SummaryUsing a monoclonal antibody (No-194) we have identified, in Xenopus laevis and other amphibia, an acidic protein of M, 40,000 (ribocharin) which is specifically associated with the granular component of the nucleolus and nucleoplasmic 65S particles. These particles contain the nuclear 28S rRNA and apparently represent the precursor to the large ribosomal subunit in nucleocytoplasmic transit. By immunoelectron microscopy ribocharin has been localized in the granular component of the nucleolus and in interchromatin granules. During mitosis ribocharin-containing particles are associated with surfaces of chromosomes and are recollected in the reconstituting nucleoli in late telophase. We suggest that ribocharin is a specific component of precursor particles of the large ribosomal subunit, which dissociates from the 65S particle before passage through the nuclear envelope, and is reutilized in ribosome biogenesis.

Upon incubation of cultured rat cells with the adenosine analogue 5,6-dichloro-l-ß-D-ribofuranosylbenzimidazole (DRB), nucleoli reversibly dissociate into their substructures, disperse throughout the nuclear interior, and form nucleolar "necklaces" . We have used this experimental system, which does not inhibit transcription of the rRNA genes, to study by immunocytochemistry the distribution of active rRNA genes and their transcriptional products during nucleolar dispersal and recovery to normal morphology . Antibodies to RNA polymerase I allow detection of template-engaged polymerase, and monoclonal antibodies to a ribosomal protein (Sl) of the small ribosomal subunit permit localization of nucleolar preribosomal particles. The results show that, under the action of DRB transcribed rRNA, genes spread throughout the nucleoplasm and finally appear in the form of several rows, each containing several (up to 30) granules positive for RNA polymerase I and argyrophilic proteins . Nucleolar material containing preribosomal particles also appears in granular structures spread over the nucleoplasm but its distribution is distinct from that of rRNA gene-containing granules . We conclude that, although transcriptional units and preribosomal particles are both redistributed in response to DRB, these entities retain their individuality as functionally defined subunits . We further propose that each RNA polymerase-positive granular unit represents a single transcription unit and that each continuous array of granules ("string of nucleolar beads") reflects the linear distribution of rRNA genes along a nucleolar organizer region . Based on the total number of polymerase I-positive granules we estimate that a minimum of 60 rRNA genes are active during interphase of DRB-treated rat cells .DNA and chromatin of interphase nuclei occur in topologically well-defined arrangements which may play an essential role in transcriptional and replicative events (for reviews see 1 and 2). The most impressive example for the constraint of genes at a specific nuclear locus is provided by the genes coding for the precursor molecules of ribosomal RNA (rRNA genes) which are clustered in a spheroidal body known as nucleolus (for a review see 3) . To gain more insight into the forces that maintain this highly ordered arrangement of the nucleolar genes and their products we have studied the druginduced nucleolar disintegration and dissociation of nucleolar subunits.A large number of chemicals are known to interfere with ribosome formation and to induce characteristic alterations ofthe nucleolar architecture (4, 5) . The halogenated adenosine analogue 5,6-dichloro-1-0-D-ribofuranosylbenzimidazole (DRB)' is especially remarkable . (a) When DRB is added to ' Ahbreviation used in this paper : DRB, 5,6-dichloro-J-ß-1>-ribofuranosylbenzimidazole.

A monoclonal murine antibody (No-I 14) is described which reacts specifically with a polypeptide of molecular weight (M,) 180000 present in low-speed nuclear pellets from oocytes and somatic cells of Xenopus laevis and X. borealis and in isolated amplified nucleoli. Two-dimensional gel electrophoresis has revealed the acidic nature of this polypeptide (isoelectric at pH of ca 4.2 in the presence of 9.5 M urea). A relatively large proportion of the protein is extracted at elevated ionic strength( i.e., at 0.4-0.5 M alkali salt) in a form sedimenting at approx. 7-8S , compatible with a monomeric state. It is also extracted by digestion with RNase but not with DNase. In immunofluorescence microscopy , antibody No-114 stains intensely nucleoli of oocytes and all somatic cells examined , including the residual nucleolar structure of Xenopus erythrocytes which are transcriptionally inactive. During mitosis the antigen does not remain associated with the nucleolar organizer regions (NOR) of chromosomes but is released and dispersed over the cytoplasm until telophase when it re-associates with the reforming interphase nucleoli. At higher resolution the immunofluorescent region is often resolved into a number of distinct subnucleolar components of varied size and shape. Immunoelectron microscopy using colloidal gold-coupled secondary antibodies reveals that the M, 180000 protein is confined to the dense fibrillar component of the nucleolus. This conclusion is also supported by its localization in the fibrillar part of segregated nucleoli of cells treated with actinomycin D. We conclude that nucleoli contain a prominent protein of M , 180000 which contributes to the general structure of the dense fibrillar component of the interphase nucleolus , independent of its specific transcriptional activity.In the past two decades considerable progress has been made in understanding the basic functional organization of the nucleolus. Combined morphological, biochemical, and auto radiographic data have established that the nucleolus is the site of the genes which code for pre-rRNAs, transcription of these genes, assembly of the pre-rRNA molecules with specific proteins into ribonucleoprotein complexes, and maturation of the preribosomal particles both at the RNA and protein level (for reviews see [1][2][3]). Based primarily on electron microscopic autoradiography of cells labeled with radioactive uridine, morphologically distinct components of nucleoli can be correlated with certain steps of the pathway of ribosome biogenesis [4][5][6][7][8][9]. Although nucleoli can display different shapes and arrangements of their compot)ept substructures, depending on the .specific cell type and metabolic condition of the cell, there is general agreement that primary transcripts containing pre-rRNA are located in the dense fibrillar part of the

Using antibodies to various nucleolar and ribosomal proteins, we define, by immunolocalization in situ, the distribution of nucleolar proteins in the different morphological nucleolar subcompartments. In the present study we describe the nucleolar localization of a specific ribosomal protein ($1) by immunofluorescence and immunoelectron microscopy using a monoclonal antibody (RS1-105). In immunoblotting experiments, this antibody reacts specifically with the largest and most acidic protein of the small ribosomal subunit ($1) and shows wide interspecies cross-reactivity from amphibia to man. Beside its localization in cytoplasmic ribosomes, this protein is found to be specifically localized in the granular component of the nucleolus and in distinct granular aggregates scattered over the nucleoplasm. This indicates that ribosomal protein $1, in contrast to reports on other ribosomal proteins, is not bound to nascent pre-rRNA transcripts but attaches to preribosomes at later stages of rRNA processing and maturation. This protein is not detected in the residual nucleolar structures of cells inactive in rRNA synthesis such as amphibian and avian erythrocytes. During mitosis, the nucleolar material containing ribosomal protein $1 undergoes a remarkable transition and shows a distribution distinct from that of several other nucleolar proteins. In prophase, the nucleolus disintegrates and protein $1 appears in numerous small grariules scattered throughout the prophase nucleus. During metaphase and anaphase, a considerable amount of this protein is found in association with the surfaces of all chromosomes and finely dispersed in the cell plasm. In telophase, protein Sl-containing material reaccumulates in granular particles in the nucleoplasm of the newly formed nuclei and, finally, in the re-forming nucleoli. These observations indicate that the nucleolus-derived particles containing ribosomal protein $1 are different from cytoplasmic ribosomes and, in the living cell, are selectively recollected after mitosis into the newly formed nuclei and translocated into a specific nucleolar subcompartment, i.e., the granular component. The nucleolar location of ribosomal protein $1 and its rearrangement during mitosis is discussed in relation to the distribution of other nucleolar proteins.The current concept of the functional organization of the nucleolus is primarily based on electron microscopy and some information at the nucleic acid level. Thus, the nucleolus is defined as a cluster of transcriptionally active rRNA genes that is associated with structures representing a stockpile of ribosomal precursor particles at various stages of processing and maturation (for reviews see references 1-3). By electron microscopy, three morphologically distinct nucleolar components have been distinguished (for reviews see references 4-6): the fibrillar center(s), the dense fibrillar component, and the granular component. Different functions have been assigned to these nucleolar substructures. Recently, RNA polymerase I, the enzyme ...

SUMMARY We have investigated the existence of structural components in the nucleus of the oocyte of Xenopus laevis and other amphibia that are insoluble in non-denaturing detergents and buffers of low and high ionic strength. These cells are particularly suitable for such studies as they have a high frequency of extrachromosomal amplified nucleoli and pore complexes of the nuclear envelope. Using biochemical and immunological techniques, we have shown these structures to contain only two major proteins. These are a polypeptide of Mr 145 000, which is located in a meshwork of filaments specific to the nucleolar cortex, and certain nucleoplasmic bodies probably derived therefrom, and a polypeptide of Mr 68 000, which is the predominant constituent of the lamina–pore complex structure. We show that the latter protein is related to, but not identical to, lamina proteins (‘lamins’) of somatic cells, indicating cell type-specificity of the expression of polypeptides of the lamin family. In addition, we describe a protein of Mr 180 000, which is the major constituent of the dense fibrillar component of the nucleolus. This can be partially solubilized in buffers of moderately high ionic strength. We interpret proteins of this category as karyoskeletal components involved in the architectural organization of specific functional topology within the nucleus. In contrast to previous reports for other cell types we have found no other prominent high-salt-insoluble structures in the nuclear interior, indicating the absence of an extended internal nuclear matrix in this kind of nucleus.

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