The isolation of nuclei from tissue-cultured plant cells

A method for the isolation of nuclei from tissue-cultured plant cells is presented. The method employs polyamines as stabilizing agent and uses Percoll for the purification of the nuclei in density gradients. It is applicable to large amounts of tissue and is reasonably quick (500 g of tissue can be processed within 2–3 h). The purified nuclei have retained their morphological characteristics as demonstrated by phase contrast, as well as electron micrographs. High molecular weight DNA can be isolated from these nuclei and histones extracted from the purified nuclei display the expected gel electrophoretic pattern. The retainment of the nucleosomal arrangement is demonstrated by digestion with micrococcal nuclease and subsequent analysis of the DNA fragments on agarose gels. The purified nuclei contain high activities of several nucleus-specific enzymes such as α-amanitin-sensitive and insensitive RNA polymerases, protein kinases, poly-ADP-ribosylating enzymes and DNA polymerases.     

Presence of histone mRNA sequences in high molecular weight RNA of HeLa cells

Pulse-labeled HeLa cell RNA centrifuged under denaturing conditions was hybridized with DNA of recombinant phages containing sea urchin histone genes. This cross-hybridization showed the presence of histone mRNA sequences in high molecular weight RNA molecules. Treatment of the cells with actinomycin to stop RNA synthesis resulted in the rapid decay of this high molecular weight RNA followed by an increase of 9S histone mRNA in the cytoplasm.The results are consistent with the presence in HeLa cells of a high molecular weight precursor to histone messengers.     

A new species of virus-coded low molecular weight RNA from cells infected with adenovirus type 2

A virus-coded low molecular weight RNA (5.2S), which migrates slightly faster on polyacrylamide gels than the well characterized adenovirus-specific 5.5S RNA, has been isolated from cells infected with adenovirus type 2. Hybridization-competition experiments and RNA fingerprints indicate that the two virus-associated (VA) RNAs differ in their primary structures. The gene for 5.2S RNA is located to the right of the gene for 5.5S RNA, on the I strand of a DNA segment which extends between positions 30.3 and 32.2 on the map of adenovirus type 2 DNA.Both 5.5S and 5.2S RNA can be detected early after infection and also in the presence of cytosine-arabinoside or cycloheximide. After the onset of viral DNA replication, the synthesis of 5.2S RNA levels off, whereas 5.5S RNA is synthesized in increasing amounts. Both 5.2S and 5.5S RNAs are synthesized in isolated nuclei by an enzyme which resembles RNA polymerase III in its sensitivity to α-amanitin. In isolated nuclei, both RNA species are labeled with β-³²P-labeled GTP, which suggests that they are initiated at separate promoter sites.     

ISOLATION AND PARTIAL CHARACTERIZATION OF MACRO- AND MICRONUCLEI FROM PARAMECIUM AURELIA

A method was developed for the isolation of macro- and micronuclei from Paramecium aurelia. This method utilized ionic and nonionic detergents to rupture the intact cells, calcium ions and spermidine were employed to protect the nuclei, and the nuclei were purified by centrifugation. Macronuclei consisted of 22% DNA, 10% RNA, and 68% protein. Micronuclei were composed of 9% DNA, 11% RNA, and 80% protein. DNA from both macro- and micronuclei had a density of 1.687 g/cc in CsCl and 1.417 g/cc in Cs₂SO₄. These values corresponded to G + C content of about 23%. The RNA of macronuclei was examined by gel electrophoresis, and two high molecular weight species were identified having molecular Weights of 1.3 x 10⁶ and 2.8 x 10⁶ daltons. Three syngens were studied, and in each case the conditions for isolation of the nuclei were the same and no differences were observed in the properties of the nuclei.     

Isolation and properties of isolated nuclei from the Florida red tide dinoflagellate Gymnodinium breve (Davis)

A simple and rapid method is described for the isolation of nuclei from the Florida red tide dinoflagellate Gymnodinium breve. The nuclei are free of cytoplasmic contamination and are active in endogenous RNA synthesis. The ratio of DNA : RNA : acid-soluble protein : acid-insoluble protein is 1:0.39:0.13:0.63, respectively, and each nucleus contains ca. 113 picograms of DNA. Electrophoretic analysis of the acid-soluble proteins reveals the presence of two histone-like proteins with molecular weights of 12,000 and 13,000.     

Analysis of RNA synthesized in isolated nuclei of Ehrlich ascites tumor cells

The molecular size and poly-A content of RNA synthesized in isolated nuclei of Ehrlich ascites tumor cells were measured. KCl was found to be essential for synthesis of high molecular weight RNA: when 0.4 M KCl was added to the reaction mixture, the average molecular size of the RNA formed was 14S; without KCl the average molecular size was 5S. A significant amount of poly-A sequences was found in RNA synthesized in the presence of alpha-amanitin, suggesting that RNA polymerase I and/or III may synthesized some RNA containing poly-A in isolated nuclei.     

A study of nucleated erythrocytes by density-gradient centrifugation in a zonal rotor

1. Several substances of high molecular weight were examined for their suitability as suspension media in the formation of density gradients for the zonal centrifugation of avian erythrocytes. None proved satisfactory. 2. The behaviour of pigeon erythrocytes in rate-sedimentation experiments in a type A zonal rotor with density gradients of sucrose was examined. The mature cells sediment more rapidly than the younger cells and have a lower RNA/DNA ratio. Maturation is accompanied by a greater loss of RNA from the nucleus than from the cytoplasm. 3. The base composition of the nuclear RNA and of the two species of cytoplasmic ribosomal RNA is reported. 4. The RNA of erythrocytes may be labelled in vivo by injection of inorganic [(32)P]phosphate. The cells most active in the synthesis of RNA sediment less rapidly than the bulk of the cells. 5. Reticulocyte nuclei sediment more slowly than those from erythrocytes. Reticulocyte nuclei have a mean volume of 35mu(3) and are isopycnic with sucrose of density 1.2871 (measured at 20 degrees ). Maturation of the nuclei causes them to shrink to a volume of 25mu(3) and the density to increase to 1.2944.     

Isolation and Properties of Isolated Nuclei from the Florida Red Tide Dinoflagellate Gymnodinium breve (Davis)1

A simple and rapid method is described for the isolation of nuclei from the Florida red tide dinoflagellate Gymnodinium breve. The nuclei are free of cytoplasmic contamination and are active in endogenous RNA synthesis. The ratio of DNA: RNA: acidsoluble protein: acid-insoluble protein is 1:0.39:0.13:0.63, respectively, and each nucleus contains ca. 113 picograms of DNA. Electrophoretic analysis of the acid-soluble proteins reveals the presence of two histone-like proteins with molecular weights of 12,000 and 13,000.     

A rapid method for the purification of extrachromosomal DNA from eukaryotic cells.

A simple and efficient procedure to purify the low molecular weight extrachromosomal DNA from eukaryotic cells is described. Gentle lysis of cells with urea and sodium dodecyl sulfate in 0.24 M phosphate buffer (pH 6.8) is followed by the removal of high molecular weight bulk DNA by centrifugation. Protein and RNA are removed from the supernatant by hydroxyapatite chromatography in urea/phosphate buffer. Urea is then removed with 0.15 M phosphate buffer and the extrachromosomal DNA, virtually free from protein and RNA, is finally eluted in 0.5 M phosphate buffer. The procedure allows the recovery of about 99% simian virus 40 (SV40) DNA from infected monkey kidney cells in the extrachromosomal fraction. In normal mouse, monkey, andhuman cells, approximately 1% of total cell DNA appears to be extrachromosomal.     

Isolation and characterization ofEuglena nuclei

Summary A novel method for isolatingEuglena gracilis Z. nuclei, based on pretreatment of cells in concentrated glycerol buffer before homogenization, is described. Such a treatment weakens the tough cell pellicle facilitating cell disruption, and avoids nuclear damage induced by detergents and by freezing and thawing the cells in aqueous media. Nuclei, purified by centrifugation in dense sucrose, are obtained with a 30% yield, and only small amounts of cell wall fragments contaminate the nuclear pellets. The purified nuclei retain their ultrastructural characteristics. High molecular weight DNA, as well as undegraded RNA species and histones, can be extracted from these nuclei. Nuclease digestions and spread preparations show an unaltered nucleosomal structure of chromatin. This method has been applied to cell samples at any stage of the cell cycle, including mitosis, since inEuglena the nuclear envelope persists during cell division.     

Biochemical effects of bleomycin A2 on Novikoff hepatoma ascites cells.

Purified nucleolar DNA was markedly degraded at a concentration of 13 mug/ml by bleomycin A2; bleomycin concentrations 20-30 times greater were required to degrade nucleoplasmic DNA. Whole nuclear DNA was degraded to only a small extent at 13 mug/ml but was markedly degraded at higher bleomycin concentrations. Treatment of the various types of DNA with high concentrations of bleomycin A2 produced low molecular weight (approximately 6S) fragments that were no longer sensitive to degradation by bleomycin A2. Hybridization studies demonstrated a loss of ribosomal DNA sequences from nucleolar DNA treated with bleomycin A2 in vitro. Studies on RNA synthesis in Novikoff hepatoma ascites cells in vitro showed there was a decreased uptake of 32Pi into high molecular weight nuclear RNA in the presence of bleomycin A2. These results indicate that nucleolar function is inhibited by a direct effect of bleomycin A2 on nucleolar DNA.     

Reinitiation of synthesis of small cytoplasmic RNA species K and L in isolated HeLa cell nuclei in vitro.

Isolated HeLa cell nuclei were used to synthesize low molecular weight RNA species in-vitro. The labelled RNA released from the nuclei during the incubation mainly consists of 5S RNA, pre-tRNA and small cytoplasmic RNA species K and L. All these low molecular weight RNA species are synthesized by RNA polymerase C (or III). The polyanion heparin was applied to study the reinitiation of these RNA molecules in-vitro. A comparison of the kinetics of RNA synthesis in the absence and in the presence of this inhibitor demonstrates a highly efficient in-vitro reinitiation of scRNA species K and L as well as 5S and pre-tRNA by RNA polymerase C. These results indicate a general competence of this enzyme to catalyze the de-novo formation of specific gene products in-vitro.     

DNA synthesis in L-cells in the presence of 5-fluorodeoxyuridine.

After 16 h of incubation with 10-minus6 M FdUrd, the rate of (32P) orthophosphate uptake into DNA isolated from L-cells amounted to 15% of that of an untreated culture, although cell division had stopped several hours earlier. All 4 deoxynucleotides were present in this DNA but its nucleotide composition, as measured by enzymatic digestion and chromatography, reflected a decreased thymidine precursor pool in FdUrd-treated cells. Sedimentation analysis in alkaline sucrose gradients revealed that the DNA formed in the presence of FdUrd had a sedimentation coefficient of 10 S which corresponded to a single-stranded molecular weight of 5.5.105. This DNA could be "chased" into a high molecular weight DNA if the FdUrd block was bypassed with added dThd or BrdUrd. Other analyses failed to detect RNA covalently linked to the DNA fragments at a level of more than 5% RNA or about 90 ribonucleotides. The accumulation of these DNA fragments could be explained by assuming that in the presence of limiting precursor pool the rate of DNA chain initiation is greater than the rate of chain elongation.     

In vitro transcription by isolated nuclei of Rhynchosciara americana salivary glands

A method for the isolation of polytene nuclei from salivary glands cells of the Diptera Rhynchosciara americana is described. The stage-specific morphological pattern of the chromosome is maintained during the isolation. The isolated nuclei show two distinct RNA polymerase activities, namely I and II, characterized on the basis of ionic requirements and -amanitin sensitivity. Studies of the product under the incubation conditions show that the system allows the synthesis of high-molecular weight RNA, beside a low molecular weight peak which may comprise pre-4S and 5S RNAs.-Autoradiographic studies carried out in the presence or absence of the toxin -amanitin showed that micronucleoli contain products of RNA polymerase type I activity (ribosomal RNA) and that the DNA puffs are engaged in -amanitin sensitive RNA synthesis and thus are sites of polymerase type II activity.     

Rapid isolation of genomic DNA from animal tissues

A simple technique for the isolation of very high molecular weight genomic DNA from animal tissues and cells is described. The method involves rapid isolation of nuclei and their embedding in agarose beads followed by extraction of lipids and proteins with SDS. The protocol does not require proteolytic digestion and the whole procedure can be completed in 1 day. The isolated DNA is digestible by restriction enzymes and free of ligase inhibitors.     

Nuclear structures in the ovarioles of the butterfly Laspeyresia pomonella. Sex chromatin in trophocytes

The nuclear structures in the ovarioles have been studied in Laspeyresia pomonella by means of light and electron microscopy, autoradiography (RNA and DNA synthesis) and molecular hybridization in situ. The karyosphere was shown to form in oocyte nuclei at the beginning of oocyte growth. Numerous protein granules appeared in close contact with the karyosphere chromosomes; the true nucleolus was absent and the whole nucleus was inactive in RNA synthesis. A special attention was paid to studying nuclear structures in trophocytes. Numerous complex nucleoli actively synthesizing RNA formed in highly endopolyploid nuclei of trophocytes. Besides, each trophocyte had a spheroid vacuolized body of DNA which developed from one of meiotic bivalents soon after trophocyte differentiation and increased in diameter up to 10-15 mu. The DNA body in trophocytes and follicle cells was in close contact with the nucleolar material. Ribosomal DNA was present in these bodies as was shown by molecular hybridization in situ. A suggestion is put forward to the effect that the DNA bodies take part in the formation of complex nucleolar apparatus of trophocytes. On the basis of both the author's and literary data, a conclusion is drawn that DNA spheres in trophocytes and follicle cells are sex chromatin bodies formed, however, by both the X- and Y-chromosomes, rather than by one Y-chromosome.