Ribosome biogenesis in man: current views on nucleolar structures and function.

Nucleoli develop when preribosomes are synthesized at the chromosomal nucleolar organizer regions. Typically they consist of at least three nucleolar subcompartments, the fibrillar center (FC), the dense fibrillar component (DF), and the granular component (GC). The understanding of the functional arrangements of these subcompartments relates to aspects in cell biology, pathology, and virus research. In the present review morphological studies are discussed in the light of molecular findings. The available data confirm the hypothesis that rDNA transcription is connected with the DF but not necessarily with the presence of an FC. Within the DF, rDNA transcription is restricted to foci, possibly representing single transcribing genes. FCs may serve to store inactive transcription factors, to initiate rDNA transcription, and may provide structural support for transcription. The GC can be interpreted as a collection of maturing preribosomes. More recently the nucleolar subcompartments were focused on in the context of virus research and tumor biology.

The nucleolus.

Nucleoli are the sites of biosynthesis of the ribosomal precursors. They contain may copies of the genes for the main rRNAs (18S- and 28 S-rRNA) in the form of tandemly arranged repeats at the chromosomal nucleolar organizer regions (NORs). They also contain the small rRNA (5S-rRNA) that is synthesized outside the nucleolus, specific nucleolar proteins, among them the factors and enzymes necessary for transcription and transcript processing, and the precursor units of the ribosomes. In man as in may vertebrate species, three main components of nucleoli, besides chromatin, can be detected: fibrillar centres (FC), dense fibrillar component (DCF), and granular component (GC). Within a nucleolus the FCs are in many cases situated in its central region. The DFc forms a network of strands surrounding the FCs, but may sometimes reach for out towards the periphery of the nucleolus. The GC is usually situated in the peripheral regions of the nucleolus. In cells with a low level of ribosomal biosynthesis the nucleoli are small, usually with a single FC and little surrounding DFC and GC ("ring-shaped nucleolus"). In active cells the DFC forms a large network enclosing several, sometimes up to hundreds of FCs, and the GC covers a large area in the periphery ("compact nucleoli"). In cells at the onset of a new stimulation, the DFC is very prominent whereas the FCs are few and small, and the GC is also not very extensive ("reticulate nucleoli"). In some special cell types that are very active other arrangements of the structural components are found. In Sertoli cells, for instance, only one nucleolus is found, or occasionally two, each with a single large FC and a distinct area of GC, both areas being engulfed by DFC intermingled with some peripheral GC. Immunocytological and in situ hybridization studies to localize the rRNA genes within the nucleolus have so far led to divergent results. Both fibrillar components, the FCs and the DFC, have been claimed as the most probable candidates. Transcription of rDNA and the subsequent early steps of ribosome biosynthesis are localized in the DFC, whereas later steps (mature rRNA, preribosomes) are localized in the GC. The FCs may also serve as sites for the preparation of the rDNA for transcription, and as a store for certain nucleolar proteins. During mitosis, parts of the nucleolar proteins remain at the NORs. A direct contact between the nucleolus and the nuclear envelope is frequently observed but is not dependent on nucleolar activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Nucleolar morphology and rDNA in situ hybridisation in monocytes.

The aim of this study was to correlate morphological changes of nucleoli of non-proliferating monocytes to their functional activity, since nucleolar morphology is currently considered as a diagnostic marker for cell proliferation. Monocytes from healthy donors were fractionated by current counterflow centrifugation and kept in culture for 6 days. Cells were stimulated by the addition of 200 units/ml interferon gamma (IFN gamma). Under this stimulus the monocytes show no proliferation but a strongly augmented expression of type I Fc IgG receptor, human leucocyte antigen DR, human leucocyte antigen DP and human leucocyte antigen DQ. Morphological changes after stimulation included the appearance of multinucleated cells, typical signs of the activation of rRNA synthesis indicated by an increase in nucleolar size, and changes in nucleolar structure such as the appearance of reticulate and compact nucleoli. The number of nucleolus organiser regions (NORs) visualised by in situ hybridisation was compared with the position and number of nucleoli visualised by silver staining in interphase cells. In comparison with control cultures, activated monocytes show a distinct increase in the number of those NORs that take part in the formation of nucleoli. Our results show that, in non-proliferating activated monocytes, the morphology of nucleoli and the increase of NOR activity are similar to those in proliferating cells. NOR activation is therefore an indicator for cellular activity, but is not necessarily correlated with proliferation.

Human ribosomal RNA gene repeats are localized in the dense fibrillar component of nucleoli: light and electron microscopic in situ hybridization in human Sertoli cells.

The distribution of the human ribosomal gene repeat within human Sertoli cell nucleoli was investigated with the help of DNA-DNA in situ hybridization at the light and electron microscopic level. Probes from both the transcribed part of the gene repeat and the "non-transcribed" spacer were found to hybridize predominantly to the dense fibrillar component of nucleoli. It therefore can be concluded that the dense fibrillar component of nucleoli is the major site of the intranucleolar location of the ribosomal DNA. This holds true not only for the dense fibrillar component adjacent to fibrillar centers, but also for the dense fibrillar component remote from the fibrillar centers.

Nucleoli, nucleolar chromosomes and ribosomal genes in the human spermatocyte.

The formation and development of nucleoli and their connections with the nucleolar chromosomes were studied in human spermatocytes using electron microscopy, silver staining of nucleolus organizer regions (NORs), high resolution autoradiography and in situ hybridization in order to localize rRNA genes and their transcription in the different stages of meiotic prophase I. At leptotene, new nucleoli were formed, consisting of a fibrillar centre surrounded by a cap of dense fibrillar component. Following [3H]uridine uptake, label was found only over the dense fibrillar component. In situ hybridization revealed rDNA mainly in the dense fibrillar component and in the chromatin. During zygotene, nucleoli increased in size. The fibrillar centre was connected with the secondary constriction region of the nucleolar bivalent and was partially surrounded by dense fibrillar component. This shell of dense fibrillar component merged into a fibrillo-granular mesh that extended away from the fibrillar centre. Autoradiography following [3H]uridine uptake again showed the label overlaying the dense fibrillar component and the proximal part of the fibrillo-granular strands. With in situ hybridization in both the light and electron microscope, signal was mainly found in the dense fibrillar component. A small quantity of label was observed in the peripheral region of the fibrillar centre and in the adjacent chromatin. From early to late pachytene segregation of nucleolar components occurred, with a reduction in the dense fibrillar component that formed a narrow rim around the fibrillar centre with small extensions along the granular component. [3H]uridine incorporation progressively decreased. In situ hybridization showed signal located mainly in the dense fibrillar component and in the chromatin corresponding to the condensed short arm of the nucleolar bivalent. Our results indicate that the majority of rDNA is located and transcribed in the dense fibrillar component; only a small amount is present in the peripheral part of the fibrillar centre and may be transcribed there. Moreover, from leptotene to zygotene, rDNA unravels from the nucleolar chromosome into the nucleolar dense fibrillar component. From zygotene to late pachytene a progressive return to the condensed acrocentric short arm is observed.

Transcribed and nontranscribed parts of the human ribosomal gene repeat show a similar pattern of distribution in nucleoli.

The distribution pattern of the transcribed and nontranscribed parts of human ribosomal RNA genes were visualized simultaneously in the same cells by nonautoradiographic in situ hybridization. DNA probes labeled with either digoxigenin or biotin were detected in the same cells by different fluorescence systems. The signals from both the transcribed and nontranscribed parts showed a similar distribution pattern. This finding is not compatible with the conclusion, suggested by earlier studies, that the transcribed and nontranscribed parts of the rRNA genes are located at different sites within the nucleoli or in different nucleolar components.

The functional significance of nucleolar structures.

The structure of nucleoli differs widely according to their functional state. Main structural components of human and mammalian nucleoli are: the fibrillar centres (FC), the dense fibrillar component (DF), and the granular component (GC). A critical review of the recent literature and of the author's results suggests that in active nucleoli the rRNA gene repeats of the nucleolus organizing regions (NORs) are localized in the DF. The DF contains also enzymes and factors necessary for transcription, RNA processing, and pre-ribosome synthesis. The FC serves possibly as a stock of proteins, among them RNA-polymerase I. The GC is made up mainly of preribosomes. Non active (non transcribed) NORs may be situated far away from nucleoli within the cell nucleus.

Electron microscopic in situ hybridization and autoradiography: localization and transcription of rDNA in human lymphocyte nucleoli.

The distribution of ribosomal DNA (rDNA) in the nucleoli of human lymphocytes was revealed by in situ hybridization with a nonautoradiographic procedure at the electron microscopic level. rDNA is located in the dense fibrillar component of the nucleolus but not in the fibrillar centers. In the same cells the incorporation of tritiated uridine takes place in the dense fibrillar component of the nucleolus as seen by autoradiography followed by gold latensification. From these findings it can be concluded that the transcription of ribosomal DNA takes place in the dense fibrillar component of the nucleolus.

Nucleolus organizer regions in human lymphocytes as studied with premature chromosome condensation.

Non-stimulated human lymphocytes from peripheral blood usually contain only one ring-shaped nucleolus. Polyethyleneglycol-mediated cell fusion with mitotic Chinese hamster ovary cells induces premature chromosome condensation in human lymphocytes. Subsequent silver staining reveals that more than one nucleolus organizer region (NOR) is silver-positive and frequently participates in the formation of "satellite associations". It can therefore be concluded that more than one NOR contributes to the ring-shaped nucleoli of lymphocytes in human peripheral blood and that they may be transcriptionally active. During phytohemagglutinin (PHA) stimulation, the number of silver-positive NORs, the number of nucleoli and the number of chromosomes participating in "satellite associations" increase.

Sequential changes in the nucleoli of human spermatogonia with special reference to rDNA location and transcription.

The nucleoli of human spermatogonia were studied using electron microscopy, silver staining, radioautography and in situ hybridization. In all types of A spermatogonia, nucleoli were consistently located at the periphery of the nucleus and contained a single fibrillar center associated with the nuclear envelope. In B spermatogonia, nucleoli were centrally located in the nuclei and showed several fibrillar centers or were found to disintegrate. Nucleolar morphology was found to be a good, though not an unequivocal indicator of spermatogonial type. The observed changes in nucleolar morphology reflect the differentiation of spermatogonia: the nucleolar disintegration seen in B spermatogonia corresponds to a pre-leptotene cessation of rDNA transcription. In radioautographs following 3H-uridine uptake, the label was consistently found over the dense fibrillar component, except in the B spermatogonia with disintegrating nucleoli, where no uptake could be detected. In situ hybridization demonstrated that the distribution of rDNA did not correspond to the site of the fibrillar center but to the dense fibrillar component. Compared with radioautographs, this finding clearly established that transcribed units of rDNA were located in the dense fibrillar component. Silver staining was strongly positive in fibrillar centers and in the dense fibrillar component. In Ap spermatogonia the silver deposit was often localized at the edge of the fibrillar threads. The relationships between silver-stained proteins and transcribed and nontranscribed portions of ribosomal genes are reevaluated.

Ribosomal DNA is located and transcribed in the dense fibrillar component of human Sertoli cell nucleoli.

The distribution of rDNA and the uptake of tritiated uridine was investigated in nucleoli of human Sertoli cells. The nucleolar components in these cells are spatially arranged in a highly ordered and invariable way and can be recognized in both light and electron microscopy. The pattern of distribution of rDNA and the pattern of uridine uptake in these nucleoli correspond to the distribution of the dense fibrillar component but cannot be correlated to the shape and size of the fibrillar centers in these cells. It therefore can be concluded that the dense fibrillar component, and not the fibrillar centers, is the site of rDNA location and transcription in nucleoli of human Sertoli cells.

Structural changes in nucleoli during inhibition of protein- and RNA-biosynthesis.

A variety of cells (unstimulated human lymphocytes, phytohemagglutinin-stimulated human lymphocytes, diploid human fibroblasts, human melanoma cells, and Hela cells) were subjected in vitro to inhibition of protein biosynthesis by puromycin. Hela cells were also treated with actinomycin D to inhibit RNA-synthesis. Under puromycin treatment, the fibrillar centers of the nucleoli were smaller in all actively dividing cell types, whereas in small inactive lymphocytes from peripheral blood the inhibition of protein synthesis had no noticeable effect. Nucleoli with nucleolonema changed into compact nucleoli under puromycin treatment. When RNA-synthesis was inhibited, the fibrillar centers remained at an approximately constant volume. These findings indicate that proteins localized in the fibrillar centers are involved in, and are used up during, rDNA-transcription and/or further steps of ribosome biogenesis. The changes in nucleolar architecture after the inhibition of protein synthesis suggest that transcriptional processes become concentrated near sites where proteins have been stored, i.e. the fibrillar centers.

On the position of nucleolus organizer regions (NORs) in interphase nuclei. Studies with a new, non-autoradiographic in situ hybridization method.

The distribution of 18S and 28S ribosomal RNA (rRNA), i.e. the chromosomal nucleolus organizer regions (NORs) was visualized in interphases and metaphases of non-stimulated and phytohemagglutinin (PHA)-stimulated human lymphocytes with a recently developed non-autoradiographic in situ hybridization method. This procedure involves mercurated RNA as a probe and a sulfhydryl-trinitrophenyl-mercury binding ligand and FITC-labelled antibodies as detection system. Silver staining was used to visualize nucleoli in interphase. In the secondary constriction of all ten acrocentric chromosomes, varying amounts of rDNA were detected. In the interphase nuclei of most of the non-stimulated human lymphocytes, only one small nucleolus could be seen. The in situ hybridization, however, revealed several agglomerations of rDNA scattered over the whole nuclear area, clearly outnumbering the number of nucleoli in these cells. This means that not all of the NORs are transcriptionally active in non-stimulated lymphocytes and that these inactive NORs lie at a distinct distance from the active ones. With PHA stimulation (transforming the small lymphocytes from peripheral blood into large, lymphoblast-like cells) the number of nucleoli increased slightly, whereas the number of separable rDNA spots decreased. This means that in the course of PHA-induced cellular activation, formerly inactive NORs become transcriptionally active and tend to associate with one another. This indicates the occurrence of movements of the NORs within the nucleus, depending on their transcriptional activity.

On the fusion of nucleoli in interphase.

By comparing unstimulated and phytohaemagglutinine (PHA)-stimulated human lymphocytes from peripheral blood it was found that the fusion of nucleoli in interphase is not merely a passive phenomenon but is strongly correlated to the metabolic activity of cells. The fusion is independent of the cell cycle (DNA-replication cycle).

Evaluation of radiation-induced chromosomal aberrations in human peripheral blood lymphocytes in vitro: result of an IAEA-coordinated programme.

The results of an IAEA coordinated programme on radiation induced chromosomal aberrations in human peripheral blood lymphocytes in vitro are presented. In a master experiment, a whole blood sample from one donor was irradiated with 200 R of X-rays. Different fixation times from 46 to 82 h were used. The progression of cells into mitosis was monitored by BrdUrd incorporation. 14 investigators took part in the scoring of chromosomal aberrations. The main conclusions of this study are: (1) The mean frequencies of aberrations changed with fixation time. (2) The number of cells scored as aberrant by different laboratories was very similar, but there was variability in the number of aberrations scored per aberrant cell. (3) The differences in the frequencies of aberrations between laboratories were minimal when the scoring was restricted to the first major peak of mitotic activity and sufficient cells were scored. It is concluded that using controlled experimentals conditions, human peripheral blood lymphocytes can effectively be used as a reliable biological dosimeter for absorbed radiation dose.

The influence of the cell cycle on structure and number of nucleoli in cultured human lymphocytes.

The nucleoli of lymphocytes undergo a typical sequence of structural changes after stimulation by phytohaemagglutinin. These changes are independent of the cell cycle. Neither the inhibition of DNA-synthesis (by adenosine and methotrexate), nor the elimination of postmitotic interphase nuclei (by a colchicine block of mitoses), nor the release from such blocks has a noticeable effect on nucleolar structure or on the sequence of nucleolar changes. The number of nucleoli per cell is clearly influenced by the cell cycle. Mitosis leads to a marked increase in the number of nucleoli, whereas in all stages of interphase a decrease occurs.