugs (T) and that of the untreated control cells (C). The IC 50 ovarian tumors and cell lines were isolated using the hot phenol method (28). Total RNA was isolated from three different cultures of each cell line.…”

Ido serves as a marker of poor prognosis in gene expression profiles of serous ovarian cancer cells

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