The ribosome cycle,nucleoli, and cytoplasmic nucleoloids in the meiocytes ofLilium

Summary During the meiotic prophase in pollen mother cells ofLilium species the ribosome population of the cytoplasm diminishes to a minimum reached during the diplotene-diakinesis interval. This fall is associated with a drop in the ribonuclease-extractable RNA present in whole, fixed cells. A rise in RNA and in number of observable ribosomes follows during the meiotic mitoses and in the very early life of the spores. Bodies with the characteristics of nucleoli—here termed nucleoloids—are released into the cytoplasm at anaphase I and anaphase II, and it is suggested that these may represent the main mechanism through which the ribosome population of the cytoplasm is restored after the prophase elimination.     

Nucleolar changes in human phytohaemagglutinin-stimulated lymphocytes

Summary The nucleoli of lymphocytes from circulating peripheral blood and from phytohaemagglutinin (PHA)-stimulated cultures (from 2 h–96 h) 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 stain 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.     

Unusual prophase structures and multiple nucleoli in male meiosis of Drosophila species of the virilis group

With silver nitrate (Ag-NOR) staining, unusual fibrillar structures, apparently coupled to the nucleolus, were found is several species of the D. virilis group. In D. littoralis, beaded strings appear in connection with these structures, whereas the late prophase is characterized by the appearance of multiple nucleoli in the nucleoplasm. In D. virilis, the nucleus has a prominent pointed protrusion in the region of the nucleolus and often a fibril protrudes from this point. Small nucleoli are budding from the nucleolus during prophase. The multiple nucleoli at late prophase are smaller and fewer. A nucleolar body with black spots appears at prometaphase and persists through metaphase and anaphase. In D. lummei, the nucleolus becomes surrounded by fibrils, which are released into the nucleoplasm and on which multiple nucleoli are synthesized.These phenomena are similar to the events described in oocyte meiosis of many animals, where rDNA amplification, coupled to the synthesis of multiple nucleoli in late prophase, has been established.     

Meiosis readiness in Lilium longiflorum “Croft”

It is proposed that anthers of Lilium longiflorum Croft approaching the end of premeiotic mitosis reach a state described as meiosis readiness after which cells in premeiotic prophase are unable to complete a mitotic division but despiralize to interphase and enter a meiotic division. Many of the laggard premeiotic cells begin despiralization before reaching an extremely contracted state of late prophase. Premeiotic despiralization is not, therefore, attributed to a deficiency in metaphase but to an inability of these cells to complete prophase. Premeiotic despiralization appears to be preceded by a slowing-down of prophase development. There is variation among anthers and anther regions in the onset of prophase retardation and meiosis readiness. It is suggested that meiosis readiness depends upon a gradual accumulation of meiosis-inducing substances in the cytoplasm of the premeiotic cells. It has not been determined whether the cells that undergo premeiotic despiralization give rise to the giant microsporocytes with shattered chromosomes observed at late prophase of meiosis.     

Microtubule organization in cultured soybean and black spruce cells: Interphase —mitosis transition and spindle morphology

Summary Microtubule (MT) distribution during the cell cycle, especially spindle organization, has been investigated using immunofluorescence light microscopy in cultured cells of two higher plant species, soybean (angiosperm) and black spruce (gymnosperm). In soybean, the prophase and metaphase spindles were different in morphology and structure. The prophase spindle covering the nucleus was barrel-shaped and MTs extended between poles. The metaphase spindle consisted mainly of short MT bundles on either side of the chromosome mass. During prometaphase, the polarity and shape of the prophase spindle disappeared, suggesting that the metaphase spindle is newly formed in prometaphase and not derived from the prophase spindle. A striking feature of MT organization in black spruce was sharply defined poles during prometaphase and anaphase. They were located close to the cell edge, suggesting that a structure in the cytoplasm or associated with the plasma membrane is responsible for their formation. In black spruce the metaphase spindle was long with pointed poles and MT fir tree structures. In contrast, the metaphase spindle of soybean was short with very broad poles and lacked MT fir trees. These results suggest that MT fir tree structure may not be necessary for a functional spindle.     

Gonoid thelytoky in soft scale insects (Coccidae: Homoptera)

Cytological analysis of the thelytokous soft-scale insects Coccus hesperidum L. (2n=14) and Saissetia coffeae (Walker) (2n=16) revealed that while in both species the chromosomes did not pair during prophase I, meiosis consisted of two divisions, the chromosome number was reduced, and diploidy was restored by the fusion of the female pronucleus with the polar nucleus II. The difficulty of trying to classify this type of thelytoky as either automictic or apomictic led to the proposal that a new criterion and new terms be used to classify thelytoky (and parthenogenesis). The new criterion is whether the number of chromosome elements present in the first (or only) metaphase of oogenesis is the same as that present in the oogonia (gonoid thelytoky) or different from it (agonoid thelytoky). The new criterion is superior to the existing criteria because it is unambiguous, and because it groups together forms with a similar tendency towards heterozygosity (or homozygosity). The possible evolution of the forms analyzed as well as the two other thelytokous forms of each species described by Thomsen (1927) are discussed. Another soft-scale insect, Physokermes hemicryphus Dalam, consisted of a diploid (2n=18) and a triploid (3n=27) form, in both of which the chromosomes also did not pair. Each of the three species contained a strain in which only a single nucleolus was present per cell. In C. hesperidum some strains with two nucleoli differed in the size of the nucleoli.     

A chromosome rearrangement in Neuvospora that produces viable progeny containing two nucleolus organizers

In rearrangement T(VLIVL)AR33 the segment of chromosome 2 bearing the nucleolus organizer is translocated to the end of chromosome 4. When AR33 is crossed by Normal sequence (N), one third of the viable progeny contain a stable nontandem duplication with two organizers per nucleus. The organizer-deficient complementary products are inviable. Chromosomes and nucleoli have been examined during meiosis and postmeiotic nuclear divisions in the ascus, comparing heterozygous AR33 × N crosses with N × N and with crosses heterozygous for other interchanges. When AR33 is heterozygous, asci are of three types having the nucleolus organizer duplicated in 0, 1 or 2 of the meiotic products. Frequencies of the ascus types are as expected from the known positions of rearrangement break points. Nucleoli formed by two organizers frequently fuse. Deficiency nuclei that contain no nucleolus organizer may form one or more small nucleolus-like bodies.     

γ-Tubulin and microtubule organization during microsporogenesis in Ginkgo biloba

This is the first report on -tubulin and microtubule arrays during microsporogenesis in a gymnosperm. Meiosis in Ginkgo biloba is polyplastidic, as is typical of the spermatophyte clade, and microtubule arrays are organized at various sites during meiosis and cytokinesis. In early prophase, a cluster of -tubulin globules occurs in the central cytoplasm adjacent to the off-center nucleus. These globules diminish in size and spread over the surface of the nucleus. A system of microtubules focused on the -tubulin forms a reticulate pattern in the cytoplasm. As the nucleus migrates to the center of the microsporocyte, -tubulin becomes concentrated at several sites adjacent to the nuclear envelope. Microtubules organized at these foci of -tubulin give rise to a multipolar prophase spindle. By metaphase I, the spindle has matured into a distinctly bipolar structure with pointed poles. In both first and second meiosis, -tubulin becomes distributed throughout the metaphase spindles, but becomes distinctly polar again in anaphase. In telophase I, -tubulin moves from polar regions to the proximal surface of chromosome groups/nuclei where interzonal microtubules are organized. No cell wall is deposited and the interzonal microtubules embrace a plate of organelles between the two nuclear cytoplasmic domains (NCDs) of the dyad. Following second meiosis, phragmoplasts that form between sister and non-sister nuclei fuse to form a complex six-sided structure that directs simultaneous cytokinesis. -Tubulin becomes associated with nuclei after both meiotic divisions and is especially conspicuous in the distal hemisphere of each young microspore where an unusual encircling system of cortical microtubules develops.     

Satellited chromosomes,systematics and phylogeny inTaraxacum (Asteraceae)

A survey is made of the occurrence, nature and frequency of satellited chromosomes in the agamospermous genusTaraxacum. Species belonging to the 10 sections thought to be most primitive in the genus lack satellited chromosomes. In most other sections, a characteristic satellited chromosome is seen with a large euchromatic region distal to the presumed nucleolar oraniser region (NOR). In sections of a precursor type, there is always one chromosome of this Taraxacum type per haploid genome. In sections thought to be of an advanced type the number of such satellited chromosomes is very unstable, sometimes even within the same tissue. In sectionHamata, two such satellited chromosomes are invariably found in triploids. This finding strongly supports the integrity of this section, suggests that the species of the section are monophyletic, and have evolved from a single ancestor subsequent to the occurrence of obligate agamospermy. In three sections of the genus, satellited chromosomes of the conventional type with a very small distal euchromatic region distal to the NOR are reported for the first time in the genus.     

Nucleolar morphology and rDNA in situ hybridisation in monocytes

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

Ultrastructure of the kinetochore inGraphosoma italicum (Hemiptera: Heteroptera)

Summary Ultrastructural analysis of the centromere in the germ-line ofGraphosoma italicum (Hemiptera: Heteroptera) (2n=12 + XY) revealed differences between mitotic and meiotic chromosomes. In mitotic spermatogonial divisions a trilaminar kinetochore plate extending almost the entire length of the chromosomes is present. Meiotic chromosomes, on the contrary, lack trilamellar kinetochore plate, although one chromosome end exhibits a round structure which is denser than the remainder chromatin and resembles a ball and cup kinetochore. These structures are orientated towards the poles and sometimes show associated microtubules. Additionally, the meiotic chromosomes are surrounded by a complex system of membranes. The possible role of the round structure and of the complex membrane system in meiotic segregation is discussed.     

Chromosome behavior in the primary nucleus ofAcetabularia calyculus as revealed by epifluorescent microscopy

Summary Chromosome behavior preceding secondary nuclei formation within a giant primary nucleus (50–100 m in diameter) inAcetabularia calyculus was observed by the fluorescence emitted from 4-6-diamidino-2-phenylindole (DAPI)-stained DNA.Throughout the period when the large nucleolus was present in the primary nucleus, thin chromonemata were observed twining around the nucleolus. Nuclear division was initiated by degeneration of the sausage-shaped nucleolus into a number of spherical subunits soon after the initiation of cap formation. On the fourth day of cap development, the chromonemata became thicker and chromomeres appeared. They accumulated adjacent to the single spherical nucleolus. The lump of chromosomes became loosened and thick chromosomes were scattered in the nucleus. The peculiar shapes of chromosomes which suggest the existence of chiasmata were frequently observed until the chromosome segregation started. This sequence of chromosome behavior seems to be the prophase of meiotic division. Chromosome segregation, the first meiotic division, occurred on the seventh day of cap development, probably being accompanied by the second meiotic division. Immediately after nuclear division of the primary nucleus, secondary nuclei were formed and cyst formation started 24 hours after repeated mitoses of the secondary nuclei.     

A chromosome rearrangement in Neurospora that produces segmental aneuploid progeny containing only part of the nucleolus organizer

In translocation T(ILVL)OY321 of Neurospora crassa a distal portion of the nucleolus organizer chromosome, including ribosomal DNA sequences and the nucleolus satellite, is interchanged with a long terminal segment of IL. When OY321 is crossed by Normal sequence, one-fourth of the meiotic products are segmental aneuploids that contain two copies of the long IL segment and that are deficient for the distal portion of the organizer. Each such product forms a nucleolus and is viable. The complementary aneuploid products are deficient for the IL segment and are therefore inviable. — In crosses of OY321xOY321, each product is capable of making two nucleoli; nucleoli formed by the separated nucleolus organizer parts usually fuse, but most 8-spored asci contain some nuclei in which two separate nucleoli can be seen. One nucleolus is then terminal on its chromosome while the second is interstitial and somewhat smaller. — In crosses of OY321 x Normal, half of the meiotic products are capable of making two nucleoli. However, only about 15% of 8-spored asci have one or more nuclei containing separate nucleoli. At pachytene and later in prophase I, the single fusion nucleolus is associated with three bivalent chromosome segments. Each nucleus of every ascus contains at least one nucleolus, even in asci where some nuclei display two nucleoli. — Crosses of Aneuploid x Normal are usually semibarren, producing a reduced number of ascospores, some of which are inviable. Some aneuploid cultures become fully fertile by reverting to a quasinormal sequence lacking a satellite. In some crosses of Aneuploid x Normal, individual asci may show at prophase I either complete loss, partial loss, or pycnosis of the translocated IL segment. This observation of pycnosis suggests chromosome inactivation. — Growth from aneuploid ascospores is initially slow, but can accelerate to the wild-type rate.     

Studies on the neurosecretory system of Iphita limbata Stal.

Summary The present paper gives an account of some experiments upon the insect Iphita limbata Stal. (Hemiptera: Pyrrhocoridae). The experiments were carried out in order to find out whether in the adult animal there is a relationship between the activity of the neurosecretory cells and the water balance. Under varying conditions one group of the neurosecretory cells of the pars intercerebralis of the brain, the so called A-cells, show histological differences. It has been seen that under conditions stimulating hydration there is a marked retention of the stainable colloids in the cytoplasm of A cells. This retention probably indicates a relationship between the secretions of A cells and the water balance of the insect.The author is indebted to Mr. N. R. Prabhoo and Miss Maya Menon of this Department for a critical discussion of the paper.     

Heterochromatin variation in Cryptobothrus chrysophorus

Northern and southern races of the Australian grasshopper Cryptobothrus chrysophorus share the same diploid chromosome number (2n=23, 24). The northern race is differentiated from the southern by fixed extra blocks of heterochromatin located distally on five of the six medium pairs of autosomes (M4, 5, 6, 8 and 9). The megameric M7 pair, which is completely heterochromatic in both races, is also frequently larger in the northern race. Additionally, while there is considerable polymorphism for the presence of supernumerary heterochromatic segments on the two smallest autosome pairs (S10, 11) in both races, the precise character of this polymorphism is strikingly different between them. That found in the north is both more extensive and more variable. An analysis of the patterns of C-banding obtained in neuroblast c-mitoses indicates even more variation within and between races than was anticipated from the patterns of heteropycnosis seen at first prophase of male meiosis. Thus, while the distal blocks on the M4, 6, 8 and 9 elements in the northern race invariably C-band those of the M5 never do. On the other hand polymorphisms for C-bands on the M5, 6, 8 and 9 are seen in some populations of the southern race but in regions which are not visibly heteropycnotic at meiosis. Polymorphisms for the pattern of C-banding also occur in the northern race populations in the M4, 6, 8 and 9 elements and some of these are associated with clear length differences in the chromosomes concerned. Others involve differences in the expression of the distal C-bands in M8 and 9 which vary from dark to intermediate. The supernumerary segments on the S10 and 11 pairs are especially variable in respect of their C-banding properties. Some are entirely C-banded, some show no C-banding whatsoever and some are composed of both banded and unhanded regions. Banding is again most pronounced, however, in the northern race. Finally the character of the megameric M7 is strikingly different in the two races not only in respect of its size, which is sometimes larger than that of the south, but also in respect of the extent of C-banding which is always more complex in the northern form irrespective of its size.     

Ultrastructural studies of late meiotic prophase nuclei of spermatocytes in Ascaris suum

Post pachytene stages of meiotic prophase in males of Ascaris suum have been analyzed with the electron microscope. No synaptonemal-like polycomplexes (PCs) have been observed in the nucleoplasm or cytoplasm during the period from pachytene to diakinesis. From Serially sectioned diplotene nuclei it was found that the bivalents are located near the periphery of the nuclei, the central part of the nuclei being vacant. Each nucleus contains one nucleolus. Up to 1 m long stretches of unpaired lateral elements (LEs) are found in some of the diplotene bivalents. These LEs are morphologically similar to unpaired LEs in early zygotene nuclei. Partial 3-dimensional reconstruction of two nuclei shows that the bivalents contain some small stretches of synaptonemal complex (SC) up to 1.9 m long. Some bivalents at diakinesis show remnants of SCs. At this stage chromosomes are fibrous, condensed, attached to the nuclear envelope and mostly with a rounded profile in cross section. The synchronous development of the spermatocytes and small bivalents at diplotene in A. suum make this system a good object for the study of localization of SC remnants.     

Chromosome banding in Amphibia

The karyotype of the marsupial frog Gastrotheca riobambae is characterized by exceptionally highly differentiated XY/XX sex chromosomes. The 18S and 28S ribosomal RNA genes were found only in the nucleolus organizer region (NOR) of the X chromosome by in situ hybridization, silver staining and mithramycin banding. This amphibian species therefore exhibits a sex-specific difference in the number of ribosomal RNA genes of about 2()1(). This constitutes an extremely rare situation in the karyotype of vertebrates. Examination of various somatic tissues from female animals showed that the NORs on both X chromosomes are always active. The results are discussed in relation to the apparent absence of dosage compensation for sexlinked genes in the Amphibia.     

The nucleolus organizer region of maize (Zea mays L.): Chromosomal site of DNA complementary to ribosomal RNA

A maize genetic marker strain (designated as the 2NOR strain) was found to carry a cytologically-visible duplication of the nucleolar organizer region (NOR). The 2 NOR condition is a simply inherited cytological marker which is transmitted normally through mega- and microsporogenesis and is associated when homozygous with a nucleolus approximately 64% greater in volume than that of two representative normal inbred lines of maize.—Utilizing DNA · rRNA hybridization techniques and the 2 NOR strain plus two unrelated inbred lines with normal NOR's, the chromosomal site of DNA complementary to rRNA was shown to be localized in the NOR. To our knowledge, this is the first report on the localization of rDNA cistrons in the NOR of plants. An estimate was made of 17,000 rDNA cistrons per diploid nucleus for normal maize.—The 2 NOR strain was shown to possess approximately twice as many rDNA cistrons (34,000) as normal maize. The usefulness of this 2 NOR condition for plant protein improvement programs is being investigated.     

Ring shaped nucleoli in liver cells of rats after treatment with Actinomycin D

Summary The incidence of ring shaped nucleoli was studied by means of light and electron microscopical methods in liver cells of rats injected intravenously with Actinomycin D in a dose of 150 g per kg body weight.Ring shaped nucleoli were most frequent 20 minutes after administration of Actinomycin D. The number of fragmented nucleoli was increased 20 minutes after administration of the drug and did not decrease even after 72 hours.Fibrillar ring like inclusions within nuclei and more perichromatin granules at the periphery of nucleoli were observed after treatment with Actinomycin D.

---

Zusammenfassung Die Wirkung von Aktinomycin D (150 g/kg) auf die Leberzellen der Ratte wurde licht- und elektronenenmikroskopisch untersucht. 20 min nach intravenöser Aktinomycin-D-Gabe wurden ringörmige Nukleolen beobachtet. Die Zahl der fragmentierten Nukleolen war 20 min nach der Applikation gesteigert. Ihre relative Zahl änderte sich nach Aktinomycingabe nicht. Nach Behandlung der Tiere mit Aktinomycin D kann man in den Kernen der Leberzellen fibrilläre ringförmige Einschlüsse und eine größere Menge von perichromatin-granules an der Peripherie der Nukleolen beobachten.

---

---

The authors are indebted to M. Mikoláová, V. Povolná, I. erníková and Dr. M. Titlbach for their help, to Dr. J. Chaloupka for providing Actinomycin D and to Professor Busch and Professor Wolf for many helpful suggestions.     

Ribonucleic acid transfer from nucleus to cytoplasm during interphase and mitosis in mouse somatic cells cultured in vitro

Summary Kidney cells from primary cultures of 15-day old mouse embryos were incubated for 2, 5 or 10 min with H³-uridine, then either fixed immediately or incubated again for various periods in a chase medium containing an excess of unlabeled uridine and cytidine. The number of grains over the non-nucleolar part of the nucleus (chromatin), the nucleolus and the cytoplasm were counted on the autoradiograms.The grain count showed that both chromatin and nucleolus incorporate very rapidly H³-uridine from the medium, whereas a time lag elapses before any H³-radioactivity above background is detected in the cytoplasm. Incorporation of H³-uridine into the RNA of the nucleus and the nucleolus is not immediately blocked after chase, suggesting that the labeled precursor pool is not completely washed out from the living cell, or diluted by the excess of unlabeled uridine present in the medium. The grain count over the nucleus and the nucleolus rises for a certain time after chase and then gradually declines; H³-radioactivity appears in the cytoplasm 10 min after chase and keeps rising through a 110-min interval. The experiment, then — even though it suggests that the bulk of cellular RNA is synthesized in the chromatin and the nucleolus and then continuously released into the cytoplasm — does not rule out the possibility that some RNA fraction, characterized by a low turnover rate, is synthesized independently in the cytoplasm.Synthesis of RNA is a continuous process throughout the cell cycle, except during metaphase and anaphase. It ceases at prometaphase after the disappearance of the nucleolus and disintegration of the nuclear membrane, and resumes in early telophase. Part of the chromosomal RNA does not remain associated with the chromosomes through division, but is suddenly released into the cytoplasm when the cell enters metaphase.