Showing papers on "Nucleolus published in 1973"

Independent control of ribosomal gene replication in polytene chromosomes of Drosophila melanogaster.

Abstract: The saturation values for ribosomal RNA hybridization have been determined for DNA from diploid and polytene tissues of Drosophila melanogaster. These values have been measured in XO and XX larvae which have, respectively, one and two nucleolus organizers in the diploid chromosome set. The results show that (1) in diploid cells the ribosomal (r)DNA is present in amounts proportional to the number of nucleolus organizers, (2) in polytene cells the rDNA is under-replicated with respect to the euchromatic DNA, and (3) in polytene cells the amount of rDNA is independent of the diploid number of nucleolus organizers. These observations suggest that somatic variations in rDNA content may involve independent polytenization of the nucleolus organizer without change in the number of ribosomal cistrons per organizer. The independent polytenization of rDNA is proposed as the chromosomal explanation for the relative increase in rDNA in flies of the XO constitution.

Differential Staining of Nucleolus Organisers in Mammalian Chromosomes

Abstract: THE in situ DNA-RNA hybridisation techniques to locate a certain genome fraction with unique nucleotide sequences1–6 have assigned ribosomal cistrons to the satellites of human D and G group chromosomes4,5. This is of particular interest because the satellites organise nucleoli in somatic7 and meiotic cells8–10. During the course of study on the Giemsa banding mechanism (unpublished) we found that the satellite bodies of human acrocentrics can be differentially stained with Giemsa after simple procedures including extraction of both nucleic acids and histones. The staining profile, herein referred to as ‘N band’, differed clearly from the Quinacrine, Giemsa, Reversed-Giemsa, Centromeric, or Giemsa 11 banding patterns11,12. In rat kangaroo chromosomes, the N bands appeared exclusively in the nucleolus organisers13,14, that is, in the secondary constriction of the X chromosome. Further application of this technique to other mammalian species strongly indicated that the N-band-positive sites coincide with nucleolus organisers.

Regulation of ribosomal rna gene multiplicity in drosophila melanogaster

Abstract: The ribosomal RNA (rRNA) genes of Drosophila melanogaster can undergo a disproportionate replication of their number. This occurs when the cluster of rRNA genes (rDNA) of one chromosome is maintained with a homologous chromosome that is completely or partially deficient in its rDNA. Under appropriate genetic conditions, it appears that disproportionate rDNA replication can be generated at the level of both somatic and germ line cells. In the latter case, mutants partially deficient for rDNA can increase their rRNA gene number to the wild type level and transmit this new genotype to successive generations.

An Ultrastructural and Radioautographic Study of Early Oogenesis in the Toad Xenopus Laevis

Abstract: SUMMARY Early oogenesis in the toad Xenopus laevis has been investigated at the ultrastructural level, with particular reference to the formation of extrachromosomal DNA. Thymidine incorporation was localized by electron microscope radioautography. In oogonia, the nucleus is irregular in outline and may contain several nucleoli. Oocytes, from premeiotic interphase to late pachytene, are found in cell nests which are estimated to consist of about 16 cells each. Adjacent oocytes within a nest are connected by intercellular bridges and develop synchronously. Each premeiotic interphase-leptotene oocyte has a round nucleus which contains one or two centrally located, spherical nucleoli. Electron-microscope radioautography showed that all nuclei in a cell nest incorporate thymidine synchronously during premeiotic S-phase. In zygotene oocytes, axial cores and synaptonemal complexes are observed in the nucleus and abut against the inner nuclear membrane in the region nearest the centre of the cell nest. The nucleolus is still moreor-less round in outline, but is asymmetrically positioned in the nucleus. It lies near the nuclear envelope on the side of the nucleus furthest away from the attachment of the chromosome ends, that is, nearest the outside of the cell nest. Each nucleolus is surrounded by a fibrillar ' halo' of nucleolus-associated chromatin into which a low level of thymidine incorporation occurs during zygotene. This is thought to represent the start of the major period of amplification of the ribosomal DNA. Pachytene is characterized by the presence of synaptonemal complexes in the nucleus. The nucleolus becomes very irregular in outline. The fibrillar area around it, which represents the extrachromosomal DNA, increases in size and thymidine is incorporated over the whole of this region. In late pachytene, many small fibrogranular bodies, the multiple nucleoli, are formed in it. The members of a cell nest become separated from one another at this time and begin to develop asynchronously. In diplotene, synaptonemal complexes are no longer observed in the nucleus. The most prominent structures in the nucleus are now the multiple nucleoli, which increase greatly in number in early diplotene. A large increase in cytoplasmic volume occurs and the oocyte grows in size.

A light- and electron-microscopic study of primordial germ cells in the early mouse embryo.

Abstract: The primordial germ cells (PGC9s) of the early mouse embryo have been identified in semi-thin and ultra-thin plastic sections on the combined bases of location and a distinctive set of morphological features. These cells originate extra-gonadally and their path of migration during the stages investigated agrees with results of previous histological and experimental studies. At 8–9 days of development, PGC9s are observed among the endoderm cells of the mid- and hindgut and the yolk stalk; at 10–11 days, PGC9s are seen in the dorsal mesentery, the dorsal coelomic lining, and in the rudimentary genital ridge; by 13 days, the gonad is abundantly populated with germ cells. During the migratory period the PGC9s appear as small (10–12 μm diameter), oval, basophilic cells which have a large nucleo-cytoplasmic ratio. These cells possess numerous free ribosomes and polysomes, a filamentous ground cytoplasm, and few profiles of endoplasmic reticulum. Mitochondria display small oval and thread-like profiles. A Golgi complex is not prominent in the PGC9s. The densely granular and fibrillar nucleus is often oval in outline but sometimes shows irregular contours. A large nucleolus is present. Although located among groups of cells that exhibit extensive cell junctions, the PGC9s have not been found to share such junctions with neighboring cells. Furthermore, the PGC9s possess small cytoplasmic processes that contain numerous microfilaments. These observations are interpreted as morphological correlates to the migratory activity of these cells. In the early PGC9s located in the gut, regions of the endoplasmic reticulum where membranes are closely apposed, perhaps fused, often present the appearance of annulate cisternae. In addition, compact aggregates of granulofibrillar material are found in the cytoplasm of these same cells. Neither of these structures is detected in the mesenteric PGC9s, the gonadal germ cells, or any other cell of the embryo at the stages studied. It is suggested that these two cytoplasmic elements may be related to further differentiation of the germ cells. The germ cells of the developing gonad are large, oval cells which possess a nucleus of very round contour. Both the nucleus and the cytoplasm retain a densely granular appearance. The expanded cytoplasm contains a large Golgi complex but still few profiles of endoplasmic reticulum. Free ribosomes and polysomes are abundant. The germ cells share extensive gap junctions with one another and with adjacent stromal cells.

A temperature-sensitive mutation affecting 28S ribosomal RNA production in mammalian cells.

Abstract: We have characterized a temperature-sensitive (ts) mutant of the hamster cell line BHK 21 that appears to have a defect in the processing of ribosomal RNA precursors at 39°. Mutant ts 422E grows at a normal rate at 33°, but upon shift to 39° growth stops after about one cell doubling. The appearance of 28S rRNA and large ribosomal subunits in the cytoplasm of ts 422E at 39° is inhibited by about 95%, when compared to wild-type BHK cells. Production of 18S rRNA and small ribosomal subunits is unaffected. Shift-up experiments show that the defect in 28S rRNA production can be detected as early as 2-3 hr after the shift to 39°. Synthesis of the larger rRNA precursor is normal at high temperature, but the processing appears to be arrested after the formation of 32S rRNA. 32S rRNA accumulates to some extent in the nucleoli of ts 422E. ts 422E cells appear to have a single mutation, directly affecting the conversion of 32S to 28S rRNA. The reduced amount of 28S rRNA in the cytoplasm of ts 422E cells at 39° seems therefore responsible for their inability to grow at this temperature.

Fine structure and peroxidase activity of circulating micromegakaryoblasts and platelets in a case of acute myelofibrosis.

Abstract: Summary. In a case of acute myelofibrosis with thrombocytosis, electron microscopical examination of the buffy coat revealed the existence of numerous mononuclear cells with a blastic appearance. They were considered blasts because nucleoli were prominent, and ribosomes, free or linked to short saccules of rough endoplasmic reticulum (RER), were numerous. The presence of peroxidase activity in the perinuclear space and RER of these cells was identical with the occurrence and location of this enzyme reaction product in normal bone marrow megakaryocytes. The results indicated that the atypical cells were circulating micromegakaryoblasts. Highly abnormal platelets were also present in the patient's peripheral blood. The peroxidase test provides a useful adjunct for distinguishing cells of the megakaryocyte line which in the past may have been mistaken for atypical lymphocytes or myeloblasts by light or electron microscopy.

The ultrastructure of the accessory sex organs of the male rat

Abstract: The fine structure of the nuclei of epithelial cells of the dorsal lobe of the rat prostate were studied 2, 3, 5, 7 and 21 days after castration. The nucleolus appears to undergo a progressive disorganisation with partial fragmentation and dispersion of its normal components. Changes in the nucleoplasm were primarily reflected by a condensation of chromatin, particularly along the nuclear membrane and adjacent to the nucleolus. Later, different types of intranuclear inclusions were observed. After 21 days, the nuclei were characterized by an irregular outline with large indentation. Within the nucleoplasm aggregates of coarse granular chromatin were found. No cell necrosis was observed, indicating that androgen deprivation results in a remodeling of the cell to a less active state with marked cellular alterations and cessation of secretion, but apparently with some of their basic functions still intact. Injections of testosterone completely reverse the castrated-induced alterations. The changes observed are assumed to be due to the withdrawal of the androgenic stimulus, with a direct influence on the secretory function of the cell. The findings support the view that the stimulating secretory effect of androgen is mediated via an intranuclear androgen receptor, probably located in the nucleolus-associated-chromatin. It is also proposed that the secretory function of the epithelial cells of the prostatic complex, initiated by androgens, may be regulated by an intranuclear secretory center.

Oogenesis in Tenebrio molitor: Histological and autoradiographical observations on pupal and adult ovaries

Abstract: (i) Nurse cells and oocytes in the telotrophic ovary of Tenebrio molitor L. (Insecta, Coleoptera-Polyphaga) are differentiated in the larval stadium. (ii) During pupation DNA synthesis occurs in the nurse cells and is probably associated with polyploidy; some of them become multinucleate. The functional significance of these events is interpreted as preparation for subsequent massive RNA synthesis. (iii) The oviducts incorporate [3H]thymidine and undergo elongation due to mitoses. This ceases at eclosion. (iv) RNA synthesis in the pupal ovary is low, but increases in the nurse cells and follicle cells just prior to eclosion. (v) In the adult ovary, once the growth phase has been initiated, a primary oocyte takes about six days to reach maturity. (vi) The nurse cells, though apparently lacking nucleoli, synthesize much stable RNA which reaches each oocyte via a trophic cord. (vii) The follicle cells undergo continuous DNA synthesis: some nuclei contain over 64 times the haploid amount of DNA. Replication is probably asynchronous within a nucleus: this may account for the phenomenon of simultaneous DNA and RNA synthesis in the follicle cells, which also lack nucleoli. (viii) Oogenesis has been divided into nine developmental stages, three of which are vitellogenic. (ix) The oocyte chromosomes are capable of RNA synthesis both when despiralized during early previtellogenesis and after karyosome formation, which occurs at stage 6. (x) The protein content of the oocyte appears to have a dual origin: at least part of the ooplasmic proteins form in situ ; while yolk proteins are derived from the haemolymph. The extra-ovarian protein reaches the oocyte via spaces which develop between the follicle cells. (xi) The nucleoplasm becomes more heavily labelled with proteins than the ooplasm. With [3H]leucine, methionine and phenylalanine but not with tryptophane or arginine, there is an increased incorporation into the karyosome. It is suggested that this karyosome-associated protein may function in gene masking. (xii) The significance of these findings is discussed with reference to the literature.

The activity of the nucleolus organising region and the origin of cytoplasmic nucleoloids in meiocytes of Lilium

Abstract: The origin of the nucleolus-like bodies (“nucleoloids”) released into the cytoplasm during the meiotic divisions in pollen mother cells ofLilium has been traced. Chains of accessory nucleoli are formed at the nucleolus organising regions (NOR) of the nucleolar chromosomes during pachytene and diplotene while the parent-cell nucleolus is undergoing dissolution. Autoradiography using3H-uridine as a tracer shows that this involves the resumption of RNA synthesis at the NOR, although no new synthesis is associated with the parent-cell nucleolus. The accessory nucleoli are released from the NOR to become distributed throughout the nucleus in late prophase; there is no evidence that they contain DNA. In division phases, their material is probably held at the chromosome surfaces as part of the metaphase sheath. After the divisions, globuli are re-formed, and these eventually appear as the nucleoloids after detachment into the cytoplasm. It seems improbable that a gene amplification phase is associated with accessory nucleolus or nucleoloid formation. Evidence from a wide range of species suggests that the production of cytoplasmic nucleoloids during microsporogenesis is a general phenomenon among angiosperms, probably linked with the rapid build-up of ribosome numbers which follows upon the period of elimination in the meiotic prophase.

Stimulation of ribonucleic acid polymerase activity in vitro by prostatic steroid–protein receptor complexes

Abstract: A system has been developed which allows the stimulation in vitro of prostatic RNA polymerase by prostatic 5alpha-dihydrotestosterone-protein receptor complexes prepared from the tissues of castrated rats. The reconstitution in vitro of such a system necessitates the purification of several subcellular components. Two 5alpha-dihydrotestosterone-receptor complexes are located in the prostatic soluble supernatant fraction, separable by selective ammonium sulphate fractionation, and one complex can be isolated from the nuclear fraction. In the presence of all these complexes, stimulation of RNA polymerase in intact nuclei and nucleoli was observed. The complexes also increased the activity of the enzyme solubilized from whole nuclei. Greater stimulation of this system was noted in the presence of prostatic chromatin as template, as compared with that observed with calf thymus DNA or liver chromatin as template. The effects of the complexes on subnuclear forms of RNA polymerase, of nucleolar and extranucleolar origin, are also described. RNA polymerase solubilized from nucleoli is more susceptible to stimulation by the 5alpha-dihydrotestosterone-receptor complexes than is the ;nucleoplasmic' enzyme. Stimulation occurs less readily in the presence of Mn(2+) and at high ionic strength than in the presence of Mg(2+) and at low ionic strength. Preliminary experiments show that prostatic nucleolar RNA polymerase transcribes prostatic chromatin poorly as compared with the nucleoplasmic enzyme. The observations reported indicate an involvement of non-histone proteins associated with DNA in the process by which stimulation of enzyme activity by the 5alpha-dihydrotestosterone-receptor complexes is achieved. The implications of these findings in the mechanism of steroid hormone action is considered.

Characteristics of nuclear-ribosomal and DNA-like ribonucleic acids differentially extracted by hot-phenol fractionation.

Abstract: The purpose of the present study was to define more exactly the conditions of the method of hot-phenol fractionation of nuclear RNA (Georgiev and associates). The thermal RNA fractions obtained were characterized by agar-gel electrophoresis, determination of the specific radioactivity and base composition analysis, both of the total and newly synthesized RNA. It was found that the fraction extracted at 0–4 °C (considered as cytoplasmic RNA in the literature) contained some nuclear ribosomal precursor RNAs, expecially 32-S and 23-S RNAs. The composition of the different thermal RNA fractions strongly depended on the extraction conditions (time, pH, volume ratios etc.). Conditions were well-defined (15-min extraction at each temperature, pH 6.2) for obtaining, as separate fractions, the nuclear RNA species of ribosomal type (40-°C RNA, i.e. that obtained at 40 °C) and two classes of DNA-like RNA (63-°C and 85-°C RNA). The 55-°C RNA fraction was found to be of mixed character (rRNA + DNA-like RNA) regardless of the extraction conditions. The 85-°C RNA contained the “giant” RNA molecules, had the highest specific radioactivity and a (G + C)/(A + U) ratio of about 0.80. These findings are in agreement with the view that 85-°C RNA is the very first product of transcription. 63-°C RNA was also DNA-like but different from 85-°C RNA with respect to electrophoretic profile, specific radioactivity and base composition. A high content of adenylic acid (above 30%) was found in the region 8 to 18 S of 63-°C RNA. It is suggested that poly(A) segments are attached at the 18-S stage of the processing of 63-°C RNA as a precursor to the cytoplasmic mRNA. The results presented demonstrate that under well-defined conditions the method of the hot-phenol fractionation of Georgiev may be used as a reproducible procedure for obtaining different types of nuclear RNA without preliminary isolation of nuclei and nucleoli.

Two-dimensional gel electrophoresis of acid-soluble nucleolar proteins of Walker 256 carcinosarcoma, regenerating liver, and thioacetamide-treated liver.

Abstract: Summary The acid-extractable proteins of nucleoli of Walker 256 carcinosarcoma, regenerating rat liver, and thioacetamide-treated rat liver were separated into 91, 94, and 92 spots, respectively, by two-dimensional polyacrylamide gel electrophoresis. Although the protein patterns from these different nucleoli have remarkable similarities, some qualitative and quantitative differences were found among these patterns. Spots A6′ and B16′ were found only in Walker 256 nucleoli, whereas C11′ and C16′ were found in regenerating and thioacetamide-treated livers. Among these nucleoli, quantitative differences were found in spots A11, A24, B1, B2, B12, B34, C8, C11, and C17.

Erythropoiesis in the Newt, Triturus Cristatus Laur II. Characteristics of the Erythropoietic Process

Abstract: In splenectomized newts ( Triturus cristatus Laur.) rendered anaemic by acetylphenylhydrazine (APH), an erythropoietic response is delayed so that the animals are completely devoid of the erythron, including erythrocytes. At 11-14 days after APH, ‘erythroid precursor cells’ (EPC) in the blood signal the occurrence of an erythropoietic response. Ultrastructural studies have shown few or no ribosomes in EPCs, but well developed nucleoli and intense RNA synthesis are seen in these cells. Correlated morphological and cytochemical data indicate the production of ribosomes in EPCs, a process culminating in the formation of basophilic erythroblasts (BE). Microphotometric studies show the accumulation of haem during this interval. Thus, in EPCs and BEs, both ribosomal (rRNA) and messenger (m) RNA are synthesized, making possible the early synthesis of haemoglobin. In subsequent stages, nucleoli exhibit a size decrease, most evident in the particulate component, while all RNA synthesis ceases during the mid-poly-chromatophilic erythroblast (MPE) stage. Coupled with the gradual loss of ribosomes characteristic of this developmental period, the results suggest that rRNA synthesis occurs in EPCs, BEs, and in early polychromatophilic erythroblasts, where it is completed. Haemoglobin mRNA is also formed in these stages since ultrastructural and microphotometric data show the accumulation of haemoglobin. Beyond the MPE, haemoglobin production is dependent upon stable messenger RNA since no RNA synthesis is detected in this period. Ultrastructural studies demonstrate that autophagy may play an important role in the loss of cytoplasmic organelles characteristic of the erythropoietic process. The occurrence of swollen cristae and outer compartment in mitochondria coupled with the presence of myelin-like membrane whorls in association with mitochondria is seen in all erythroid cells except EPCs. Membrane profiles derived from endoplasmic reticulum are frequently encountered in the vicinity of mitochondria and appear to encircle these organelles and adjacent cytoplasm to form autophagic vacuoles. In the process, the limiting membranes assume a dense laminar appearance. Elements of the Golgi complex also display morphological alterations suggestive of degeneration. Cytoplasmic bodies of varying appearance and content, some of which are similar to lysosomes in other cell types, are seen in all erythroid cells. Ferritin is seen in some bodies, in vacuoles, or in aggregates, but does not appear in micropinocytotic vesicles.

Repopulation of postmitotic nucleoli by preformed RNA. II. Ultrastructure.

Abstract: The reconstruction of the nucleolus after mitosis was analyzed by electron microscopy in cultured mammalian (L929) cells in which nucleolar RNA synthesis was inhibited for a 3 h period either after or before mitosis. When synchronized mitotic cells were plated into a concentration of actinomycin D sufficient to block nucleolar RNA synthesis preferentially, nucleoli were formed at telophase as usual. 3 h after mitosis, these nucleoli had fibrillar and particulate components and possessed the segregated appearance characteristic of nucleoli of actinomycin D-treated cells. Cells in which actinomycin D was present for the last 3 h preceding mitosis did not form nucleoli by 3 h after mitosis though small fibrillar prenucleolar bodies were detectable at this time. These bodies subsequently grew in size and eventually acquired a particulate component. It took about a full cell cycle before nucleoli of these cells were completely normal in appearance. Thus, nucleolar RNA synthesis after mitosis is not necessary for organization of nucleoli after mitosis. However, inhibition of nucleolar RNA synthesis before mitosis renders the cell incapable of forming nucleoli immediately after mitosis. If cells are permitted to resume RNA synthesis after mitosis, they eventually regain nucleoli of normal morphology.