On the composition of the nucleolus with special reference to its filamentous structure

Summary Different staining procedures, various digestion methods and autoradiographic techniques were employed to study the structure and composition of the nucleolus and of the nucleolonema, after unmasking the latter by adenosine treatment. The presence of DNA, RNA, protein and lipid in these structures has been shown. It has been demonstrated that the filamentous structure within the nucleolus — the nucleolonema— has a core of DNA, around which RNA and protein have accumulated. The structure of the nucleolonema suggests that it is in a highly active state, in synthesizing ribosomal RNA and protein.We take the opportunity to express our gratefulness to the Director, Prof. Dr. Hans Lettré, for providing facilities to work in this Institute. We like to thank our other colleagues, particularly Dr. N. Paweletz, for their valuable help during the course of the investigations.     

Oogenesis in the malaria mosquito Anopheles gambiae

Summary Oogenesis has been followed with the electron microscope in 2 strains of the malaria mosquito Anopheles gambiae, from the emergence of the adult (oocytes at leptonema) till shortly before the oocytes are ready for oviposition. After pachynema the chromosomes form a karyosphere and a fibrous capsule develops around it. Work on other mosquitoes suggests that the capsule may be related to the synaptonemal complexes. Both Anopheles strains contain at some time an extrachromosomal (not DNA-containing) body comparable to the karyosphere in size. Clusters of granules are present at the surface of the nucleolus and free in the nucleoplasm. Tentative results indicate that they may contain DNA. During oogenesis the nucleolus becomes very large, mainly because of proliferation of the nucleolonema. Towards the end of oocyte development the nucleus assumes the large canoe-shape also seen in Aedes and Culex. Nucleolonema traverse the entire nucleus, and modified granular clusters are found throughout.     

Cytochemical and ultrastructural studies of ribonucleoprotein containing structures in oocytes of Acheta domesticus

Summary Two distinct types of ribonucleoprotein containing structures are found in oocytes of the house cricket, Acheta domesticus, a large secondary or accessory nucleolus and many small primary nucleoli. The secondary nucleolus increases in size during oocyte development and is similar in appearance to the nucleolus of somatic cells. The primary nucleoli are intimately associated with a large, extrachromosomal DNA containing body. The DNA body is no longer visible in nuclei of late diplotene stage cells when the primary nucleoli are dispersed within the nucleoplasm. Both types of nucleoli contain cytochemically detectable RNA and acid protein, little or no DNA and basic protein, and particulate structures similar to but smaller than cytoplasmic ribosomes.The authors acknowledge the technical assistance of Miss Celeste Malinoski and Mrs. Marcia Andrews. This work was supported by a U.S.P.H.S. grant, number GM-16440-01 and grants number L-16 and J-1 from the Health Research Services Foundation.     

Fine structure of the nucleolus in the young oocyte of a centipede

Summary The young oocyte of the centipede Scutigera forceps possesses numerous nucleoli which vary considerably in size. The electron microscope reveals them to be composed of small ribosome-like granules (probably RNP), relatively dense matrix, and, in some cases, a poorly defined nucleolonema.The appearance of lamellae at the surface of the nucleoli is of special interest. These consist of nucleolar material, are about 70 m in thickness, and of much greater width. They encircle the nucleolus, forming a relatively large concentric body, and in some instances probably extend from one nucleolus to another. In that case, the lamellae may represent a means of transfer from one nucleolus to another.This research was supported by grants from the National Institutes of Health (RG-4706, 5479) and the National Science Foundation (G-9879).     

Some observations on the mode of division of somatic nuclei of Mucor and Allomyces

Summary A series of successive photographs of the division of the living nucleus in a germinating sporangiospore of Mucor fragilis has been obtained. In this sequence the nucleus is seen to divide directly by elongation and constriction. The nucleolus divides at the same time and in the same way. These observations agree with the finding, first made by Léger (1896) and several times confirmed since then, that the nuclei of Mucorales apparently divide without first arranging their chromosomes in a metaphase plate and without the help of a spindle apparatus.In stained preparations of Mucor chromosomes are not normally visible as separate entities but they can be clearly seen in Feulgen preparations of dividing somatic nuclei of Allomyces arbuscula. In contrast to Mucor the nucleolus of Allomyces is dissolved during division. The chromosomes seem to sort themselves out on their own and form new nucleoli. Metaphase plates and spindles have not been encountered.To Professor Dr. E. G. Pringsheim, teacher and friend, on his 80th bithday.     

Ultrastructural localization of RNA in cryptomonads

Summary In a previous study, DNA was localized in cells of two cryptomonads,Pyrenomonas sp. andCryptomonas ovata, by use of immuno-gold technique. Of particular interest was the ultrastructural localization of DNA in the nucleomorph, supposed to be a vestigial nucleus of a former endosymbiont [Hansmann Pet al. (1986) Eur J Cell Biol 42: 152–160]. In the present paper, distribution of RNA in the same two organisms is reported. RNA was detected by the specific and very sensitive RNase-gold method. RNA could be demonstrated in all of the four plasmatic compartments of cryptomonad cells (cytoplasm, periplastidal compartment, mitochondrion, and plastid), although the amounts differed greatly in the respective compartments. In the nucleus, the condensed chromatin and the nucleolus were preferentially labeled. Intense labeling could also be found over the fibrillogranular region of the nucleomorph. This fact lends strong support to the supposition that the fibrillogranular body represents the structural and functional equivalent of a nucleolus and thus again supports the hypothesis that the nucleomorph represents a vestigial eukaryotic nucleus. InPyrenomonas sp., gold-particle density over the nucleolus and the fibrillogranular body was quantitatively evaluated in order to compare their respective RNA synthesizing activities. Labeling density over the nucleolus was found to be 2.7 times higher and thus, on account of its greater volume, the nucleolus may contain 17 times more RNA than the fibrillogranular body of the nucleomorph.Abbreviations BSA bovine serum albumin - ER endoplasmic reticulum - GA glutaraldehyde - SSC standard saline citrate - SSCB SSC containing BSA     

Multiplication of nucleolar fibrillar centres and absence of rDNA amplification in mouse ooctye during meiotic prophase I

During meiotic prophase I the nucleolus of the mouse oocyte assumes a reticulate structure of ‘nucleolonema’ type. This change coincides with the appearance of several secondary fibrillar centres. The number of these centres at diplotene (97–113), largely exceeds that of nucleolar organizers (4c DNA = 20 NORs). The quantitatative analysis of autoradiographs after hybridization in situ with -³H-uridine labelled rRNA, enabled us to demonstrate that the multiplication of the fibrillar centres in mouse oocyte nucleolus during meiotic prophase I is not the result of an amplification of the rDNA. The number of silver grains in pachytene and diplotene nuclei was twice that counted for somatic cell and oogonium nuclei (2c DNA).     

Analysis of nucleolus organizer regions (NORs) in mitotic and polytene chromosomes ofPhaseolus coccineus by silver staining and Giemsa C-banding

Chromosomes with active nucleolus organizer regions (NORs) were visualized in root tip metaphases ofPhaseolus coccineus using the silver staining technique. A mean number of 5.5 Ag-NORs per cell was observed in 54 cells from eight plants. In the endopolyploid nuclei of the suspensor the silver technique did not demonstrate the reported specificity for nucleolus organizer activity, because there was usually pale staining of nucleoli and preferential staining of heterochromatic regions in the polytene chromosomes including pericentromeric material, telomeres and NORs. The mean number of NORs per nucleolus as detected by this method was 5.8 (28 nucleoli analysed). Using a modified preparation technique, giant chromosomes stained pale, but nucleoli of suspensor cells displayed darkly silver staining internal domains, each of which originating from a nucleolus organizer.—Giemsa C-banding of endopolyploid suspensor nuclei revealed C-positive nucleolus organizers with darkly staining intranucleolar fibrils. The latter were frequently involved in inter-NOR associations. In 34 nucleoli analysed, the mean number of Giemsa C-positive NORs per nucleolus was 6.0.Dedicated to Professor Dr.Lothar Geitler on the occasion of his 80th birthday.     

Replication of autosomal heterochromatin in man

Summary In interphase nuclei of leukocytes and oral mucosa cells of normal human males and f males, two types of heterochromatin can he distinguished according to their location in the nucleus. Firstly, nucleolus-associated heterochromatin which consists of one large mass of autosomal segments surrounding the nucleolus, or several large masses if there appears to be more than one nucleolus in the same nucleus. Secondly, scattered heterochromatin composed of a large number of positively heteropycnotic bodies scattered throughout the nucleus and not directly associated with the nucleolus. The correspondence of this type of heterochromatin with chromosome segments is obtained at late prophase where several positively heteropycnotic regions belonging to the autosomes are found scattered throughout the nucleus.In human females sex-chromatin is present in addition to these two types. In leukocytes the sex-chromatin cannot be easily identified due to the large size and number of the scattered heterochromatic bodies, but in oral mucosa cells such a distinction is more easily achieved due to the smaller amount of autosomal heterochromatin.Nucleolus-associated and scattered heterochromatin from leukocytes of both sexes synthesized their DNA at a different period of time from the euchromatin. The asynchrony of replication observed in the heterochromatin at interphase is in agreement with the asynchrony between autosomes and within autosomes described by many authors at metaphase. This does not mean, however, that every segment or chromosome found replicating asynchronously at metaphase contains necessarily heterochromatin.Dedicated to Professor H. Bauer on the occasion of his 60th birthday. — This investigation was supported by a research grant to A. Lima-de-Faria from the Swedish Natural Science Research Council.     

Heterochromatin and histone phosphorylation

Histone phosphorylation and nuclear structure have been compared in cultured cell lines of two related species of deer mice, Peromyscus crinitus and Peromyscus eremicus, which differ greatly in their heterochromatin contents but which contain essentially the same euchromatin content. Flow microfluorometry measurements indicated that P. eremicus contained 36% more DNA than did P. crinitus, and C-band chromosome staining indicated that the extra DNA of P. eremicus existed as constitutive heterochromatin. Two striking differences in interphase nuclear structure were observed by electron microscopy. Peromyscus crinitus nuclei contained small clumps of heterochromatin and a loose, amorphous nucleolus, while P. eremicus nuclei contained large, dense clumps of heterochromatin and a densely structured, well defined, nucleolonema form of nucleolus. Incorporation of ³²PO₄ into histones indicated that the steady-state phosphorylation of H1 was identical in P. crinitus and P. eremicus cells. In contrast, the phosphorylation rate of H2a was 58% greater in the highly heterochromatic chromatin of P. eremicus cells than in the lesser heterochromatic chromatin of P. crinitus cells, suggesting an involvement of H2a phosphorylation in heterochromatin structure. It is suggested that the three histone phosphorylations related to cell growth (H1, H2a, and H3) may be associated with different levels of chromatin organization: H1 interphase phosphorylation with some submicroscopic (molecular) level of organization, H2a phosphorylation with a higher level of chromatin organization found in heterochromatin, and H3 and H1 superphosphorylation with the highest level of chromatin organization observed in condensed chromosomes.     

Histochemical study on localization of adenosine triphosphatase and 5-nucleotidase in the olfactory bulb of the rat

Summary In the present paper, a histochemical study of the localization of adenosine triphosphatase and 5-nucleotidase in fixed frozen sections of rat olfactory bulb has been carried out by Wachstein and Meisel's method. The structures showing positive reaction are the neurons and blood vessels. The neurons show a wide range of variation of reaction in both intensity as well as distribution. The nucleolus shows an intense and variable reaction and the significance of these have been discussed. The reaction in the cytoplasm is diffuse when it is mild but tends to concentrate toward the cell wall as the intensity increases. It is suggested that the nucleolus may play an important role in the synthesis of the enzymes which can take part in providing the energy needed for the molecular transport concerned with the coduction of nerve impulses.     

Different physiological functions of free D- and L-alanine in three body parts of the intertidal sipunculid Phascolosoma arcuatum

Free D- and L-alanine contents were comparable in the body wall and introvert cum retractor muscles of Phascolosoma arcuatum. In contrast, the content of free D-alanine in the internal organs was twice that of free L-alanine. Since alanine aminotrans-ferase from P. arcuatum was L-alanine specific, D-alanine appeared to be synthesized from L-alanine through the action of alanine racemase. Alanine racemase activity was higher in the D-alanine-forming direction in the three body parts of P. arcuatum. In addition, the ratio of DL/LD racemase activity in the internal organs was the lowest among the body parts studied. These results indicate that free D-alanine might be of lesser importance than the free D-isomer to the internal organs as compared to the body wall and introvert cum retractor muscles. Indeed, L-alanine inhibited pyruvate kinase from the body wall and introvert cum retractor muscles but had no effect on the pyruvate kinase from the internal organs. Furthermore, the activity of alanopine dehydrogenase present in the internal organs was significantly lower than those of the body wall and introvert cum retractor muscles. L-Alanine was an essential substrate for alanopine formation in the body wall and introvert cum retractor muscles during hypoxia since alanopine dehydrogenases from these body parts were L-alanine specific. When P. arcuatum was confronted with hypo-osmotic stress, the free D-alanine/total free alanine ratio in the internal organs increased approximately from 0.6 to 0.8 as the total free alanine content decreased. In comparison, those ratios in the body wall and introvert cum retractor muscles remained relatively constant. It was concluded that D- and D-alanine had different physiological functions in the three body parts of P. arcuatum.Abbreviations ADP adenosine-5-diphosphate - ADH alanopine dehydrogenase - ALT alanine aminotransferase - AOD amino acid oxidase - BW body wall - EDTA ethylenediaminetetra-acetic acid - EGT A ethylene glyco-bis (-aminoethyl ether) - N,N,N,N tetra-acetic acid - ICRM introvert cum retractor muscles - IO internal organs - I ₅₀ inhibitor concentration producing 50% inhibition of enzyme activity - -KG -ketoglutarate - LDH lactate dehydrogenase - NAD nicotinamide adenine dinucleotide - NADH nicotinamide adenine dinucleotide (reduced form) - PEP phosphoenolpy-ruvate - PEPCK phosphoenolpyruvate carboxykinase - PK pyruvate kinase - PMSF phenylmethylsulphonyl fluoride - SE standard error - SW sea water - TCA trichloroacetic acid     

Ultrastructural localization ofl -fucose residues in nuclei of root primordia of the green peaPisum sativum

Summary Sugars have been demonstrated in animal cell nuclei, but only a few studies have mentioned their presence in plant cell nuclei. In this studyl-fucose residues were localized at the ultrastructural level, usingUlex europeaus agglutinin I lectin, during the early stages of germination ofPisum sativum and in mature root tip cells. This sugar was present after 1 h of germination, and its concentration was found to vary during 3 to 6 h imbition; after 72 h of imbition its concentration had more than doubled. Furthermore, labelling was particularly abundant in the nucleolus, nucleolus-associated bodies and dense nuclear bodies. The possibility that some of thel-fucose residues are associated with proteins is discussed.     

Three-dimensional architecture of the higher plant nucleolonema disclosed on serial ultrathin sections

Summary— The three-dimensional architecture of the nucleolonema of Vicia faba has been studied by applying a silver impregnation technique to serial ultrathin sections. This technique disclosed lateral and transverse segments of the nucleolonema which were heavily impregnated with silver. The lateral profiles of the nucleolonema segments were classified into three main categories; a segment made up of one to several rod-like filaments (type I); a ladder-like segment consisting of two parallel and of transverse filaments (type II); and a last type constructed from two parallel filaments (type III). Tracing of the lateral segments through serial sections has indicated that type I first appears, then either type II or III and finally type I reappears at the corresponding sites on sections. Types II and III remained constant in width, about 1.0 μm, along their longitudinal axes whereas the width of type I was significantly smaller than that of the two former. The lateral filaments of both types II and III showed heterogeneity in width on account of the presence of knobs intermittently distributed along them. The thickness of these knobs was about 0.35 μm. Combining the observations on serial ultrathin sections and the morphometrical data it is very probable that the elementary structure of the nucleolonema is a 0.35-μm thick filament that tightly coils up into a solenoid structure with a thickness of approximately 1.0 μm. This model can explain the appearance of open- and closed-argyrophilic rings in serial sections since transverse segments of the solenoid are expected to show the argyrophilic rings. The elementary filament of the nucleolonema solenoid was sometimes loosened. Judging from our cytochemical data at the electron microscope level, some argyrophilic proteins appear to reside in the axial space of the solenoid but both DNA and RNA were not detectable in this space.     

Differences in the fine structure and chemical constitution of nucleolar bodies and the true nucleolus in Xenopus laevis embryos

Numerous bodies resembling nucleoli, named “prenucleolar bodies”, were seen in the interphase nucleus of Xenopus laevis embryos between stages 7 and 11 of Nieuwkoop and Faber (1956) but not at stage 12. These bodies are composed of thick strands, 200 A in diameter, and apparently differ from the fibrillar component of the true nucleolus which consists of thin fibrils, 50 A in diameter. The granular component of the true nucleolus consists of fibers and granules which are both also 150–200 A in diameter, but which differ in chemical nature from the prenucleolar bodies. The granular component and fibrillar component are readily digested by RNase with or without pretreatment with trypsin, while the prenucleolar body is only digested with RNase after pretreatment with trypsin. This suggests that the prenucleolar body consists of strands of RNA coated with protein. At stage 9, another type of nucleolus-like body is formed, which is larger (2–2.6 μ in diameter) than the prenucleolar body (0.2–1 μ) and consists of thin fibrils of 50 A. This body resembles the fibrillar component of the true nucleolus in the size of the elemental fibrils as well as in its susceptibility to actinomycin D, RNase and trypsin. It seems to be a precursor of the true nucleolus and for this reason was named the “primary nucleolus.” From stage 9 to stage 10, each nucleus in the presumptive ectodermal and mesodermal areas contains 2 primary nucleoli together with multiple prenucleolar bodies. At stage 12, the prenucleolar body is not seen at all, but a new type of nucleolus-like body appears. There are usually 2 of these bodies in each nucleus, and they consist of 2 components: a network of 50 A fibrils, and a group of strands, 150–200 A in diameter, containing some granule-like elements. The former has the same susceptibility to actinomycin D, RNase and/or trypsin as the fibrillar component of the definitive nucleolus and the primary nucleolus, while the latter has the same susceptibility as the granular component of the definitive nucleolus. Thus, this body may     

Structure of the nucleoli of developing microsporangiate strobili and root tips of scotch pine

Summary The characteristics of the nucleoli of the microsporangiate strobili and the root tips of Scotch pine (Pinus sylvestris L.) vary both during the course of the cellular cycle and, with regard to the pattern and stage of organ and tissue differentiation. Nucleologenesis takes place in interphase and the nucleoli last until prophase. Several types of nucleoli occur during the nucleolar cycle, the pattern and age of tissues determining which type or types dominate. In the strobilus primordia collected at the end of July and in August, the mitotic frequency is high. Nucleoli remain small throughout the nucleolar cycle, and at the electron microscopic level, they display intermingled fibrillar and fibrillogranular components. Strobilus primordia collected in September contain larger nucleoli in the sporogenous nuclei than in the nuclei of the tapetum or of the wall cells. Amongst the nucleoli with completely intermingled fibrous and granular material, nucleoli with nucleolonema or with vacuoles occur frequently. Small balls of fibrous material are seen on the nucleolar surface and in the nucleoplasm. In October, the mitotic frequency of strobilal cells is low. Nucleoli with completely intermingled fibrillar and granular components have vanished whereas a new, compact type of nucleolus with a dense fibrillogranular main portion and with nucleolonema, has developed. The nucleoli of the sporogenous cells have enlarged continuously whereas those of the wall cells are small. The nucleoli of the root tip cell resemble, to a certain extent, those of the strobilus primordia collected in September. In squashed preparations, the nucleoli of the strobilal cells bind the common nucleolar stains poorly whereas the nucleoli of the root cells can be stained with all the methods used. In certain cases, DNase treatment improves the stainability of the strobilal nucleoli. AgNO₃-staining is successful after acetic acid: alcohol fixation but not after formalin: hydrochinone fixation.     

The effects of oestrogens and other steroid hormones on the ultrastructure of the liver of Xenopus laevis Daudin

Summary Administration of oestradiol-17 to Xenopus laevis Daudin results in profound modifications of hepatic ultrastructure. In all liver cells there is a considerable proliferation of the granular endoplasmic reticulum and an increase in the electron density of the nucleolus. The amount of stored glycogen is depressed. Injection of pregnant mare serum gonadotrophin causes a similar response in intact females but not in males or ovariectomized females, suggesting an indirect effect of this hormone by stimulation of ovarian oestrogen secretion. Oestradiol benzoate, oestrone and oestriol are also able to effect the described response, though in the last case the changes are less complete. Testosterone propionate, progesterone, and cortisone acetate are inert in this respect.Supported by a grant from the Agricultural Research Council (A. G. 24/28) to Professor J. M. Dodd.     

Orchid embryology: Pollen tetrads of Epidendrum scutella in the anther and on the stigma

Summary The pollinium of Epidendrum scutella, both in the anther and on the stigma, was examined with the electron microscope. The sporoderm of the outer tetrads has a sexine and an intine while that of the inner tetrads lacks a sexine, and an intine is formed only after the pollinium is on the stigma. A fibrous wall layer apparently holds the tetrads together. The cytoplasm is filled with plastids, mitochondria, polysomes, vacuoles and vesicles of various sizes, and endoplasmic reticulum (ER) with narrow cisternae. The vegetative nucleus is oval in form and contains a large nucleolus. The generative nucleus is deeply lobed and contains a well-developed nucleolus. The generative cytoplasm lacks both plastids and mitochondria and has little ER. Dictyosomes are present as well as assorted vesicles. A pocket is present between the plasma membrane of the generative cell and the wall; it contains assorted membranes and ribosome-like particles. After the pollen is on the stigma the wall surrounding the generative cell begins to disappear and gaps develop in it.This investigation was carried out during the tenure of a research fellowship from the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.Research supported by grants from National Science Foundation (GB-3460) and the Miller Institute for Basis Science at the University of California, Berkeley. The authors would like to thank Miss Mary Ashton for her assistance in the research.