Intact Nucleoli Research Articles

Isolation of transcriptionally active chromatin from mammalian nucleoli.

Nucleoli isolated from HeLa cells are functionally active but contain large amounts of RNA and proteins (RNA/DNA ratio 1:1; protein/DNA ratio 7:1). We have isolated from the nucleolus a DNA-protein complex that has the characteristics of nucleolar chromatin (RNA/DNA ratio less than 0.05:1; protein/DNA ratio 1.7:1). This nucleolar chromatin has most of the transcriptional activity of the intact nucleolus and, as assayed by circular dichroism and dye binding, has largely preserved its structure. The isolation of a transcriptionally active, fragment of chromatin, which constitutes only a small part of the total genome and codes for only one recognizable product, offers several advantages for the study of chromatin structure and function.

Nuclear thyroid hormone receptors: Evidence for association with nucleolar chromatin

When hypothyroid rat liver nuclei labeled in vivo with [125 I]L-triiodothyronine are incubated with micrococcal nuclease, the nuclear chromatin is digested and chromatin particles are released into the medium. The nuclease-treated nuclei contain intact nucleoli and a residual chromatin fraction. When this residual chromatin is purified, it contains only a small percentage of the initial nuclear DNA but is strikingly enriched in [125 I]L-triiodothyronine. This chromatin fraction has many of the characteristics of nucleolar chromatin including a high protein to DNA ratio, an abundance of nonhistone proteins, and a relatively high RNA to DNA ratio. An association of thyroid hormone receptors with a nucleolar component implicates this organelle in the early events of thyroid hormone action.

Comparative effect of heparin on RNA synthesis of isolated rat-liver nucleoli and purified RNA polymerase A.

The polyanion heparin has been employed to study the interaction of rat liver DNA-dependent RNA polymerase A and its template under various conditions. Heparin very efficiently inhibits polymerase molecules, which are not bound to DNA or are associated with the template in a loose, i.e. non-specific fashion. Purified nucleoli, isloated from rat liver nuclei, contain RNA polymerase A in abundant quantities of which only a portion is bound in a transcriptional complex. Excess enzyme, which is contained in the nucleolus in a quasi free form, can be transferred to an exogenously added template and can be completely inhibited by the prior addition of heparin. The enzyme contained in a transcriptional complex, however, initiated in vivo and completing these RNA chains in vitro, is fully resistant to heparin. In contrast to these results it has been found that RNA polymerase A extracted from nuclei and purified by various chromatographic steps does not form heparin-resistant complexes, even after the enzyme has been bound to the DNA template. Moreover it has been found that purified RNA polymerase A transcribes truly native DNA extremely poorly, indicating that the enzyme is highly deficient in the act of initiation on duplex DNA. It is therefore questionable whether the interaction of the purified enzyme and isolated DNA represents binding to true initiation complexes as is observed in the intact nucleolus.

Isolation and some characteristics of nucleoli from the brain

A method is described for isolating pure and intact nucleoli from pig and young rat brain cortex using the procedure of Muramatsu and Busch slightly modified. The isolated nucleolar preparation possesses the following properties. (1) Morphological characteristics, as revealed by phase-contrast and electron microscopy, show pure and intact nucleoli. (2) RNA/DNA ratio is about 1.2–1.3. (3) Electrophoretic patterns of nucleolar RNA show evidence of high molecular RNA (45 S, 36 S, 32 S and 28 S), unlike those of extranucleolar RNA (28 S and 18 S). (4) Polyacrylamide gel electrophoresis of 8M urea buffer soluble proteins shows evidence of specific patterns. (5) The nucleolar preparation exhibits RNA synthetic activity. (6) The nucleolar preparation has some ribonuclease activity but no glutamic dehydrogenase activity.

Isolation and Some Characteristics of Nucleoli from the Brain

Abstract A method is described for isolating pure and intact nucleoli from pig and young rat brain cortex using the procedure of Muramatsu and Busch slightly modified. The isolated nucleolar preparation possesses the following properties. (1) Morphological characteristics, as revealed by phase-contrast and electron microscopy, show pure and intact nucleoli. (2) RNA/DNA ratio is about 1.2–1.3. (3) Electrophoretic patterns of nucleolar RNA show evidence of high molecular RNA (45 S, 36 S, 32 S and 28 S), unlike those of extranucleolar RNA (28 S and 18 S). (4) Polyacrylamide gel electrophoresis of 8M urea buffer soluble proteins shows evidence of specific patterns. (5) The nucleolar preparation exhibits RNA synthetic activity. (6) The nucleolar preparation has some ribonuclease activity but no glutamic dehydrogenase activity.

Meiotic cells in situ of Lilium longieflorum as seen with the scanning electron microscope.

After fixation in an acid fixative, 20 μm paraffin sections of microsporocytes of Lilium longiflorum showed a number of details when observed by scanning electron microscopy. These were: (1) the surface of meiotic prophase chromosomes, (2) chromatids of synapsed pachytene chromosomes, (3) chromosome-nuclear boundary associations, (4) chromosome-nucleoli associations, (5) the surface of intact nucleoli, and (6) the interior of sectioned nucleoli. The most suitable fixative appears to be unbuffered 10% formalin, but 10:5:85 formalin-acetic-alcohol or 1:3 acetic-alcohol could be used successfully, since they solubilized the nucleoplasm. Critical point drying technique, compared with simple air drying, yielded no difference in the image of the chromosomes. The procedure described is useful for observing meiotic prophase from preleptotene through pachytene stages.

Protein synthesis by isolated pea nucleoli.

A new method is described for the preparation of active, nucleus-free nucleoli and chromatin in relatively high purity and in sufficient quantities to permit biochemical and electron microscopic investigation. This method consists of disintegrating previously isolated nuclei by grinding with glass beads in an isotonic medium thus liberating structurally intact nucleoli and chromatin threads. Nucleoli and chromatin are then purified by differential centrifugation in Ficoll solutions. A study of the chemical composition, submicroscopic structure, and biological activity of the nucleolar preparation has been made. An equivalent study of the chromatin material has also been carried out in order to assess the significance of chromosomal contamination in nucleolar protein synthesis. The isolated nucleoli rapidly incorporate leucine-C(14) into acid and base stable compounds in vitro. Such incorporation lasts for 20 minutes at 37 degrees C and is enhanced by the addition of an energy-regenerating system and a complete amino acid mixture. It is independent of the nuclear Ph 5 enzymes. The bulk of the incorporated label is recovered in the residual, ribosome-like nucleolar protein fraction and a small percentage is found in the acid-extractable basic proteins. The rate of protein synthesis by isolated nucleoli is more rapid than that occurring in the chromatin fraction. This is taken as an additional proof that the nucleolus is the principal site of protein synthesis in the interphase pea nucleus.