Specific Staining of Nucleolar Substance with Aceto-Carmine

A method is described for staining nucleoli intensely by treating tissues with formaldehyde, hydrolysing in normal HC1 at 60°C. and staining with aceto-carmine. With correct hydrolysis time, chromosomes and cytoplasm are almost colorless.

Formaldehyde increases the acidity of cell parts, especially the nucleolus, presumably by neutralizing the basic protein groups, and increases the resistance to hydrolysis, perhaps by protecting the phospholipoprotein complexes which are most abundant in the nucleolus.

Hydrolysis reduces the acidity of cell parts, chiefly by removal of nucleic acids.

Aceto-carmine stains cell structures which are weakly acid in character (about pH 4-5) probably by precipitating as large dye aggregates.

The technic appears to be highly specific for nucleoli and related cell bodies.     

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.     

On the nucleoli of the dinoflagellate Prorocentrum

To date no nucleolus had been observed in Prorocentrum under the light microscope. The author failed to show the nucleoli of P. micans and P. cassubica with eosin in 70% alc or with methyl green-pyronin. But when these dinoflagellates were treated with an Ag-1 technique which had been improved for demonstrating NORs in unicellular organisms, nucleoli were stained dark brown or black, while all other parts showed no colour. When the materials were stained well, only the central part of the nucleolus was stained. Under the electron microscope, it was observed that all the silver grains were concentrated in the pars fibrosa of the nucleolus. P. cassubica had only one small oblate nucleolus attached to the nuclear envelope, with NOR usually in the shape of the letters O or C. P. micans had 1–7 nucleoli of various sizes and shapes with NORs in various complicated forms. The number of nucleoli bore a certain relationship to the living state of the dinoflagellate. One day after fresh medium was added, cells with 3 nucleoli were most common, and 28.5% of the individuals had 4–6 nucleoli. Cells having only one nucleolus accounted for 8.6%. 3 days after, cells with 2 nucleoli became dominant, and those with 4–6 decreased to 18.4%. After a month, cells with 1 nucleolus became most abundant, cells having 4 nucleoli decreased to 2.4%, and no cells had 5 or 6 nucleoli.     

Nucleolar silver-staining patterns related to cell cycle phase and cell generation of PHA-stimulated human lymphocytes

Silver staining (Ag-I) was used to investigate changes in the nucleolar structure of PHA-stimulated human lymphocytes through the phases of the cell cycle, G1, S and G2. Ag-I patterns and cell cycle phases of individual cells were assessed by sequential silver staining, Feulgen staining, DNA microdensitometry and 3H-thymidine autoradiography. The morphology and number of Ag-I nucleoli in a particular cell depended upon the phase of the cell cycle reached and on the number of generations the cell had passed through in culture. Resting, unstimulated cells usually had one small silver positive nucleolus. During blast transformation, the silver stained nucleoli increased in number and size, and then fused to form one very large, rounded or irregular-shaped nucleolus which was present through all cell cycle phases of the first reproductive cycle. Many lymphocytes developed a band-shaped nucleolus during their first S phase in culture. Lymphocytes at all cell cycle stages of the second and third generations after PHA-stimulation had multiple nucleoli whose combined areas approximated that of the single large nucleolus observed in first generation cells.     

Relative position of nucleolar chromatin and nucleolar components in ciliate <Emphasis Type="Italic">Didinium nasutum</Emphasis> somatic nuclei

According to our computer modeling data obtained earlier, nucleoli in interphase ciliates Didinium nasutum are complex netlike structures, in which the trabeculumor lamella-shaped fibrillar component is located on the periphery, and the granular component in the central part of the nucleolus. Chromatin bodies connected with nucleoli act as the nucleolar organizers in D. nasutum. In the present work, the arrangement of all chromatin bodies, which could correspond to nucleolar organizers by morphological criteria, is studied by means of a 3D-reconstruction. It is shown that all of these chromatin bodies are localized outside the nucleoli, on the fibrillar component’s periphery. Even those chromatin bodies which appeared to be completely surrounded by the fibrillar nucleolar component on single ultrathin sections are actually settled down in nucleolus cavities open to the nucleoplasm. This proves that the RNA processing in D. nasutum nucleoli is directed toward the center of nucleoli, where the granular component is located. The analysis of the nucleolar chromatin distribution made it possible to conclude that different parts of the complex interfase netlike nucleoli of D. nasutum have approximately the same activity.     

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.     

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.     

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.     

The genetic control of nucleolus formation in wheat

The wheat variety Chinese Spring has four pairs of nucleolus organisers of known rDNA content. The genetic control of these has been investigated in root tip cells by cytologically scoring the number of nucleoli per cell in (a) aneuploid derivatives each having a different dosage of a particular chromosome or chromosome arm and (b) in substitution lines where nucleolus organiser chromosomes have been replaced by homologues possessing different amounts of rDNA. It has been assumed that nucleolus organiser activity is correlated with nucleolus size and thus with the presence of a cytologically visible nucleolus. Those nucleolus organisers on chromosomes 1A and 5D, which together possess only 10% of the rDNA form a visible nucleolus only infrequently in the presence of the larger nucleolus organisers on chromosomes 1B and 6B. When a major pair of organisers on chromosomes 1B or 6B is deleted, the smaller nucleolus organisers form a visible nucleolus more frequently. Similarly, when the major nucleolus organisers are replaced by organisers with less rDNA, the smaller nucleolus organisers form visible nucleoli more frequently. When a small nucleolus organiser is replaced by one with much more rDNA, a larger nucleolus is formed. These and other findings lead to the general conclusions that there is a frequently, but not invariably, seen correlation between rRNA gene number and nucleolus size. However the relative size of the nucleolus formed depends principally upon the proportion of the total active rRNA genes in the cell which are localised at the nucleolus organiser in question. Varying the dosage of at least 13 non nucleolus organiser chromosomes also resulted in changes in the number of visible nucleoli per cell. This implies the genetic control of individual nucleolus organisers is complex. Inclusion in the wheat genome of the nucleolus organiser chromosome from Aegilops umbellulata, causes suppression of the wheat nucleolus organisers, the Aegilops umbellulata organiser remaining active. This suppression is similar to that observed in many interspecific plant and animal hybrids.     

扁蓿豆冷诱导基因差异表达分析

以直立型扁蓿豆幼苗为试验材料,采用cDNA-AFLP技术分析扁蓿豆在低温胁迫诱导下的基因表达差异.结果显示,利用筛选的64对引物组合,对0℃低温处理3～5叶期扁蓿豆幼苗的叶片cDNA进行扩增,共获得549条差异表达的转录衍生片段(TDFs).选取上调表达较好的43条片段进行克隆、测序、Blastx比对和功能分析,其中32个TDFs的蛋白序列与基础代谢、信号转导、转录因子、防御等功能有关,11个TDFs为假设蛋白、未知蛋白或没有找到一致序列.利用荧光定量PCR对3种不同上调表达差异片段进行验证,结果可从数值上更准确地显示差异片段在低温胁迫过程中的相对表达量.     

低氧对小麦根端分生细胞核仁结构和功能的影响

为了探讨低氧对小麦根端分生细胞核仁结构和功能的影响,本实验以普通小麦为材料,用低氧水处理其根尖,按常规细胞制片、银染、电镜观察、间接免疫荧光染色和半定量PCR分析等手段开展研究.观察发现:(1)低氧水处理后小麦核仁结构发生膨胀、突出、进而凝集、内部出现空泡、细微结构消失、核仁通道结构异常、甚至解体等一系列变异现象.(2)间接免疫荧光染色技术观察看到,低氧水处理后小麦核仁内的核磷蛋白B23向核质甚至胞质扩散.(3)半定量PCR分析显示,低氧处理后rRNA基因的表达量较对照明显降低,而且C23的表达信号几乎检测不到,表明核糖体RNA和核仁蛋白C23基因的表达均显著下调,低氧严重抑制它们的转录.研究证明,低氧除了对小麦根端分生细胞核仁结构有破坏作用外,还严重抑制核仁的功能.     

Heterochromatin,repetitive DNS und Nukleolen in Leberzellen von Microtus agrestis

Zusammenfassung Die Zellstruktur von Leberzellen der Erdmaus, Microtus agrestis, wurde nach Giemsafärbung, Feulgenbehandlung, Behandlung mit Ribonuklease und nach Färbung des konstitutiven Heterochromatins untersucht. Das konstitutive Heterochromatin ist in Leberzellen nicht heteropyknotisch, das fakultative Heterochromatin ist im weiblichen Geschlecht als Sexchromatinkörperchen sichtbar. Bestimmungen des relativen DNS-Gehalts ergaben, daß die Zahl der Sexchromatinkörperchen der Ploidie der Zellkerne proportional ist. Die Nukleolen liegen in Hepatozyten oft randständig; in 59% der diploiden Zellkerne sind 2 Nukleolen enthalten. Nach Anfärbung der repetitiven DNS werden oft auch die Nukleolen gefärbt, nach Ribonukleasebehandlung tritt dieser Effekt nicht auf. Das konstitutive Heterochromatin wird in Form von 2 langen fädigen Strukturen sichtbar.

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Heterochromatin, repetitive DNA and nucleoli in liver cells of Microtus agrestis

Summary The nuclear structure of parenchymal liver cells of embryo and adult Microtus agrestis was studied in smear and section preparations after staining with Giemsa solution and treatment according to Feulgen, after treatment with ribonuclease and after specific staining of constitutive heterochromatin. In liver cell nuclei only the facultative heterochromatin is heteropycnotic, a sex chromatin body is observable in female but not in male animals. Constitutive heterochromatin is not heteropycnotic in liver cells. Measurements of the relative DNA content showed that nuclei with one sex chromatin body are diploid; tetraploid nuclei possess two and octoploid nuclei four sex chromatin bodies. Solely in the diploid cell nuclei of the intrahepatic gall ducts two large chromocenters are found. The nucleoli in hepatocytes often lie at the perimeter of the nucleus. 17% of the diploid nuclei contain one nucleolus, 59% two nucleoli, 23% three and 1% four. After staining of repetitive DNA, the nucleoli often become stained as well; after treatment with ribonuclease this effect does not occur. The constitutive heterochromatin becomes visible in form of two long, threadlike structures. After longer periods of dissociation the sex chromatin body ceases to be visible. Sex chromatin and constitutive heterochromatin are contiguous to the nucleoli.

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Mit dankenswerter Unterstützung durch das Bundesministerium für Bildung und Wissenschaft der Bundesrepublik Deutschland.     

Evidence from studies on segregated nucleoli that nucleolar silver staining proteins C23 and B23 are in the fibrillar component

Several procedures for the silver staining of nucleoli have been evaluated at the electron microscopic level to determine optimal conditions for ultrastructural preservation and staining specificity. The present study shows that a brief fixation with 1% buffered formaldehyde followed by methanol: acetic acid (3 : 1) fixation yielded optimal preservation and silver staining of nucleoli. Using this procedure for electron microscopic studies of interphase nucleoli, it was found that the punctate silver grains observed by light microscopy were composed of fine silver granules, of approx. 100 Å diameter, organized in discrete clusters. In similar studies on adriamycin-induced segregated nucleoli, it was observed that the silver staining reaction was mainly limited to the fibrillar portion of the nucleolus. Accordingly, nucleolar proteins C23 and B23, found earlier to be the major silver binding proteins of the nucleolus, are mainly concentrated in the fibrillar nucleolar component.     

Cell cycle analysis and drug inhibition studies of silver staining in synchronous HeLa cells

Cytological staining with silver nitrate is specific for a protein associated with chromosomal nucleolus organizer regions and interphase nucleoli. At metaphase the amount of staining present is usually much less than that at interphase. During the transition from mitosis to G1, as seen in synchronized HeLa cells, the amount of silver staining increases and, by late G1, is located discretely and completely over the nucleolus. Such staining remains constant through G2. Towards late G2 a slight disorganization of the silver staining material is observed, possibly in preparation for the upcoming mitosis. Cells synchronized at mitosis and treated with either actinomycin D (AMD) or 2-mercapto-1-[2-(4-pyridyl)-ethyl]-benzimidazole (MPB), at concentrations which inhibit ribosomal RNA (rRNA) synthesis, show nucleolar fragmentation and little, if any, apparent increase in silver staining at early G1. After removal of the MPB, the nucleolar fragments reform nucleoli and the staining increases to control levels. Treatment of mitotic cells with puromycin dihydrochloride does not effect nucleolar morphology or the increase in silver staining. These results directly demonstrate that silver staining is associated with rRNA synthesis.     

Number of nucleoli in various cell types of the mouse

The nucleoli of cells of the adult mouse were examined by staining with toluidine blue after removal of deoxyribonucleic acid from tissue sections by deoxyribonuclease treatment. The nuclei of each cell type examined contained one or more nucleoli. This was observed even in lymphocytes and neuroglia, although these cells have occasionally been described as anucleolated. In mature spermatids and spermatozoa, however, it was not possible to detect a nucleolus. The distribution of the number of nucleoli in many diploid cells exhibited a mode of two or three nucleoli per nucleus, and a range from 1 to 6 nucleoli. In presumedly diploid hepatic nuclei, the maximum number of nucleoli was six; but in presumedly tetraploid hepatic nuclei, it was 11. Thus, nearly twice as many nucleoli are present when the chromosome number is doubled. In view of this observation, it is suggested that six nucleolar organizers are present in the diploid chromosomal complement of the mouse. However, through failure of some nucleolar organizers or more probably through fusion of nucleoli, the number of these organelles in most nuclei is less than six.     

The effect of the Bacillus thuringiensis exotoxin on the fine nucleolar morphology and ultrastructure

Depending on the dose administred to the experimental mice, the Bacillus thuringiensis exotoxin produces striking changes in the nucleolar morphology of hepatocytes such as the formation of ring-shaped nucleoli, micronucleoli and the segregation of nucleolar components. Such changes are apparently related to the decrease and inhibition of nucleolar biosynthetic activities in the production of the nucleolar RNA. In addition, the Bacillus thuringiensis exotoxin causes the formation of nucleolar peripheral dense plaques and, at higher concentrations, the segregation of two distinctly separated granular areas in the nucleolus. Both these light and dense granular areas showed positive staining with Bernhard's EDTA procedure for the preferential demonstration of RNA-containing structures. In some segregated nucleoli the granular components of light granular areas seemed to leave the nucleolus. The presence of discontinuous filamentous shell around micronucleoli produced by the high dose of exotoxin suggests the nucleolar origin of nuclear granular bodies which are surrounded by similar but continuous filamentous shell characteristic of these structures.     

Multiple nucleoli and enhanced nucleolar activity in the nurse cells of the insect ovary

Origin and function of the nucleolar apparatus in nurse cell nuclei of Calliphora erythrocephala have been investigated by cytological and autoradiographic methods in some inbred lines of laboratory blowflies with well paired polytene chromosomes in the nurse cell nuclei. Besides the nucleolus at chromosome VI large numbers of multiple free nucleoli develop in the highly polyploidized nurse cells during oocyte growth. The nucleoli incorporate H³-uridine in a considerable amount producing a homogeneous and RNase-sensitive label even after short time incubation. Their capacity of RNA synthesis is independent of their spatial relationships to other nuclear components. DNA particles in the nucleoli could be identified by the Feulgen reaction and by fluorescence staining with N,N'-diethylpseudoisocya-ninchloride, which also demonstrates the existence of own templates for autonomous RNA synthesis. There are indications that the nucleolus' own DNA is produced by gene amplification beyond the level of endomitotic polyploidization in the nurse cell nuclei. A quantitative estimation of grain density in the autoradiograms shows a rigorous shift of rRNA synthesis: at least 72% of all newly synthesized macromolecular RNA in nurse cell nuclei as contrasted to 13 % of nucleolar RNA synthesis in bristle forming cells with a similar degree of polyploidy. It seems that the nurse cell nuclei of Calliphora in addition to polyploidization increase their template capacity for synthesizing rRNA in a similar way as has repeatedly been demonstrated for Amphibia. Cytological and physiological peculiarities of the nurse cells have been discussed from the viewpoint of their functional similarity to the oocyte nucleus.     

Isolation and characterization of different-sized nucleoli from Ehrlich ascites tumor cells : Evidence of different nucleolar ribosomal cistrons

A procedure was developed for isolation of variously sized nucleoli in order to study the mechanism of nucleolar formation from multiple nucleolar organizers and to compare the compositions of different-sized nucleoli from Ehrlich ascites tumor cells. Relatively small nucleoli and large nucleoli from Ehrlich ascites tumor cells were separated by centrifugation at 400 g for 5 min in a layer of 0.34 M sucrose over 0.88 M sucrose. Small nucleoli remained in the 0.34 M sucrose layer while the large nucleoli accumulated in the 0.88 M sucrose.Three fractions, provisionally named small, intermediate and large nucleoli, containing 0.33, 0.41 and 0.84 pg DNA/nucleolus, respectively, were separated. Unfractionated nucleoli contained 0.59 pg DNA/nucleolus. The RNA content also increased with the size of the nucleolus and no significant difference was observed in the RNA/DNA ratios in the three fractions. Large nucleoli incorporated more [³H]uridine and [³²P]orthophosphate into RNA than did small nucleoli, but the base compositions of the RNAs extracted from the different-sized nucleoli were similar. No significant fragmentation occurred on sonication of large nucleoli for 3 min, so the observed difference in the DNA contents was not due to mechanical damage of the nucleoli.The DNAs of these different-sized nucleoli were analysed on CsCl gradients. The nucleoli contained similar percentages of satellite DNA (20–22%) which were also similar to those of total, unfractionated nucleoli. Approx. 10% of the extranucleolar DNA is satellite DNA—thus the nucleolar fractions were probably not appreciably contaminated with extranucleolar DNA. The DNA of small nucleoli contained a slightly lower percentage (0.058%) of ribosomal cistrons than large nucleoli (0.081%). This means that the higher content of DNA in the large nucleoli is not merely due to longer sized chromatin with extra regions of the vicinity of nucleolar organizers. Thus these results suggest that the total content of ribosomal cistrons/nucleolus is roughly proportional to the DNA content of the nucleoli, at least in Ehrlich ascites tumor cells. Namely, the number of ribosomal cistrons per nucleolus for small, intermediate and large nucleoli is 40, 60 and 130, respectively.     

An inventory of yeast proteins associated with nucleolar and ribosomal components

Background

Although baker's yeast is a primary model organism for research on eukaryotic ribosome assembly and nucleoli, the list of its proteins that are functionally associated with nucleoli or ribosomes is still incomplete. We trained a naïve Bayesian classifier to predict novel proteins that are associated with yeast nucleoli or ribosomes based on parts lists of nucleoli in model organisms and large-scale protein interaction data sets. Phylogenetic profiling and gene expression analysis were carried out to shed light on evolutionary and regulatory aspects of nucleoli and ribosome assembly.

Results

We predict that, in addition to 439 known proteins, a further 62 yeast proteins are associated with components of the nucleolus or the ribosome. The complete set comprises a large core of archaeal-type proteins, several bacterial-type proteins, but mostly eukaryote-specific inventions. Expression of nucleolar and ribosomal genes tends to be strongly co-regulated compared to other yeast genes.

Conclusion

The number of proteins associated with nucleolar or ribosomal components in yeast is at least 14% higher than known before. The nucleolus probably evolved from an archaeal-type ribosome maturation machinery by recruitment of several bacterial-type and mostly eukaryote-specific factors. Not only expression of ribosomal protein genes, but also expression of genes encoding the 90S processosome, are strongly co-regulated and both regulatory programs are distinct from each other.