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Directed proteomic analysis of the human nucleolus.

The argyrophilic proteins of the nucleolar organizer region (Ag-NOR proteins) were specifically localized at the optical level with a modified one-step silver technique performed at 20° C.

Improvement in the staining and in the visualization of the argyrophilic proteins of the nucleolar organizer region at the optical level.

DNA REPLICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 TRANSCRIPTION AND PRE-mRNA PROCESSING .... . . ) ....... ,.... 279 Localization of Transcriptionally Active Nuclear Regions by [ HJ-Uridine Incorporation or In Situ Nick Translation . . . . . . . . . . . . . . . . 279 Pre-mRNA Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 An Arginine/Serine-Rich Domain Targets Proteins to Nuclear Speckles . . . . . . 284 Effect of RNA Polymerase II Transcription on the Localization of Splicing Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,.... 285 Distribution of hnRNP Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Localization of Splicing Factors in the Amphibian Germinal Vesicle . . . . . . . . 289 Localization of Splicing Factors in Yeast 290 Distribution of Poly(A)'t RNA . . . . . . ... . . . . ..... . ..... . . . . .... 291 Distribution of Specific Cellular Transcripts . . . . . . . . . . . . . . . . . . . . . . . 291 NUCLEOLAR ORGANIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

Macromolecular domains within the cell nucleus.

During mitosis in cultured newt pneumocytes, one or more chromosomes may become positioned well removed (greater than 50 microns) from the polar regions during early prometaphase. As a result, these chromosomes are delayed for up to 5 h in forming an attachment to the spindle. The spatial separation of these chromosomes from the polar microtubule-nucleating centers provides a unique opportunity to study the initial stages of kinetochore fiber formation in living cells. Time-lapse Nomarski-differential interference contrast videomicroscopic observations reveal that late-attaching chromosomes always move, upon attachment, into a single polar region (usually the one closest to the chromosome). During this attachment, the kinetochore region of the chromosome undergoes a variable number of transient poleward tugs that are followed, shortly thereafter, by rapid movement of the chromosome towards the pole. Anti-tubulin immunofluorescence and serial section EM reveal that the kinetochores and kinetochore regions of nonattached chromosomes lack associated microtubules. By contrast, these methods reveal that the attachment and subsequent poleward movement of a chromosome correlates with the association of a single long microtubule with one of the kinetochores of the chromosome. This microtubule traverses the entire distance between the spindle pole and the kinetochore and often extends well past the kinetochore. From these results, we conclude that the initial attachment of a chromosome to the newt pneumocyte spindle results from an interaction between a single polar-nucleated microtubule and one of the kinetochores on the chromosome. Once this association is established, the kinetochore is rapidly transported poleward along the surface of the microtubule by a mechanism that is not dependent on microtubule depolymerization. Our results further demonstrate that the motors for prometaphase chromosome movement must be either on the surface of the kinetochore (i.e., within the corona but not the plate), distributed along the surface of the kinetochore microtubules, or both.

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Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells.

A nuclear framework structure termed the nuclear matrix has been isolated and characterized. This matrix forms the major residual structure of isolated nuclei and consists largely of protein with smaller amounts of RNA, DNA, carbohydrate, and phospholipid. The nuclear matrix can be further resolved by combined treatment with DNase and RNase. The remaining nuclear protein structure, after extraction of 90 percent of the nuclear protein, 99.9 percent of the DNA, and 98 percent of the RNA and phospholipid, is termed the nuclear protein matrix. Electron microscopy of this final nuclear protein matrix reveals an interior framework structure composed of residual nucleolar structures associated with a granular and fibrous internal matrix structure. The internal matrix framework is derived from the interchromatinic structures of the nucleus, and is connected to a surrounding residual nuclear envelope layer containing residual nuclear pore complex structures. Sodium dodecyl sulfate-acrylamide gel electrophoresis of the nuclear matrix proteins demonstrates three major polypeptide fractions, P-1, P-2, and P-3, with average molecular weights of approximately 69,000, 66,000 and 62,000, as well as several minor polypeptides which migrate at approximately 50,000 and at higher molecular weights (>100,000). Polypeptides with molecular weights identical to those of P-1, P-2 and P-3 are also components of isolated nuclear envelopes and nucleoli, whereas isolated chromatin contains no detectable matrix polypeptides. This suggests that the major matrix polypeptides are localized in specific structural regions of the nucleus, i.e., nuclear envelope, nucleoli, and interchromatinic structures. The presence of cytochrome oxidase activity in the isolated nuclear matrix indicates that at least some integral proteins of the nuclear membrane are associated with the matrix.

Nuclear matrix: Isolation and characterization of a framework structure from rat liver nuclei

Publisher Summary The nucleolus is organized at the nucleolus-organizing region of the chromosomes, which are generally visible as secondary constriction regions in metaphase chromosomes. The chromatin within the constriction region is lost at interphase inside the nucleolar mass. The chromatin is highly extended at this stage. At the ultrastructural level, the nucleolus has at least three components: (1) a granular component consisting mainly of ribonucleoproteins (RNP) granules—pars granulosa, (2) a fibrillar component, consisting of RNP fibrils—pars fibrosa, and (3) chromatin elements. Chromatin elements may be present in three forms: (1) nucleolus-associated chromatin, which most likely does not take part in nucleolus formation; however, the possibility of its association with condensed inactive ribosomal cistrons, at least in some cells, cannot be overruled at present, (2) septalike intranucleolar chromatin, and (3) isolated or dispersed intranucleolar chromatin threads. Intranucleolar chromatin is often associated with the pars fibrosa. Identical components can also be found in isolated nucleoli. Studies on the nucleoli in giant chromosomes indicate that the intranucleolar chromatin in these nucleoli is present as puffs of different sizes. The nucleolar chromatin is not an autonomous structure of the nucleolus but is a continuous structure and part of the nucleolar chromosome.

The nucleolar structure.

Cytological identification and characterization of the nuclear matrix.

The orientation of Giemsa C-bands has been studied in mitotic and interphase cells of Allium cepa, A. sativum and of Aloe vera. The C-bands in these three species are located at the telomeres, secondary constriction region of the nucleolar chromosomes and the centromeric regions, respectively. Observations in A. cepa and Aloe indicate clearly that the interphase chromosomes are non-random in their orientation and possibly maintain their telophase configuration through the attachment of telomeres and perhaps of kinetochores with the nuclear membrane. Electron micrographs of onion cells also reveal that certain heterochromatic segments are associated with the nuclear membrane. — The nucleolar interstitial C-bands in A. sativum remain free in the nucleoplasm and may come close to each other due to heterochromatic attraction. Such a heterochromatic attraction is also evident between telomeric regions and between centromeres. However, a two by two attachment could not be noticed. A diagrammatic representation of the orientation of interphase chromosomes has been presented.

Sibdas Ghosh

Papers

The nucleolar structure.

Cytological identification and characterization of the nuclear matrix.

Orientation of interphase chromosomes as detected by Giemsa C-bands.

Failure of kinetochore development and mitotic spindle formation in okadaic acid-induced premature mitosis in HeLa cells.

Okadaic acid inhibits sister chromatid separation in mammalian cells.