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).

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)

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.

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.

[Cell aging in vitro (author's transl)]. / Zellalterung in vitro

Human diploid fibroblast cultures in the phase of degeneration were compared with logarithmically-growing young cultures. Old cultures contain cells of increased size showing all signs of intact protein synthesis by means of the light and the electron microscope. DNA synthesis and cell division do not occur in old cultures. Premature chromosome condensation is still possible in old cells after fusion with mitotic cells of a fast-growing culture.

[Ultrastructure of human chromosomes]. / Feinstruktur der menschlichen Chromosomen.

The structural elements of chromosomes are chromatin fibrils of about 100 A diameter (see the preceeding paper by NOLL). The chromosomes of most eukaryontic species are single stranded. Each chromatid consists of only one chromatin fibril and thus only of one DNA-double helix. In interphase the chromatin fibril is loosely and irregularly folded. With the onset of mitosis it becomes more densely but still irregularly packed to form the long and thin prophase chromosome. Metaphase chromosomes are formed by a coiling of the thin prophase chromatids (major coils). Chromosome banding can be produced with the aid of specific fluorescent dyes or by special pretreatments before staining. The banding is due to many factors but an important one is the different AT- ad GC-content of bandlike regions. In very thin chromosomes such as premature condensed chromosomes the bands are very fine and can be correlated in size to the chromomeres of polytene giant chromosomes.

Modern ideas on chromosome structure.

The structural element of an eukaryotic chromosome is the so-called chromatin fibre. It is a DNA-protein complex of about 100-200 A thickness and most probably running through from one end of a chromatid to the other. The fine structure of this DNA-protein fibre suggests a core of globular histone subunits around which the DNA-molecule is wound. The single strandedness of chromatids is suggested by the structure of premature condensed chromosomes. The course G-banding seen in metaphase chromosomes is presumably caused by groups of much finer bands seen in decondensed chromosomes. The number of such fine bands in the human genome is estimated to be 10 000-100 000, figures which are in the range of the number of genes in man.

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.

Comparison of silver staining of nucleolus organizer regions in human lymphocytes and fibroblasts.

Ag-staining of the nucleolus organizer region (NOR) was studied in the acrocentric chromosomes identified by Q-banding in repeated lymphocyte and skin fibroblast cultures from three different individuals. A similar pattern of Ag-stainability of NORs was found in the two tissues in each individual. Small differences concerning, in each case, only one of the acrocentric chromosomes were found between repeated lymphocyte cultures, as well as between lymphocyte and fibroblast cultures of the same individual without indication of any prevalence of one tissue type in a certain direction. The possibility that these differences are caused by different stages of NOR activation is discussed.

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.

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.

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.

Random position of human heterochromatin-bearing chromosomes in first and third mitoses of lymphocyte cultures.

The position of chromosomes 1, 9, and 16 in first and third mitoses of lymphocyte cultures was measured in BUdR-labelled air-dried chromosome preparations. No significant deviation from a random distance could be found between the two homologues of each chromosome, either in the first or in the third mitoses after phytohemagglutinin stimulation. Colchicine treatment also had no influence.

Satellite association frequency and number of nucleoli depend on cell cycle duration and NOR-activity. Studies on first, second, and third mitoses of lymphocyte cultures.

In human lymphocyte cultures the frequencies of satellite associations in first, second, and third mitoses were investigated using the BUDR-method. A marked decrease of the association frequency with increasing numbers of cell cycles was found. The number of nucleoli seen in interphase is correlated with the satellite association frequency in the respective metaphase. Satellite association is positively correlated to Ag-staining intensity of the NORs. Individual differences in satellite association are due to differences in NOR activity and in lymphocyte activation. BUDR diminishes somewhat the Ag-staining intensity of the NORs but has no effect on satellite association frequencies. The main reason for the decrease of satellite association frequency in second and third lymphocyte mitoses is presumably a certain dislocation of the original chromosome position during mitosis and a decreased possibility of association during the short interphases. The high association frequency in first mitosis resembles the chromosome position in the long interphase of G0-lymphocytes.

[New findings on the structure of chromosomes (author's transl)]. / Neue Befunde zur Struktur der Chromosomen

The structural element of eukaryotic chromosomes is the chromatin fibre consisting of histones and DNA. The chromatin fibre is about 100--200 A thick. One chromatid is built up from one chromatin fibre running through from one end to the other and laid in numerous irregular foldings. The chromatin fibre is a chain of nucleosomes. These are globular histone bodies around which the DNA winds. Nucleosomes can be observed in isolated chromatin fibrils as well as in thin sections of chromosomes after different modes of fixation. Prophasic chromosomes or early premature condensed chromosomes are thin uncoiled threads. With chromosome condensation a major coiling is seen. No constant regular arrangement of the chromatin fibril besides the major coils is observed. A rather diffuse decondensation takes place in ana- and telophase.

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.

Nucleolar changes in human phytohaemagglutinin-stimulated lymphocytes.

The nucleoli of lymphocytes from circulating peripheral blood and from phytohaemagglutinin (PHA)-stimulated cultures (from 2 h-96h) were studied using a silver method, RNA-specific fluorescent staining, and electron microscopy of ultrathin sections. In peripheral blood about 75% of the lymphocytes have one "ring-shaped" nucleolus composed of a distinct fibrillar centre surrounded by a dense pars fibrillaris and little granular material; the remaining lymphocytes showing two or more small "ring-shaped" nucleoli. With PHA stimulation, the number of cells with several nucleoli increases first (from 2 h--12 h). Next, cells containing one or, at most, two large nucleoli with nucleolonema devoid of fibrillar centers are seen (from 4 h on). 34 h after PHA, nucleoli of the "compact" type containing one or more fibrillar centres appear and comprise about 60% of the cells after 72 h. The appearance of more than one nucleolus per cell shortly after PHA administration suggests an activation of additional nucleolar organizer regions (NOR), which fuse to form one or two large nucleoli with nucleolonema. These are then transformed into "compact" nucleoli. The fibrillar centers stasin preferentially with silver. They contain nonchromosomal proteins and may serve as stores for nucleolar proteins. The fusion of activated NORs during the first cell cycle explains the relatively high frequency of satellite associations in first mitoses compared to later mitoses after stimulation.

Fine structure of 33 258 H-treated chromosomes.

Metaphase chromosomes of mouse strain L cells show strikingly uncondensed pericentric heterochromatic regions after treatment of living cells with the benzimidazol-derivative 33 258 Hoechst. In electron micrographs of total preparations after G-band staining the chromosomes are seen to be made up of irregularly folded fibrils of 200-400 A in diameter. In the uncondensed regions only very few fibrils laid loose loops are present, making it probable that only one fibril forms one chromatid.