Nucleolar and nuclear envelope proteins of the yeast Saccharomyces cerevisiae.

We have developed a fast and reliable purification protocol to obtain yeast nuclei in intact and pure form and in a reasonable yield. The purified nuclei appear homogeneous at the light and electron microscopic level, are highly enriched in the nuclear marker histone H2B and devoid of mitochondrial, vacuolar and cytosolic marker proteins. On sodium dodecyl sulfate (SDS)-polyacrylamide gels, the nuclear fraction contains unique proteins which distinguishes them from the major yeast subcellular fractions. Yeast nuclei were separated by detergent/salt extraction into soluble, insoluble and membrane fractions. Antibodies raised against subnuclear fractions lead to the identification of an integral nuclear membrane protein and a high-abundance 38-kDa protein which is located in the yeast nucleolus.

Electron microscopic localization of silver staining NOR-proteins in rat liver nucleoli upon D-galactosamine block of transcription.

The method for electron microscopy of the Ag-staining of NOR-specific proteins was adapted to tissue sections from solid organs. The distribution of the Ag-staining proteins in rat liver nucleoli after complete block of transcription caused by D-galactosamine was investigated. In control animals, the Ag-staining proteins are associated with the fibrillar components of nucleoli. After block of transcription, positive Ag-staining is observed in the condensed fibrillar components of segregated nucleoli and later in the derived dense nucleolar fibrillar remnants. The granular components and the spherical bodies in segregated nucleoli are negative. It is concluded that in interphase nucleoli the Ag-staining NOR proteins are associated with the fibrillar components and with the derived nucleolar fibrillar remnants. The positive Ag-staining does not reflect the actual transcription of rRNA genes since it is present in both transcribed and non-transcribed r-chromatin.

Appearance of host-specific nucleolar proteins in intranuclear "dense bodies" following herpes simplex infection.

Nucleolar modifications induced by herpes simplex virus type 1 (HSV1) infection were studied at the ultrastructural level with special attention to the fate of a family of proteins serologically related to the nucleolar 100 kDa protein. Immunocytochemical techniques revealed that antigenic sites related to these proteins were associated with nucleoli in a pattern similar to that observed with non-infected cells. In addition, the "dense bodies" induced by HSV infection were heavily decorated by antibodies to the 100 kDa protein. Neither DNA nor RNA was detectable in the latter by cytochemical techniques. Therefore, it appears that "dense bodies" are exclusively proteinaceous and contain at least one host protein implicated in ribosomal RNA synthesis. An accumulation of 100 kDa protein in extra-nucleolar structures might account for previously reported defects in ribosomal RNA expression during HSV infection.

The relationship between aging and ribosomal gene activity in humans as evidenced by silver staining.

Silver selectively stains nucleolar organizer regions (NORs) distributed on the D and G chromosomes of man. The number of NORs is fairly constant for a given individual but is highly variable within populations. When 2540 metaphases from lymphocytes were examined from 127 normal subjects, a mean NOR number of 7.3 was obtained with a mode of 7. No significant difference was found in mean NOR number between females and males. However, regression lines do show a decrease in NOR number with aging for both sexes but the rate of decline is more evident in females. Also, G chromosome NORs appear more stable than those in D chromosomes. Since silver--NOR staining is indicative of ribosomal gene activity, it is proposed that lymphocyte rDNA becomes less sensitive to phytohemagglutinin stimulation during aging. It is unique that this gene repression can be visualized with conventional staining and light microscopy.

Silver staining of nucleolar organizer regions in malignant melanoma and melanotic nevi.

The recently described method of staining nucleolar organizer regions (NOR) with colloidal silver nitrate was applied to the paraffin sections of five junctional nevi, 13 compound nevi, seven Spitz nevi, nine cellular blue nevi, 11 dysplastic nevi, seven malignant lentigines, 12 superficial spreading melanomas, and 14 secondary melanomas. There was a significant difference between the pooled silver-NOR (AgNOR) numbers of the 45 benign lesions (mean, 1.22; SD, 0.51) and the 33 melanomas (mean, 9.18; SD, 4.05) by t test analysis (P less than .01). The difference was striking enough to be recognized on casual microscopic examination, suggesting that AgNOR staining may be a useful technique to help separate melanocytic nevi from malignant melanomas.

Nucleolar organiser region associated proteins in cutaneous melanotic lesions: a quantitative study.

Using a silver staining technique, nucleolar organiser regions were identified in routinely processed paraffin sections of a range of dermal melanotic lesions. The technique shows argyrophilic NOR associated proteins (AgNORs), which are seen in nuclei as black dots. Although the nuclei of melanocarcinoma in situ, melanocarcinoma per se, and lentigo maligna contained similar numbers of AgNORs, in melanocarcinomas a mean of 7.9 AgNORs per nucleus was found, while in naevocellular naevi, this figure was 1.2. The AgNOR method, which is unknown to most histopathologists, could perhaps be used quantitatively or semiquantitatively to assist in the diagnosis of melanotic lesions in the skin (and elsewhere).

Recognition of the retrograde reaction in motoneurons using an image analysing system.

An automatic procedure is described which allows recognition of sectional profiles of alpha-motoneurons of the facial nucleus of the rat. The system is able to distinguish nucleus and nucleolus from the cytoplasm of the nerve cell profile and to characterize those structures by suitable parameters. The region of the nucleus of the facial nerve (FNN) has been measured 7 days after unilateral partial nerve transsection. Based on the resulting data a multivariate analysis was performed to distinguish between normal and retrograde reacting cells. It is shown that this automatic cell characterisation technique is highly sensitive and reliable. The data were used to describe comprehensively the retrograde reaction of the cell.

Differential silver-positive nucleolus organizer region activity in normal and malignant murine tissues.

Transcriptionally active nucleolus organizer regions (NORs) were analyzed using a silver-staining technique. The levels of silver-positive NOR activity for normal murine bone marrow and thymus cells were established, and significant differences in ribosomal RNA gene activity were observed. When tumor cells originating from these two tissue types were also studied, significant differences were seen not only between the normal and malignant tissues, but between the two tumor types as well. These differences in ribosomal RNA gene activity with respect to cell type and malignancy may be useful diagnostically.

Silver colloid staining technique for analysis of glioma malignancy.

A silver colloid staining technique for identifying nucleolar organizer region-associated proteins (Ag-NOR's) was applied to 51 human gliomas. These comprised 20 glioblastomas multiforme, 15 anaplastic astrocytomas, and 16 astrocytomas, in which the mean numbers of Ag-NOR's per cell (+/- standard error of the mean) were 2.51 +/- 0.12, 2.01 +/- 0.10, and 1.76 +/- 0.06, respectively. Significant differences among these were recognized, and the mean number of Ag-NOR's paralleled the degree of histopathological malignancy. In 16 cases, studies were performed of the number of Ag-NOR's and the S-phase fraction by in vitro labeling using antibromodeoxyuridine monoclonal antibody. A linear relationship was demonstrated between these two factors (r = 0.857, p less than 0.001), although some scatter was seen. In 32 adult patients, the correlation between the number of Ag-NOR's and the prognosis was estimated. The results demonstrated that the group containing patients with less than 1.80 Ag-NOR's per cell had a better prognosis than the group with 1.80 Ag-NOR's or more. Thus, the number of Ag-NOR's reflected the degree of histopathological malignancy, S-phase fraction, and prognosis. Silver colloid staining for Ag-NOR's is a simple, rapid, and reproducible method for estimating the proliferative potential of human gliomas without requiring a complicated technique.

The nucleolar organizer regions associated protein (Ag-NORs) in salivary gland tumors.

A silver colloid technique used to identify nucleolar organizer regions associated protein (Ag-NORs) has been applied to 20 salivary gland tumors. The method was readily applicable to the preparations of paraffin-embedded sections and the Ag-NORs were enumerated with ease. A significant difference was found between the numbers of Ag-NORs in the nuclei of malignant salivary gland tumors, such as adenoid cystic carcinoma, mucoepidermoid tumor and adenocarcinoma (with a mean of from 2.05 to 2.78 per nucleus) and those of benign salivary gland, such as pleomorphic adenoma, adenolymphoma (Wartin tumor) and clear cell adenoma (with a mean of from 1.47 to 1.72 per nucleus). It is proposed that the Ag-NORs technique, which is rapid, simple, and inexpensive, may be useful in the differential diagnosis of malignant and benign salivary gland tumors.

Ag-stained nucleolus organizer regions (Ag-NORs): expression in lymphocytes of patients with endometrial carcinoma.

The authors have studied the nucleolus organizing regions (NORs) from short term cultures of lymphocytes of 15 women with endometrial adenocarcinoma and 15 healthy females. In contrast with the results of Literature, the frequence of NORs in patients with endometrial adenocarcinoma does not show any significative difference.

Presence of argyrophilic cytoplasmic and nuclear proteins in the spermatic cells of Nucella lapillus (Gastropoda, Prosobranchia).

The technique for ultrastructural localization of argyrophilic proteins was modified and NOR-Silver staining methods were applied to the study of the distribution of these proteins in the spermatic cells of Nucella lapillus (Gastropoda, Prosobranchia). Two types of selective silver deposits were found during the different phases of spermiogenesis and in mature spermatozoa. Argyrophilic nuclear, nucleolar and cytoplasmic proteins were simultaneously detected by improvement of a modified one-step silver technique.

Variation within and between nucleolar organizer regions in Australian hylid frogs (Anura) shown by 18S + 28S in-situ hybridization.

Five distinct classes of secondary constriction are found in the hylid frogs from the genera Litoria and Cyclorana, each of which is defined by its C-banding pattern and morphology (King, 1980, 1987). In-situ hybridization experiments utilizing 18S + 28S copy RNA probes derived from Xenopus and Drosophila rDNA templates, were made on nine species of frogs possessing the major constriction types. Types 1, 2, 4, and 5 are confirmed as being NORs. These results also indicate that type 1 and 2 constriction types are not differentially despiralized as previously suggested, but show absolute differences in the quantity of ribosomal DNA present. This variation took two forms, deletion polymorphism and amplification polymorphism. These differences were observed between homologues within cells and between cells within individuals. Animals possessing these 'despiralized' constrictions are therefore mosaics for both deletion and amplification polymorphisms. Polymorphism frequencies vary greatly between constriction types. Some specimens have a higher level of presence/absence heterozygosity, (L. moorei, type 2, L. nannotis type 5, L. raniformis (animal A, pair 8 type 2), than do others (L. peronii, L. rothii, L. caerulea). The above species also vary markedly in the degree and frequency of amplification of the NORs. The type 4 constrictions analysed (L. coplandi, L. lesueuri and C. novaehollandiae) have a particularly low frequency of presence/absence heterozygosity, and they have fewer size heteromorphisms between homologues. The type 3 ephemeral constrictions did not hybridize to cRNA probes at any stage. In all but one of the species studied, a single pair of chromosomes possessed an NOR. However, in L. raniformis these occurred on two pairs of chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of NORs in spermatogonial metaphase chromosomes of six species of grasshoppers.

The silver staining technique was employed to locate Nucleolar Organiser Regions (NORs) in six species of grasshoppers viz. Aiolopus thalassinus F. (Tryxalinae); Oeodaleus abruptus Thunb., Gastrimargus transversus Thunb., Heteropternis respondens Walk. (Oedipodinae); Parahieroglyphus biliniatus Bol. and Spathosternum prasiniferum Walk. (Catantopinae). Usually the NORs were located on the larger elements of the chromosomal complement. However, in O. abruptus NORs were found on autosomes S8 and S9. The salient observations were: (1) NORs were seen in only a few of the several spermatogonial metaphases examined; (2) Active NORs were mostly located either on one chromatid of the homologues or on the homologue depicting heteromorphism; (3) NORs showed either proximal, subproximal or interstitial locations. However, in O. abruptus and P. bilineatus NORs were located at two positions. Distribution of NORs in different species and their probable role in tracing the evolutionary pathways in Acridoidea are discussed.

Sequential estimation of nuclear DNA and silver staining of nucleoli in plant cells.

A method for sequential estimation of nuclear DNA and silver staining of nucleoli in plant cells is described. Feulgen staining is done first and nuclear DNA estimated by absorption cytophotometry. Following this, the slides are stained with AgNO3. The method has been used to study the process of nucleolar fusion in garlic (Allium sativum L.) meristem root tip cells. It was found that during interphase nucleoli rarely fused, thus most fusion must have occurred before the G1 phase of the cell cycle.