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 Ultracytochemical Localzation of Acid Phosphatase Activity in Wheat During Fertilization

No acid phosphatase activity was observed in the mature embryo sac of wheat (Triticum aestivum) except the chalazal cytoplasm Of the central cell before fertilization. During fertilization, acid phosphataseactivity was observed in the following loci: part of chromatin of the egg nucleus and most of the mitochondria in the egg cytoplasm; the perinuclear spaces of the egg and sperm nuclei at the fusion of the egg and sperm nuclei; the chalazal cytoplasm and some vacuoles of the degenerated synergid; two sperm nuclei within the cytoplasm of female cells; the cell wall of each cell of the embryo sac and that of the nucellar cells surrounding the embryo sac. No acid phosphatase was observed in the two-celled proembryo. Dense enzyme reaction product was localized in the chromatin of the free nuclei at early stage of the endosperm. The characteristic of acid phosphatase distribution during fertilization may be associated with the physiological change of the egg Cell, the reorganization of mitochondria in the egg cell cytoplasm, the degeneration of one of the two synergids, the physiological state of the sperm nuclei and the nuclear membrane fusion of the egg and sperm nuclei.     

小麦受精过程中酸性磷酸酶的超微细胞化学定位

小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ ）受精前成熟胚囊,除胚囊中央细胞的合点端细胞质中有酸性磷酸酶外,其余部位均未发现酸性磷酸酶。受精时期,以下部位存在酸性磷酸酶活性：卵细胞的细胞核内一部分染色质和细胞质中大部分线粒体;精、卵核融合时两核的核周腔内;退化助细胞合点端细胞质和一些液泡内;进入雌性细胞中的两个精核;胚囊各成员细胞的细胞壁及胚囊周围珠心细胞的细胞壁。二细胞原胚中未见有酸性磷酸酶。早期胚乳游离核染色质上有酸性磷酸酶。小麦受精过程酸性磷酸酶的分布特点可能与卵细胞生理状态的变化和细胞质中线粒体的改组、助细胞的退化、精核的生理状态以及精核与卵核的核膜融合等有关。     

Interspecific transformation and DNA characteristics in Allomyces

Summary Heterologous deoxyribonucleic acid treatment in Allomyces has been shown to transfer epigynous versus hypogynous character in the recipient species.A certain proportion of inverted sexual arrangements have been consistently detected in the acceptors.The uptake of native DNA was demonstrated using labelled ³²P-nucleic acid. The uptake was found to be higher in homologous (controls) than heterologous receptors.Chromatographic fractionation of the total DNA reveals 3 types differing in their Tm values and therefore GC ratios; these appear to be localized in nuclei, mitochondria and residual cytoplasm.     

An improved acriflavine-Feulgen method.

The acriflavine-Feulgen method for the histochemical demonstration of deoxyribonucleic acid was modified by staining hydrolyzed cells with 0.01% acriflavine dissolved in 90% ethanol. This method offered the following advantages: (a) it simplified the preparation of the acriflavine-Feulgen reagent; (b) it left the cytoplasm essentially unstained while staining the nuclei bright green in hydrolyzed cells and left the cytoplasm and nuclei essentially unstained in unhydrolyzed cells; (c) it eliminated poorly defined reagents from the staining solutions. Because of these staining properties, this technique may be especially useful in the quantitative determination of deoxyribonucleic acid by cytofluorometry.     

Scanning Feulgen-deoxyribonucleic acid cytophotometry of Papanicolaou destained preparations.

Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou destained specimens revealed a differential loss in Feulgen reactivity among human buccal and cervical smears, cultured embryonic lung fibroblasts and invasive cervical carcinoma cells. Loss in Feulgen reactivity in Papanicolaou destained fibroblasts and polyploid nuclei of malignant lesions was observed to result in underestimates of relative Feulgen deoxyribonucleic acid and nuclear area values using scanning integrating microdensitometry. Thus, Papanicolaou stained preparations may not be suitable for deoxyribonucleic acid quantification of high ploidy lesions since distributional absorption error is unpredictably influenced by such factors as ploidy level, nuclear size, chromatin dispersion and differential aldehyde loss during destaining. Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou stained preparations can be useful for differentiating benign from malignant lesions if extent of aneuploidy (as reflected in abnormal deoxyribonucleic acid frequency distribution profile) is used as a diagnostic indicator.     

The synthesis and distribution of ribonucleic acid in developing archegonia of Pteridium aquilinum

Summay Feeding gametophytes of Pteridium aquilinum with tritiated uridine, followed by autoradiography, revealed two waves of incorporation of the nucleoside into nuclei during oognesis. The first, affecting the nucleus of the primary cell of the archegonium, is interpreted as indicating the activation of the genes initiating oogenesis. The second, seen in the nucleus of the maturing egg, is believed to be concerned with the differentiation of the egg itself.The results of the autoradiography, corroborated by enzyme digestion and fluorescence microscopy, also demonstrated a high concentration of ribonucleic acid towards the periphery of the mature egg, some possibly located in nucleolus-like bodies found only in this region of the cytoplasm. It is suggested that part of this cytoplasmic ribonucleic acid may represent genetic information remaining untranslated until after fertilization.No evidence was found of any asymmetry in the distribution of the ribonucleic acid in mature eggs which might account for the polarity of developing zygotes.     

A CHROMATOGRAPHIC STUDY OF HYDROLYSIS IN THE FEULGEN NUCLEAL REACTION

In a study on Feulgen hydrolysis of frozen-dried alcohol-fixed lily anthers, a chromatographic technique was developed to analyze the acid hydrolysate for some of the degradation products of nucleic acid. Hydrolysis was accomplished by 10 per cent perchloric acid at 20°C., and a typical hydrolysis time-Feulgen intensity curve was obtained, with maximum staining occurring at 19 hours. Microphotometric measurements indicated that the amount of stain per nucleus was no different from amount in nuclei fixed and hydrolyzed by more conventional procedures. Uracil-containing material (from ribonucleic acid) was almost completely separated from thymine-containing material (deoxyribonucleic acid) of tissue sections by acid treatment for 1½ hours. Adenine (purines), as the base, was effectively all removed from the deoxyribonucleic acid at the time of optimum hydrolysis. Detectable amounts of thymine-containing material appeared in the hydrolysate shortly after the onset of hydrolysis; and the amount increased rapidly with increased hydrolysis time. At the time of optimum hydrolysis approximately two-thirds of the total deoxyribonucleic acid thymine was lost. The removal of these thymine-containing fragments was linear with respect to time during the first 24 hours and occurred at a relatively high rate. Removal after 24 hours was also linear but was at a markedly lower rate. These results would suggest that two kinds of deoxyribonucleic acid exist in lily anthers; an acid-labile fraction amounting to approximately three-fourths of the total, and an acid-resistant fraction making up the remainder. In the Feulgen procedure much of the labile fraction is lost by the time of optimum hydrolysis and is not stained; most of the stable fraction remains in the tissue and is stained. In light of these findings the use of the Feulgen method as a means of determining cytochemically relative amounts of deoxyribonucleic acid in nuclei by measuring their Feulgen dye content was discussed.     

Individual Nuclei Differ in Their Sensitivity to the Cytoplasmic Inducers of DNA Synthesis: Implications for the Origin of Cell Cycle Variability

Nuclei of multinucleate cells generally initiate DNA synthesis simultaneously, suggesting that the timing of DNA synthesis depends upon the appearance of a cytoplasmic signal. In contrast, intact nuclei from quiescent mammalian cells initiate DNA synthesisasynchronouslyin cell-free extracts ofXenopuseggs, despite the common environment. Here we show that the two nuclei of permeabilized binucleate cells enter DNA synthesis coordinately in egg extracts, as they doin vivo,with different pairs of nuclei initiating replication at different times. This indicates that the two nuclei of a binucleate cell are identical in their sensitivity to the inducers of DNA synthesis in egg extracts; this sensitivity varies in general between the nuclei of unrelated cells. The asynchrony of DNA synthesis shown by unrelated nuclei in egg extracts is therefore not an artifact of the cell-free system but a reflection of genuine differences preexisting within the intact cell. Evidence that these differences between nuclei are responsible for a substantial fraction of G₁variability in living cells is presented.     

Evidence for insulin synthesis in normal mouse seminal vesicle based on in situ RNA-DNA hybridization

Cultured seminal vesicle epithelial cells exhibited cytoplasmic immunoreactivity following treatment with anti-insulin antisera. In addition, these cultured epithelial cells were found, by in situ hybridization with a radiolabeled insulin complementary deoxyribonucleic acid (cDNA) probe, to contain an insulin or insulin-like messenger ribonucleic acid (mRNA). Autoradiograms of the hybridized cells exhibited heavy labeling over the cytoplasm and minimal distribution of grains over the nuclei and background areas. These observations indicate that cultured mouse seminal vesicle epithelium contains an insulin or insulin-like peptide as well as the mRNA that is required for its synthesis.     

Studying nuclear disassembly in vitro using Xenopus egg extract

Xenopus egg extract provides an extremely powerful approach in the study of cell cycle regulated aspects of nuclear form and function. Each egg contains enough membrane and protein components to support multiple rounds of cell division. Remarkably, incubation of egg extract with DNA in the presence of an energy regeneration system is sufficient to induce formation of a nuclear envelope around DNA. In addition, these in vitro nuclei contain functional nuclear pore complexes, which form de novo and are capable of supporting nucleocytoplasmic transport. Mitotic entry can be induced by the addition of recombinant cyclin to an interphase extract. This initiates signaling that leads to disassembly of the nuclei. Thus, this cell-free system can be used to decipher events involved in mitotic remodeling of the nuclear envelope such as changes in nuclear pore permeability, dispersal of membrane, and disassembly of the lamina. Both general mechanisms and individual players required for orchestrating these events can be identified via biochemical manipulation of the egg extract. Here, we describe a procedure for the assembly and disassembly of in vitro nuclei, including the production of Xenopus egg extract and sperm chromatin DNA.     

Transport of DNA from Lymphocytes to Fibroblasts in Culture

The exchange of radioactivity between lymphocytes, labelled with (³H) thymidine after stimulation with Concanavalin A, and recipient V79 fibroblasts in culture was studied. The radioactive material involved in this exchange was macromolecular deoxyribonucleic acid as well as its breakdown products. This deoxyribonucleic acid from lymphocytes localised in the nuclei of the host cells soon after contact between donor and recipients. This occurred even when the V79 fibroblasts were confluent at high cell density, and thus in a steady, non-growing state with respect to cell numbers.
The fate of the radioactive donor lymphocyte deoxyribonucleic acid, substituted with bromodeoxyuridine, was followed in the recipient cells by analysing its buoyant density in caesium chloride gradients. This deoxyribonucleic acid was found to become associated with the nuclear deoxyribonucleic acid of the host cells, involving both retention of relatively intact donor deoxyribonucleic acid as well as its breakdown and re-utilisation for host cell deoxyribonucleic acid synthesis. Nongrowing recipient cells were found to retain the donor deoxyribonucleic acid in relatively intact form for much longer periods than when the same cells were in logarithmic growth phase.     

Replication of Nuclei from Cycling and Quiescent Mammalian Cells in 6-DMAP-TreatedXenopusEgg Extract

Nuclear membrane permeabilization is required for replication of quiescent (G0) cell nuclei inXenopusegg extract. We now demonstrate that establishment of replication competence in G0 nuclei is dependent upon a positive activity present in the soluble egg extract. Our hypothesis is that G0 nuclei lose the license to replicate following growth arrest and that this positive activity is required for relicensing DNA for replication. To determine if G0 nuclei contain licensed DNA, we used the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), to prepare egg extracts that are devoid of licensing activity. Intact nuclei, isolated from mammalian cells synchronized in G1-phase (licensed), G2-phase (unlicensed), and G0 were permeabilized and assayed for replication in 6-DMAP-treated and untreated extracts supplemented with [α-³²P]dATP or biotinylated-dUTP. Very little radioactivity was incorporated into nascent DNA in each nuclear population; however, nearly all nuclei in each population incorporated biotin in 6-DMAP extract. The pattern of biotin incorporation within these nuclei was strikingly similar to the punctate pattern observed within nuclei incubated in aphidicolin-treated extract, suggesting that initiation events occur within most replication factories in 6-DMAP extract. However, density substitution and alkaline gel analyses indicate that the incorporated biotin within these nuclei arises from a small number of active origins which escape 6-DMAP inhibition. We conclude that 6-DMAP-treated egg extract cannot differentiate licensed from unlicensed mammalian somatic cell nuclei and, therefore, cannot be used to determine the “licensed state” of G0 nuclei using the assays described here.     

Violacein cytotoxicity and induction of apoptosis in V79 cells

Summary Violacein, a pigment produced by Chromobacterium violaceum, is reported to be a potential drug for the treatment of Chagas' disease. Violacein is also effective against leukemia and lymphoma cells in culture (IC₅₀ 10⁻⁸ M). Changes in the nuclear acid content, 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide reduction and neutral red uptake in these cells were used to evaluate the cytotoxicity of violacein in V79 Chinese hamster (M-8) fibroblasts. Violacein was highly cytotoxic to V79 fibroblasts (IC₅₀ 5–12 μM). Using the TUNEL method and the Feulgen reaction coupled to image analysis, violacein (5 and 10 μM) was found to trigger apoptosis but not necrosis in V79 cells. The morphological changes seen in the nuclei of these cells included chromatin condensation and a decrease in deoxyribonucleic acid content. These results demonstrating that violacein induces apoptosis in V79 cells strengthen its potential as a therapeutic agent.     

Lebendbeobachtungen der Chromosomen-Aufregulation bei der Gallmücke Heteropeza pygmaea (Cecidomyiidae,Diptera)

In male-determined, paedogenetically developing eggs of Heteropeza pygmaea a restitutive fertilization takes place after meiosis. Two small nuclei of maternal origin (somatic nuclei) and the egg nucleus migrate to the center of the egg chamber. Their chromosomes then form the metaphase plate of the primary cleavage nucleus. The in vitro observations and the analysis of photomicrographs and time lapse films revealed that the metaphase stage can be reached in three different ways: 1. The egg nucleus and the two somatic nuclei form one common spindle. 2. The egg nucleus forms a spindle and the two somatic nuclei together form another one. The two spindles then fuse in late prometaphase and form a single spindle. 3. The egg nucleus alone forms a spindle. The chromosomes of the somatic nuclei migrate to the equator of this spindle. This variation in the restitutive fertilization is explained by an increasing asynchrony between the development of the egg nucleus and the slower somatic nuclei from the first to the third type.     

Initiation of DNA replication in Saccharomyces cerevisiae G1‐phase nuclei by Xenopus egg extract

Xenopus egg extracts initiate replication at specific origin sites within mammalian G1‐phase nuclei. Similarly, S‐phase extracts from Saccharomyces cerevisiae initiate DNA replication within yeast nuclei at specific yeast origin sequences. Here we show that Xenopus egg extracts can initiate DNA replication within G1‐phase yeast nuclei but do not recognize yeast origin sequences. When G1‐phase yeast nuclei were introduced into Xenopus egg extract, semiconservative, aphidicolin‐sensitive DNA synthesis was induced after a brief lag period and was restricted to a single round of replication. The specificity of initiation within the yeast 2 μm plasmid as well as in the vicinity of the chromosomal origin ARS1 was evaluated by neutral two‐dimensional gel electrophoresis of replication intermediates. At both locations, replication was found to initiate outside of the ARS element. Manipulation of both cis‐ and trans‐acting elements in the yeast genome before introduction of nuclei into Xenopus egg extract may provide a system with which to elucidate the requirements for vertebrate origin recognition. J. Cell. Biochem. 80:73–84, 2000. © 2000 Wiley‐Liss, Inc.     

Reactivation of DNA Replication in Nuclei from Terminally Differentiated Cells: Nuclear Membrane Permeabilization Is Required for Initiation inXenopusEgg Extract

We have usedXenopusegg extract to investigate the requirements for reactivation of DNA replication in nuclei isolated from terminally differentiated chicken erythrocytes. Previous work has shown that reactivation of erythrocyte nuclei in egg extract is accompanied by chromatin decondensation, nuclear envelope reformation, and the accumulation of egg lamin, L_III. However, in those studies, erythrocyte nuclei were prepared by methods that were not designed to maintain the selective permeability of the nuclear membrane, and as such, it is not clear if loss of nuclear membrane integrity played a role in the reactivation process. Therefore, the purpose of this study was to determine if changes in nuclear membrane permeability are required for reactivation of erythrocyte nuclei in egg extract. Nuclei with intact nuclear membranes were prepared from erythrocytes with streptolysin O and permeable nuclei by treatment of intact nuclei with the detergent Nonidet-P40. Like permeable nuclei, most intact nuclei decondensed, imported nuclear protein, and accumulated lamin L_IIIfrom the extract. However, unlike permeable nuclei, which replicated extensively in the extract, few intact nuclei initiated replication under the same conditions. These data demonstrate that permeabilization of the nuclear membrane is required for reactivation of DNA replication in terminally differentiated erythrocyte nuclei by egg extract and suggest that loss of nuclear membrane integrity may be a general requirement for replication of quiescent cell nuclei by this system.     

Daunomycin disrupts nuclear assembly and the coordinate initiation of DNA replication in Xenopus egg extracts

We have used Xenopus egg extracts to investigate the effects of the antitumor drug daunomycin on DNA replication in vitro. Xenopus sperm nuclei replicated nearly synchronously in our egg extracts, thereby allowing us to determine the effects of the drug on both replication initiation and elongation. Titration experiments demonstrated that daunomycin effectively inhibited replication in the extract, with 50% inhibition at a total drug concentration of 2.7 μM. However, a high concentration of daunomycin 150 μM) also inhibited nuclear envelope assembly, a prerequisite for the initiation of replication in this system. Therefore, to bypass the effects of daunomycin on nuclear envelope assembly, sperm nuclei were preassembled in extract prior to drug addition. Initiation of replication in preassembled nuclei was also inhibited by daunomycin, with 50% inhibition at a drug concentration of 3.6 μM. At low drug concentrations, where replication did occur, the synchrony of initiations within individual nuclei was lost. This drug-induced disruption of initiation events may provide important clues regarding the mechanism(s) by which these events are coordinated in eukaryotic cells. Daunomycin also inhibited replication elongation in preassembled, preinitiated nuclei. However, the concentration of drug required for 50% inhibition of elongation was nearly fourfold higher than that required for inhibition of initiation. Taken together, these data demonstrate that Xenopus egg extract can be used to investigate the effects of DNA-binding antitumor drugs on a number of interrelated cellular processes, many of which are less tractable in whole cell systems. J. Cell. Biochem. 64:476–491. © 1997 Wiley-Liss, Inc.     

Mating Reaction in Saccharomyces cerevisiae. IV. Retardation of Deoxyribonucleic Acid Synthesis

When a and a type haploid cells of Saccharomyces cere-visiae were mixed and cultured, deoxyribonucleic acid synthesis was retarded but ribonucleic acid and protein syntheses were not. It was found that culture filtrate of a type cells inhibited deoxyribonucleic acid synthesis of a type cells and that of a type cells inhibited that of a type cells. Thus, sex-specific diffusible substances secreted by opposite mating type cells are thought, at least partly, to be responsible for the retardation of deoxyribonucleic acid synthesis.