Changes in nuclear and nucleolar protein content during the growth and differentiation of root parenchyma cells in plant species with different DNA-endoreplication dynamics

Using cytophotometric procedures, we measured the nuclear and nucleolar protein content of successive zones of growth and differentiation in consecutive (1-7 mm) root segments obtained from eight species of the Angiospermae after staining the preparations with Feulgen-Naphthol Yellow S (F-NYS). In meristematic cells the nuclear and nucleolar protein content was found to double during the cell cycle. In species in which differentiation occurs at the same time as nuclear DNA endoreplication, i.e. Vicia faba subsp. minor, V. faba subsp. major, Pisum sativum, Hordeum vulgare and Amaryllis belladonna, the pool of nuclear proteins observed during the G2 phase of the cell cycle was seen in the differentiated zone in nuclei containing 8C DNA. Species in which differentiation is not accompanied by the process of nuclear DNA endoreplication, i.e. Levisticum officinale, Tulipa kaufmanniana and Haemanthus katharinae, exhibited the highest nuclear proteins content during the G2 phase of the cell cycle; comparably high values were not found in the differentiated zone. A decrease in nucleolar protein content was observed during the process of differentiation, this tendency being more evident in the studied species that do not exhibit endoreplication.     

Role of RNA and protein synthesis and turnover in the heat-induced increase in nuclear protein

The proteins which become associated with nuclei during hyperthermic exposure were characterized by labeled amino acid incorporation. Actinomycin-D (Act-D) or cycloheximide (CHM) pretreatment was used to determine whether concurrent RNA or protein synthesis is required for hyperthermia to induce the increase in nuclear protein content. Prior to heat exposure exponentially growing HeLa cells were (i) pulse labeled for 1 h, (ii) labeled for 36 h, or (iii) labeled for 24 h followed by 17 h chase. The nuclear specific activity (CPM/microgram protein) of [3H]lysine-labeled proteins did not change under any of the labeling conditions, whereas that of [3H]leucine-containing proteins increased significantly with (i) but not with (ii) or (iii), while that of [3H]tryptophan-labeled protein increased significantly with (i) and (ii) but not with (iii). Act-D treatment 1 h prior to and during heating did not affect nuclear protein increase, while CHM-treated cells showed generally less nuclear protein content (70% of control at 60 min) but nevertheless significant nuclear protein increase upon heating (60% increase at 60 min from 0 min). These results suggest that those proteins associated with nuclei following heat exposure are nonhistones with a high turnover rate, and the process dose not require the synthesis of RNA or proteins.     

hnRNP G: sequence and characterization of a glycosylated RNA-binding protein.

The autoantigen p43 is a nuclear protein initially identified with autoantibodies from dogs with a lupus-like syndrome. Here we show that p43 is an RNA-binding protein, and identify it as hnRNP G, a previously described component of heterogeneous nuclear ribonucleoprotein complexes. We demonstrate that p43/hnRNP G is glycosylated, and identify the modification as O-linked N-acetylglucosamine. A full-length cDNA clone for hnRNP G has been isolated and sequenced, and the predicted amino acid sequence for hnRNP G shows that it contains one RNP-consensus RNA binding domain (RBD) at the amino terminus and a carboxyl domain rich in serines, arginines and glycines. The RBD of human hnRNP G shows striking similarities with the RBDs of several plant RNA-binding proteins.     

Cytoplasmic assembly and nuclear accumulation of mature small nuclear ribonucleoprotein particles

The assembly pathway of small nuclear ribonucleoprotein (snRNP) particles in the cytoplasm of L929 mouse fibroblasts was analyzed by observing the nuclear accumulation of snRNP proteins. Immunoprecipitations of nuclear and cytoplasmic fractions after a pulse label and chase indicate that the snRNP D, E, F, and G proteins assemble first, followed by the small nuclear RNA (snRNA), then the snRNP B protein and, in the case of the U1 snRNP, the A and C proteins. The snRNP B' protein is not detected in the L929 cells. The U1-specific A and C proteins can enter the nucleus in the absence of snRNP assembly, suggesting that these proteins exchange on the mature nuclear snRNP particles. Two-dimensional electrophoresis using nonequilibrium pH gradient electrophoresis identifies the A, B, B", C, D, E, F, and G proteins in a distribution similar to that reported previously by immunoprecipitation (Sauterer, R. A., and Zieve, G. W. (1989) J. Biol. Chem., submitted for publication). The D protein appears in multiple isoelectric variants in the cytoplasm and shifts toward more basic variants during maturation. Kinetic experiments analyzed by two-dimensional electrophoresis indicate a quantitative maturation of the cytoplasmic B protein into nuclear particles. Quantitative densitometry of immunoprecipitated stable nuclear snRNPs labeled with [35S] methionine corrected for the published methionine content of the A, B, C, D, and E proteins indicates that the mature nuclear U1 snRNP probably contains four copies of D, two copies each of B, C, and A, and one copy of E.     

Effects of irradiation on nuclear protein synthesis in G2 phase of the cell cycle

A method was developed to determine the synthesis of nuclear proteins throughout the cell cycle which was resolved into six compartments on the basis of DNA and nuclear protein content (i.e., early and late G1, early and late S, etc). Using this technique cell-cycle-specific synthesis of certain nuclear proteins was observed. Of particular interest was a 170-kDa protein(s) whose synthesis was initiated in early S phase and reached a maximum rate in late G2. Following irradiation with 6.8 Gy of 137Cs gamma rays the synthesis of the 170-kDa protein(s) declined in the G2 population with near total inhibition seen by 24 h. Synthesis of the 170-kDa protein(s) appeared to be slightly enhanced, and the postirradiation inhibition of its synthesis was reversed, in the presence of 3 mM caffeine. Also, the synthesis of 55-kDa nuclear protein(s) was stimulated throughout the cell cycle in the presence of 3 mM caffeine. These observations suggest new possibilities regarding the mechanism of the X-ray-induced G2 block and its reversal by caffeine. However, the exact role of these nuclear proteins in cellular events remains to be ascertained.     

Protein isoprenylation in suspension-cultured tobacco cells.

Many mammalian and yeast proteins, including small ras-like GTP binding proteins, heterotrimeric G protein gamma subunits, and nuclear lamins, have been shown to be covalently linked to isoprenoid derivatives of mevalonic acid. Isoprenylation of these proteins is required for their assembly into membranes and, hence, for their biological activity. In this report, it is shown that cultured tobacco cells, when pretreated with an inhibitor of endogenous mevalonic acid synthesis (lovastatin), incorporate radioactivity from 14C-mevalonic acid into proteins. Most of these proteins are membrane associated, and many are similar in mass to mammalian ras-like GTP binding proteins and nuclear lamins. Furthermore, it is shown that tobacco cell extracts catalyze the transfer of radioactivity from 3H-farnesyl pyrophosphate and 3H-geranylgeranyl pyrophosphate to protein substrates in vitro. These studies indicate the presence of at least two distinct prenyl:protein transferases in tobacco extracts: one that utilizes farnesyl pyrophosphate and preferentially modifies a substrate protein with a CAIM carboxy terminus (farnesyl:protein transferase) and one that utilizes geranylgeranyl pyrophosphate and preferentially modifies a substrate protein with a CAIL carboxy terminus (geranylgeranyl:protein transferase type I). This work provides a basis for future work on the role of protein isoprenylation in plant cell growth, signal transduction, and membrane biogenesis.     

The relationship of DNA base composition and individual protein composition in micro-organisms

Summary To investigate the dependence of protein composition on DNA base composition, a set of data on individual proteins with known amino acid compositions from a spectrum of bacterial species has been compiled. It is found that similar relationships of amino acid frequency to G + C content exist for these proteins as for the bulk proteins studied by Sueoka (1961). The data are analysed by linear and cubic regression, and a measure of the proportions of A + T-rich and G + C-rich codons in the underlying messenger RNAs is put forward. The theoretical limits on the G + C content of coding DNA are discussed, and inference are made about the various selective forces acting on DNAs of different G + C contents.     

New developments in abscisic acid perception and metabolism

Abscisic acid is a powerful signaling molecule that accumulates in response to abiotic stress. However, no potential receptors that could perceive this increase in abscisic acid had been identified until recent reports of three abscisic acid binding proteins: the nuclear protein Flowering Time Control Locus A, the chloroplast protein Magnesium Protoporphyrin-IX Chelatase H subunit, and the membrane-associated protein G Protein Coupled Receptor 2. Abscisic acid metabolism also has a new and prominent component with the identification of a beta-glucosidase capable of releasing biologically active abscisic acid from inactive abscisic acid-glucose ester in a stress-inducible manner. These observations refocus our attention on the metabolism underlying abscisic acid accumulation, sites of abscisic acid perception, and delivery of abscisic acid to those sites.     

Tracking and elucidating alphavirus-host protein interactions

Viral infections cause profound alterations in host cells. Here, we explore the interactions between proteins of the Alphavirus Sindbis and host factors during the course of mammalian cell infection. Using a mutant virus expressing the viral nsP3 protein tagged with green fluorescent protein (GFP) we directly observed nsP3 localization and isolated nsP3-interacting proteins at various times after infection. These results revealed that host factor recruitment to nsP3-containing complexes was time dependent, with a specific early and persistent recruitment of G3BP and a later recruitment of 14-3-3 proteins. Expression of GFP-tagged G3BP allowed reciprocal isolation of nsP3 in Sindbis infected cells, as well as the identification of novel G3BP-interacting proteins in both uninfected and infected cells. Note-worthy interactions include nuclear pore complex components whose interactions with G3BP were reduced upon Sindbis infection. This suggests that G3BP is a nuclear transport factor, as hypothesized previously, and that viral infection may alter RNA transport. Immunoelectron microscopy showed that a portion of Sindbis nsP3 is localized at the nuclear envelope, suggesting a possible site of G3BP recruitment to nsP3-containing complexes. Our results demonstrate the utility of using a standard GFP tag to both track viral protein localization and elucidate specific viral-host interactions over time in infected mammalian cells.     

Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication mitiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.     

Nuclear protein following heat shock: protein removal kinetics and cell cycle rearrangements

Nuclear protein and DNA content of HeLa cells was determined as a function of time following hyperthermia by staining isolated nuclei with two fluorescent dyes: fluorescein isothiocyanate (FITC) for protein content and propidium iodide (PI) for DNA content. Bivariate FITC and PI histograms were obtained by flow cytometry. Univariate flow cytometric analysis was shown to be inadequate for this study, because some of the nuclear protein changes were due to cell cycle redistribution. Posthyperthermia cell kinetics could be divided into two distinct phases: an early phase characterized by the removal of heat-induced excess nuclear proteins with little or no cell progression through the cell cycle; and a late phase characterized by a redistribution of cells in the cell cycle resulting in an accumulation of cells in G2. The duration of these phases was dependent upon the hyperthermia dose. In the early phase, the rate of removal of excess nuclear protein was found to vary with heating time and temperature for time-temperature combinations which resulted in the same amount of excess nuclear protein. In the late phase, the cells blocked in G2 did not reduce their nuclear protein levels back to control values.     

Nuclear lamina assembly in the first cell cycle of rat liver regeneration

The nuclear lamina is a fibrous structure at the nucleoplasmic surface of the inner nuclear membrane. Its assembly state is regulated by phosphorylation of its protein components, the lamins A, B, and C. The isoprenylation of the lamins is essential for their proper membrane anchoring and functionality. The content and the membrane association of nuclear lamins and the subcellular localization at light and electron microscopical levels were studied at different times of rat liver regeneration. This model for the good synchrony of the first cell cycle is particularly suited for the study of cell-cycle-dependent modifications and is particularly interesting for the increased protein prenylation found in S phase. The biochemical results show an increased lamin content in nuclear proteins in G1 phase and a decreased content in M phase, along with an enhanced cytosolic localization of A and C lamins at later stages. The morphological results show in M phase, also in nondividing cells, a decreased lamin-like immunoreactivity around the nucleus with an apparent nuclear lamina disassembly. These data emphasize the dynamic organization of nuclear lamina not only in mitosis but also in interphase. The reduction and partial solubilization of nuclear lamina in M phase suggest a reorganization of the nuclear envelope also in those cells that do not appear in mitosis but have replicated their DNA content that will result in a higher degree of polyploidy. J. Cell. Physiol. 171:135–142, 1997. © 1997 Wiley-Liss, Inc.     

Deficiency in nuclear accumulation of G22p1 and Xrcc5 proteins in hyper-radiosensitive Long-Evans Cinnamon (LEC) rat cells after X irradiation

The DNA-dependent protein kinase (DNA-PK) complex has been implicated in the repair of DNA double-strand breaks (DSBs). DNA-PK is a heterotrimeric protein complex comprised of two components: a large catalytic subunit, Prkdc, with serine/threonine kinase activity and a DNA-targeting component, G22p1 and Xrcc5. In previous report, we showed that approximately 80% of the G22p1 and Xrcc5 proteins were observed in the cytoplasm of rat fibroblasts, and that nuclear translocation of the proteins from the cytoplasm is important for the repair of DNA DSBs. In the present study, we showed that nuclear accumulation of the G22p1 and Xrcc5 proteins was not observed in fibroblasts from a mutant strain of Long-Evans Cinnamon (LEC) rat that has an enhanced radiosensitivity and a reduced level of repair of DSBs after X irradiation. Nuclear translocation of the proteins was observed in both LEC rat cells and control rat cells with normal radiosensitivity at 5 min after X irradiation. Although high levels of G22p1 and Xrcc5 proteins were observed in the nuclei of control rat cells until 60 min postirradiation, the amounts of the proteins decreased rapidly in the nuclei of LEC rat cells in the first 10 min after X irradiation. These findings suggest that there are some defects in maintaining the levels of G22p1 and Xrcc5 proteins in the nuclei of LEC rat cells. An analysis of fibroblasts from backcross rats showed that the deficiency in nuclear accumulation of G22p1 and Xrcc5 proteins is genetically linked to enhanced radiosensitivity. Since the nucleotide sequences of the G22p1 and Xrcc5 genes of the LEC rats coincided with those of the control rats, the deficiency in nuclear accumulation may not be caused by mutations of the G22p1 and Xrcc5 proteins.     

Two-dimensional electrophoresis of proteins of citric acid nuclei prepared with aid of a Tissumizer

Nuclei prepared from normal rat liver and Novikoff hepatoma ascites cells with the aid of a Tissumizer® in media containing 0.5 and 5 % citric acid were compared on the basis of electron microscopic appearance, DNA, RNA and protein content. Electron microscopy revealed better preservation of the nucleolar and nuclear morphology in the nuclei isolated in 0.5 % citric acid than in nuclei isolated in 5 % citric acid. Moreover, losses of protein and DNA from liver nuclei prepared by the sucrose-Ca²⁺ procedure were significantly less in nuclei treated with 0.5 % citric acid than in nuclei treated with 5 % citric acid. The preservation of nuclear morphology and the retention of the majority of types of nuclear protein were significantly better with the procedure using 0.5 % citric acid than with the procedure using 5 % citric acid. The 5 % citric acid treatment was found to alter nuclear morphology and extract specific nuclear proteins, as demonstrated by two-dimensional polyacrylamide gel electrophoresis of the proteins.     

Proteins in nucleocytoplasmic interactions. 3. Redistributions of nuclear proteins during and following mitosis in Amoeba proteus

The behavior of nuclear proteins in Amoeba proteus was studied by tritiated amino acid labeling, nuclear transplantation, and cytoplasmic amputation. During prophase at least 77% (but probably over 95%) of the nuclear proteins is released to the cytoplasm. These same proteins return to the nucleus within the first 3 hr of interphase. When cytoplasm is amputated from an ameba in mitosis (shen the nuclear proteins are in the cytoplasm), the resultant daughter nuclei are depleted in the labeled nuclear proteins. The degree of depletion is less than proportional to the amount of cytoplasm removed because a portion of rapidly migrating protein (a nuclear protein that is normally shuttling between nucleus and cytoplasm and is thus also present in the cytoplasm) which would normally remain in the cytoplasm is taken up by the reconstituting daughter nuclei. Cytoplasmic fragments cut from mitotic cells are enriched in both major classes of nuclear proteins, i.e. rapidly migrating protein and slow turn-over protein. An interphase nucleus implanted into such an enucleated cell acquires from the cytoplasm essentially all of the excess nuclear proteins of both classes. The data indicate that there is a lack of binding sites in the cytoplasm for the rapidly migrating nuclear protein. The quantitative aspects of the distribution of rapidly migrating protein between the nucleus and the cytoplasm indicate that the distribution is governed primarily by factors within the nucleus.     

Identification and localisation of a nucleoporin-like protein component of the plant nuclear matrix

Salt-detergent extraction of purified plant nuclei yields a fraction enriched in putative structural proteins known as the nuclear matrix. Compared with mammalian nuclear matrices, which contain three major proteins, plant nuclear matrices are complex, containing at least 100 polypeptides. In order to characterise more fully the plant nuclear matrix we have used antibodies raised against both yeast (Saccharomyces cerevisiae) and mammalian (rat) nuclear pore proteins. We have shown that the nuclear matrix of carrot (Daucus carota L.) contains at least one nucleoporin-like protein of about 100 kDa which is immunologically related to both the yeast nuclear pore protein NSP1 and mammalian nucleoporins (p62). Antibody labelling of a variety of plant cells at the light-microscope and electron-microscope levels confirms that this antigen is located at the nuclear pores. This, to our knowledge, is the first identification of a nuclear pore protein in plants.Abbreviations IgG immunoglobulin G - kDa kilodaltons - DAPI 4,6-diamidino-2-phenylindole - FITC fluorescein isothioganate The authors would like to thank Dr. E. Hurt (European Molecular Biology Laboratory, Heidelberg, FRG) for antibodies against yeast nucleoporins, and Dr. L. Davis (Whitehead Institute for Biomedical Research, Cambridge, Mass., USA) for the monoclonal antibodies MAb 414 & 350. We thank Brian Wells for useful advice on electron microscopy. We also thank Peter Scott, Andrew Davis, and Nigel Hannant for photography, and Sue Bunnewell for development and printing of electronmicrographs.     

Studies on gene structure, enzymatic activity and regulatory mechanism of acetohydroxy acid isomeroreductase from G2 pea

Using cDNA representational difference analysis (cDNA RDA), a cDNA whose expression was induced by gibberellins was cloned in our lab from G2 pea[1]. Sequence analysis showed that it shares high homology with acetohydroxy acid isomeroreductase (also known as ketol-acid reductoisomerase, EC1.1.1.86) in branched-chain amino acids biosynthetic pathway. Previous experiments confirmed that when expressed in E. coli cells, it was able to catalyze the reduction of AHB (2-aceto-2-hydroxybutyrat…     

Molecular Cloning of a Murine cDNA Encoding a Novel Protein, p38-2G4, Which Varies with the Cell Cycle

Proliferating cells express genes active in cell cycle control. The modulation of control genes and factors are required to maintain critical cell cycle activities. We used a set of monoclonal antibodies prepared against DNA-binding proteins from Ehrlich ascites tumor cells in immunofluorescent microscopy to screen for proteins showing cell cycle-specific staining patterns. Here, we report cloning and characterizing of a novel mitogen-inducible gene from murine macrophages that predicts a cell cycle-specifically modulated nuclear protein of 38 kDa, designated p38-2G4. p38-2G4 displayed a speckled pattern of varying fluorescence intensity confined to the nucleus, but sparing the nucleoli. Strongly stained granules were observed between G1 and mid S phase, followed by a less abundant punctated arrangement toward the end of S phase, and negative fluorescence at the S/G2 transition. Thereafter, the nuclear staining reappeared. Additionally, p38-2G4 expression vanished in G0-arrested cells and was restored after release from growth arrest. p38-2G4 conserved in vertebrates by means of immunofluorescence data contains a number of putative phosphorylation sites, a cryptic nuclear localization signal, and an amphipathic helical domain. Our cDNA and its deduced amino acid sequence is related to a Schizosaccharomyces pombe gene encoding a 42-kDa protein that associates with curved DNA, suggesting that we have cloned the murine homologue of the S. pombe gene which defines a novel cell cycle-specifically modified and proliferation-associated nuclear protein in mammals.     

The modulating effect of isoproterenol on DNA replication and protein synthesis. Synthesis patterns of the HMG proteins from electrostatically sorted salivary gland nuclei during the in vivo cell cycle

Within 96 h after initial isoproterenol administration, DNA replication and cell cycling were activated, as reflected in the bimodal distribution of nuclear fluorescence determined by flow-microfluorometric techniques. A group of proteins, the cetyltrimethylammonium bromide extractable nuclear proteins (CTAB-proteins), isolated form electrostatically sorted nuclei of rat salivary glands, was shown by staining and autoradiography after two-dimensional electrophoresis to undergo differential synthesis during various phases of the in vivo cell cycle after isoproterenol administration. Stained chromatographs revealed quantitative differences in protein synthesis. Gel autoradiography was a more sensitive technique than staining for detecting nuclear protein synthesis during cell cycling. As observed in the autoradiographs of the CTAB-proteins, isoproterenol initiated two distinct periods of protein synthesis in the salivary gland cell cycle: one during the 2C population G0/G1), and one during the 4C population (G2/M). Protein synthesis after isoproterenol administration was much more dramatic in the 2C (isoproterenol) population, where five new spots were seen. There was less radioactive incorporation in the 4C (isoproterenol) population. Two spots 'a' and 'b' that demonstrate differential protein synthesis in stained gel chromatographs and gel autoradiographs were shown to have electrophoretic mobilities, molecular weights and amino acid compositions highly similar to those of HMG1 and HMG2, respectively. A positive correlation could also be drawn between quantitative levels of 'a' and 'b' and their levels of incorporation during cellular activity with HMG (high mobility group) proteins. For example protein 'b' (HMG2) was consistently more abundant in proliferating cell populations than in the quiescent ones. Autoradiographic patterns of the CTAB-proteins indicated that proteins 'a' and 'b' were synthesized during the G0/G1 phase of the cell cycle, as were the majority of CTAB-proteins.