RNA TRANSPORT FROM NUCLEUS TO CYTOPLASM IN CHIRONOMUS SALIVARY GLANDS

The fine structure and cytochemistry of the extremely large RNA puffs, or Balbiani rings, in salivary gland nuclei of midge, Chironomus thummi, larvae have been investigated. The Balbiani rings are composed of a diffuse mass of electron-opaque 400 to 500 A granules, short threads about 180 to 220 A in diameter and associated fine chromatin fibrils. These components appear to be organized into brushlike elements which form the ring. Electron microscope cytochemistry has shown that the granules and short threads contain RNA. After ribonuclease digestion, only 50 to 100 A chromatin fibrils were apparent in the Balbiani ring, and the granules were no longer demonstrable. Deoxyribonuclease digestion had no apparent effect on these structures. Observations indicate that the granules are formed from the short threads and released into the nucleoplasm in which they are evenly distributed. At the nuclear envelope, many granules have been observed partially or completely within the nuclear pores. These granules become elongated and are shown to penetrate the center of the pore in a rodlike form, about 200 A in diameter. The Balbiani ring granules are not normally visible within the cytoplasm adjacent to the nuclear envelope, but have been rarely found in this region. It is suggested that the granules represent the product of the Balbiani ring, possibly a messenger RNA bound to protein, and that they regularly pass into the cytoplasm through a narrow central channel in the pores of the nuclear envelope.     

Heat-shock induced puffing changes in Balbiani rings

A 5 minutes exposure of Chironomus larvae to near-lethal temperatures (39–40° C) produces a characteristic sequence of puffing changes in the Balbiani rings of the 4th salivary gland chromosomes: An initial phase of complete puff regression is followed, after a variable time lag, by a phase of rapid recovery to overnormal puff sizes. This is accompanied by RNP droplet formation. RNA synthesis at Balbiani rings during the initial puff regression still occurs. Regression is inhibited by 2,4-dinitrophenol, while recovery can be prevented by both 2.4dinitrophenol and actinomycin D. Regression and recovery are insensitive to cycloheximide. RNP droplets, as observed in Balbiani rings, in the nuclear sap and at the nucleolar rim, are composed of a fine fibrillar matrix which is covered by Balbiani ring granules in various phases of assembly. The results are discussed in terms of a model of puffing based on an equilibrium between RNA synthesis, RNA processing and RNP release from the puff.

     

Heat-shock induced puffing changes in Balbiani rings

A 5 minutes exposure of Chironomus larvae to near-lethal temperatures (39–40° C) produces a characteristic sequence of puffing changes in the Balbiani rings of the 4th salivary gland chromosomes: An initial phase of complete puff regression is followed, after a variable time lag, by a phase of rapid recovery to overnormal puff sizes. This is accompanied by RNP droplet formation. RNA synthesis at Balbiani rings during the initial puff regression still occurs. Regression is inhibited by 2,4-dinitrophenol, while recovery can be prevented by both 2.4dinitrophenol and actinomycin D. Regression and recovery are insensitive to cycloheximide. RNP droplets, as observed in Balbiani rings, in the nuclear sap and at the nucleolar rim, are composed of a fine fibrillar matrix which is covered by Balbiani ring granules in various phases of assembly. The results are discussed in terms of a model of puffing based on an equilibrium between RNA synthesis, RNA processing and RNP release from the puff.     

Phosphorus distribution in perichromatin granules and surrounding nucleoplasm as visualized by electron spectroscopic imaging

Summary— The in situ distribution of phosphorus in perichromatin granules (PCGs), and in the surrounding nucleoplasm was investigated in rat liver cells by means of electron spectroscopic imaging of unstained preparations. A 2–3 nm fibril containing high concentration of phosphorus was found to be the main substructural feature of the PCGs revealed in the maps of phosphorus. This fibril is folded within the PCG with no apparent order. Fibrils of similar diameter and phosphorus content were also found in both the halo surrounding the PCG and dispersed in the nucleoplasm. Some of such fibrils are in continuity with those occurring within PCGs. Sometimes these fibrils are grouped forming a stalk connecting the PCG to chromatin. Some stalked PCGs are U-shaped or kidneyshaped, resembling Balbiani ring granules in the process of formation as observed in Chironomus salivary gland cell nuclei. The external fibrils are interpreted as perichromatin fibrils considered to be precursors of PCGs.     

Nup153 affects entry of messenger and ribosomal ribonucleoproteins into the nuclear basket during export

A specific messenger ribonucleoprotein (RNP) particle, Balbiani ring (BR) granules in the dipteran Chironomus tentans, can be visualized during passage through the nuclear pore complex (NPC). We have now examined the transport through the nuclear basket preceding the actual translocation through the NPC. The basket consists of eight fibrils anchored to the NPC core by nucleoprotein Nup153. On nuclear injection of anti-Nup153, the transport of BR granules is blocked. Many granules are retained on top of the nuclear basket, whereas no granules are seen in transit through NPC. Interestingly, the effect of Nup153 seems distant from the antibody-binding site at the base of the basket. We conclude that the entry into the basket is a two-step process: an mRMP first binds to the tip of the basket fibrils and only then is it transferred into the basket by a Nup153-dependent process. It is indicated that ribosomal subunits follow a similar pathway.     

Observation of the nuclear behavior in Streptomyces cinnamonensis

The nuclear region in the hyphae of Streptomyces cinnamonensis consists of two phases: a dense fibrillar phase and a less dense phase in which the fibrillar phase is embedded. The fibrillar phase occurs in two states of aggregation, a dispersed condition in which a network of fine fibrils is apparent and a more organized condition in which the fibrils appear thicker and more compact and exhibit a variety of configurations. The fibrils may be dispersed throughout the nuclear region, either in a whorled pattern or in a more or less straightened manner aligned along the nuclear axis. They may aggregate toward the center of the nuclear region as a dense mass which may be separated into two daughter groups of fibrils. Often the nuclear region between the two daughter groups of fibrils is subdivided by cytoplasmic invagination. Conversely, the two daughter groups of fibrils divide a second time resulting in four masses of fibrils. Thus several nuclear regions may be located in a common cytoplasmic field. The significance of these findings is discussed.     

ELECTRON MICROSCOPY OF THE VOLVOCACEAE AND ASTREPHOMENACEAE

Lang, Norma J. (U. Texas, Austin.) Electron microscopy of the Volvocaceae and Astrephomenaceae. Amer. Jour. Bot. 50(3): 280-300. Illus. 1963.—Clonal cultures of Gonium sociale, G. pectorale, Pandorina morum, Eudorina elegans, Eudorina sp., Volvulina steinii, V. pringsheimii, Platydorina caudata, Pleodorina illinoisensis, P. californica, Volvox aureus, V. tertius, V. globator, V. barberi, and Astrephomene gubernaculifera representing the Volvocaceae and Astrephomenaceae in the Volvocales were examined with the electron microscope and their ultrastructure compared. The ultrastructure of the various organelles is basically similar in the species studied and no increase in cellular complexity is found to accompany the evolutionary trends evidenced in the Volvocaceae. The ultrastructure of a colonial cell is basically that of Chlamydotnonas. A cytoplasmic membrane having a unit membrane structure encompasses a cell and is continuous with the double-membraned flagellar sheaths. The flagella contain the typical 9 + 2 fibril arrangement with the 2 axial fibrils terminating in a cylinder at the flagellar base and the 9 peripheral pairs continuing into the cytoplasm as a basal body. The organelles comprising the cytoplasm are: mitochondria with plate-like cristae; dictyosomes composed of stacks of agranular cisternae; small, rough or smooth-surfaced vesicles; an endoplasmic reticulum of granule-bearing and agranular tubules, lamellae and broad cisternae; vacuoles which are either contractile, contain fine granular and fibrillar material, or have dense contents probably representing polyphosphate; lipid bodies; and dense granules 100–150 A which have been called ribosomes. The finely granular nucleoplasm is surrounded by a porous, double-membraned nuclear envelope and contains a centric nucleolus composed of dense, spherical granules. The outer membrane of the nuclear envelope bears granules and may have granular extensions into the perinuclear cytoplasm. Each extension appears to encompass one or several dictyosomes and has been termed an “amplexus.” The amplexi are agranular on the surface contiguous to a dictyosome. A double-membraned chloroplast envelope is continuous around the single, cup-shaped chloroplast. The basic chloroplast units are discs closed at each end, occurring in stacks of varying number parallel to the envelope. The presumed proteinaceous matrix of the basal pyrenoid is penetrated by elongated, tubular elements which connect with the lamellar discs. Multiple rows of granules, associated with individual discs, form the anterior stigma within the chloroplast envelope. The colonial matrix is not a structureless, mucilaginous material uniting the cells in colonies, but it has rather a highly complex structure especially around the periphery of the colony and the flagellar channels. The apparent substitution of a fibrillar layer of the colonial matrix for the discrete compact cell wall, such as is found in Chlamydomonas, implies a greater degree of complexity in the evolution of these colonial genera than is generally assumed.     

Fine structural organization of a non-reticulate plant cell nucleus

We studied the fine structural organization of the meristematic nucleus in roots of Lycopesicon esculentum (tomato) using ultracytochemical and immunocytochemical approaches. The nucleus has a non-reticulate (i.e. low DNA content) structure whose supramolecular organization differs in some respects from that in reticulate nuclei, principally in the organization of the chromocentres associated with the nuclear envelope, with which centromeric structures appear to be associated. The main difference at the nucleolar level is found in the fibrillar centres, which have a low amount of DNA labelling and in which inclusions of condensed chromatin are present only very rarely. The distribution of nucleolar DNA amongst the nucleolar compartments is similar to that in reticulate nucleoli as demonstrated using an anti-DNA monoclonal antibody. Tomato nuclei have nucleolus-associated bodies or karyosomes, like other plant species with a low DNA content and non-reticulate nuclear organization. The nuclear ribonucleoprotein structures in the inter- and perichromatin regions, namely inter- and perichromatin fibrils and granules, show similar ultrastructural and cytochemical characteristics in both types of nuclei.Abbreviations NAC nucleolus associated chromatin - CES centromeric structures - NOR nucleolar organizing region - NAB nucleolus associated body - IG interchromatin granules - RNP ribonucleoprotein - Mab monoclonal antibody by M.F. Trendelenburg     

Fine structure of the macronucleus during the cell division cycle of Euplotes eurystomus,a Ciliate Protozoon

This paper reports new observations obtained from a study of macronuclear fine structure throughout various stages of the cell division cycle of Euplotes. Study of the ultrastructural organization of the macronuclear chromatin indicates that much of the chromatin is organized into continuous masses, portions of which appear to be attached to the nuclear envelope. The macronuclear envelope appears unchanged in the region of a replication band, and apparent attachments of the chromatin to the inner membrane of the nuclear envelope are maintained in the reticular and diffuse zones. Intranuclear helices were never observed in the diffuse zone. During macronuclear division, linear elements (fibrils or microtubules) were observed in close association with both chromatin bodies and nucleoli. The ultrastructural data suggest that the intranuclear linear fibrils have two functions: elongation of the dividing nucleus, and attachment of chromatin bodies and nucleoli to the envelope. The significance of these observations for macronuclear division and chromatin segregation is considered.     

Fine Structure of the Female Intoshia variabili(Alexandrov & Sljusarev) (Mesozoa: Orthonectida)

The morphology and fine structure of female Intoshia variabili, new combination for Rhopalura variabiliAlexandrov & Sljusarev, 1992, were studied with transmission electron microscopy. The body surface is covered with a 3-layered cuticula, under which is a layer of ciliated + non-ciliated cells arranged in alternating rings around the body. Ciliated cells have lateral extensions that intercalate with the non-ciliated cells. The kinetosome of each cilium has two longitudinally oriented cross-striated rootlets. The outer surface of the ciliated cells is covered with small tubercles, and the cytoplasm of these cells contains granules, vacuoles, mitochondria, fibrillar structures and lamellary bodies. A band of dense fibrils passes through the upper part of each ring of cells, going from one cell junction to another, encircling the entire body. Between the layer of ciliated + non–ciliated cells and the oocytes, elongated contractile cells from 4–5 longitudinal columns and 1 ring, the latter at the level of ciliated rings 7–9. The contractile cells contain thick and thin longitudinally oriented fibrils. The oocytes contain a large nucleus, numerous mitochondria, electron–dense granules and 1–2 spherical structures. An anteriorly situated, ciliated goblet–like receptor, not described for any other orthonectids, consists of three closely apposed cells, the upper part of which contains densely packed cilia. The genital pore opens through a non–ciliated cell and is surrounded by several cells with granules.     

Delayed flow-through cytoplasm of newly synthesized Balbiani ring 75S RNA.

With a nonaqeous microdissection technique, the cytoplasm of Chironomus salivary gland cells can be separated into concentric zones situated at increasing distances from the nuclear envelope. This dissection technique is used here to investigate the cytoplasmic distribution of 75S RNA of Balbiani ring origin. The Balbiani ring 75S RNA has properties of a messenger RNA coding for secretory proteins. After a pulse of RNA precursor to the living animal, labeled Balbiani ring 75S RNA is found mainly in the cytoplasm located closer to the nuclear envelope, with smaller amounts toward the periphery of the cell. This gradient, initially very steep, lasts for a least 2 days, but less than 6 days. Experiments with 5-fluorouridine indicate that the formation of the gradient does not depend upon simultaneous export of ribosomal subunits. After a pretreatment of the animals with the protein synthesis inhibitor cycloheximide, however, newly synthesized 75S RNA distributes evenly in the cytoplasm-that is, this treatment prevents the formation of the 75S RNA gradient. The gradient in salivary glands of normally cultured animals is therefore likely to be the result of diffusion restriction of the labeled 75S RNA. Thus the 75S RNA located closer to the nuclear envelope is the most recently exported 75S RNA. An explanation of these results is the the 75S RNA associates with the membranes of the endoplasmic reticulum early or immediately after nuclear release. This association should occur in the cytoplasm surrounding the nucleus and may occur either as single particles and/or as parts of polysomes.     

Osmium tetroxide/p-phenylenediamine staining of nucleoli and balbiani rings in Chironomus salivary glands

Summary This paper deals with the application of the osmium tetroxide fixation followed by p-phenylenediamine treatment to salivary gland cells from Chironomus larvae. After this procedure, cytoplasm, nucleoli and Balbiani rings show a high degree of staining both in light and electron microscopy, while chromatin remains unstained. Ethanol fixation followed by osmium tetroxide/p-phenylenediamine does not modify the above mentioned staining pattern. Under these conditions, extractive procedures for lipids do not affect the osmiophilia of nucleoli and Balbiani rings, while RNase or trichloroacetic acid treatment decreaes the staining degree of these structures. In osmium tetroxide/p-phenylenediamine treated salivary glands, the highest contrast within nuclei is seen to occur in the pars granulosa from normal or segregated nucleoli, as well as in Balbiani ring granules, which appear either as hollow granules or with a bipartite or horseshoe-like structure.     

Osmium ammine-B and electron spectroscopic imaging of ribonucleoproteins: Correlation of stain and phosphorus

Summary— Ultra-thin sections of Chironomus salivary glands were stained in a non-Feulgen procedure with osmium ammine-B and imaged at several electron energy-loss windows. For two types of RNP-containing structures (ie Balbiani ring granules and endoplasmic reticulum), a significant spatial correlation was observed between stain distribution and net phosphorus distribution. Non-Feulgen osmium ammine-B staining does not require the use of ultra-thin sections and can approximate the distribution of nucleic acid phosphorus.     

Previtellogenic oocytes of South African largemouth bass Micropterus salmoides Lacépède 1802 (Actinopterygii,Perciformes) - the Balbiani body,cortical alveoli and developing eggshell

The ovaries of the largemouth bass Micropterus salmoides, an alien and invasive species in South Africa, contain a germinal epithelium which consists of germline and somatic cells, as well as previtellogenic and late vitellogenic ovarian follicles. The ovarian follicle consists of an oocyte surrounded by follicular cells and a basal lamina; thecal cells adjacent to this lamina are covered by an extracellular matrix. In this article, we describe the Balbiani body and the polarization and ultrastructure of the cytoplasm (ooplasm) in previtellogenic oocytes. The nucleoplasm in all examined oocytes contains lampbrush chromosomes, nuclear bodies and several nucleoli near the nuclear envelope. The ultrastructure of the nucleoli is described. Numerous nuage aggregations are present in the perinuclear cytoplasm in germline cells as well as in the ooplasm. Possible roles of these aggregations are discussed. The ooplasm contains the Balbiani body, which defines the future vegetal region in early previtellogenic oocytes. It is comprised of nuage aggregations, rough endoplasmic reticulum, Golgi apparatus, mitochondria, complexes of mitochondria with nuage-like material, and lysosome-like organelles. In mid-previtellogenic oocytes, the Balbiani body surrounds the nucleus and later disperses in the ooplasm. The lysosome-like organelles fuse and transform into vesicles containing material which is highly electron dense. As a result of the fusion of the vesicles of Golgi and rough endoplasmic reticulum, the cortical alveoli arise and distribute uniformly throughout the ooplasm of late previtellogenic oocytes. During this stage, the deposition of the eggshell (zona radiata) begins. The eggshell is penetrated by canals containing microvilli and consists of the following: the internal and the external egg envelope. In the external envelope three sublayers can be distinguished.     

Demonstration of a 7-nm RNP fiber as the basic structural element in a premessenger RNP particle

Balbiani ring granules in Chironomus salivary glands represent premessenger ribonucleoprotein (RNP) particles, each containing a 35- to 40-kb message for a secretory polypeptide. Their gross structure can be described as an RNP ribbon bent into a toroid. We now demonstrate that an unfolded, thin RNP fiber is observed after low salt treatment of isolated Balbiani ring granules. Moreover, the thin RNP fiber, 7 nm in diameter, can be revealed as the main structural element in Balbiani ring granules studied in situ in 3-D with electron microscope tomography. It is proposed that the thin RNP fiber consists of a premessenger RNA molecule coiled around a filamentous core of polymeric proteins, which has functional implications for processes such as assembly of RNP, intranuclear degradation of RNA, and delivery of RNA through the nuclear pores.     

Fine structural localization of peroxidase in the rat thyroid

Summary The fine structural localization of a peroxidase activity in the rat thyroid follicular epithelial cell was studied by histochemistry at electron microscopic level. The reaction product is recognized chiefly in the cisternae of the elements of granular endoplasmic reticulum and of nuclear envelope. Golgi vesicles or apical small vesicles, mitochondria, and dense granules are sometimes positive for this reaction. The relationship between the fine structural localization of peroxidase and the site of the iodination of thyroglobulin is discussed.     

Observations on the postacrosomal region and the neck membranes of rabbit spermatozoa stained en bloc with uranyl acetate

Summary The postacrosomal region (PAC) of the head and the neck membranes of rabbit spermatozoa have been reinvestigated with the use of en bloc uranyl acetate staining. The fine structure of the PAC and neck membranes is visualized with great clarity and departs from that seen in rabbit sperm prepared by other methods. In cells so treated, the PAC contains a heavily enfolded nuclear envelope, a dense lamina attached to the plasmalemma by periodic connecting links and scattered dense material between the lamina and nuclear envelope. Evidence is presented that the dense lamina is a discrete structure, separated from the plasmalemma by the connecting links. The latter may be of a different composition from both the lamina and the plasmalemma. The lamina is a homogenous structure which resists degeneration under conditions which affect other components of the PAC. The membranes of the neck are a complex labyrinth of nuclear envelope, individual membranes, and membranes coursing through a matrical gound substance.This investigation was supported by Ford Foundation Grant 67–650.     

Electron Microscopy of Neurospora crassa Mycelia

The fine structure of vegetative mycelia of the filamentous Ascomycete, Neurospora crassa, has been investigated by the standard techniques of electron microscopy. Addition of uranyl nitrate to the methacrylate-embedding medium minimized disruption of the specimens—an accident often observed in the preparation of microbial material. This report describes the presence of a chitinous polysaccharide wall containing fine fibrils embedded in a homogeneous matrix. A sinuate plasma membrane lies adjacent to the inner wall surface. This membrane is often closely associated with the endoplasmic reticulum of the cytoplasm. Numerous mitochondria of the classical type, and dense particles of 10 mµ diameter occur throughout the cytoplasm. The nuclear region is surrounded by a double membrane with pore openings. Associated with the nuclear envelope is a dense area, the nucleolus. The significance of these observations and their relationship to other forms is discussed.