Nucleoprotein Fraction Research Articles

Detection of concentration-dependent conformational changes in SARS-CoV-2 nucleoprotein by agarose native gel electrophoresis

The nucleoprotein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is abundantly expressed during infection, making it a diagnostic target protein. We analyzed the structure of the NP in solution using a recombinant protein produced in E. coli. A codon-optimized Profinity eXact™-tagged NP cDNA was cloned into pET-3d vector and transformed into E. coli T7 Express. The recombinant protein was first purified via chromatographic step using an affinity tag-based system that was followed by tag cleavage with sodium fluoride, resulting in proteolytic removal of the N-terminal tag sequence. The digested sample was then loaded directly onto a size exclusion chromatography run in the presence of L-Arg-HCl, resulting in removal of host nucleic acids and endotoxin. The molecular mass of the main NP fraction was determined by mass photometry as a dimeric form of NP, consistent with the blue native PAGE results. Interestingly, analysis of the purified NP by our newly developed agarose native gel electrophoresis revealed that it behaved like an acidic protein at low concentration despite its alkaline isoelectric point (theoretical pI = 10) and displayed a unique character of concentration-dependent charge and shape changes. This study should shed light into the behavior of NP in the viral life cycle.

IMMUNE CELLS FUNCTIONING FEATURES IN INDIVIDUALS WITH AGGRESSIVE - AND DEPRESSIVE-LIKE BEHAVIORS

Background: Integrative interaction of basic homeostatic systems of the body suggests the influence of the immune system on the nervous system functions, including the formation of a certain behavioral stereotype. In this connection, the purpose of the study was evaluation functional activity of immune system’s cellular elements in experimental animals with various behavioral statuses. Methods: Experiments were held on 3-month-old male (CBAxC57BL/6) F1 mice with aggressive-like and depressive-like types of behavior. “Open field” test was used for the study the behavior in mice. Phenotype of spleen cells was measured by flow cytofluorometry with monoclonal antibodies against CD4, CD8, CD45, CD116, labeled by fluorochromes with different emission spectra. Proliferative response of splenocytes and thymocytes were evaluated by the standard method for inclusion in the nucleoprotein fraction of cells radiolabel (3H-thymidine); RT-PCR was used for determine cytokines mRNA level in splenocytes, splenocytes’ s cytokines intracellular level and extracellular production were research by ELISA. Results: It was shown, that (CBAxC57BL/6) F1 mice with different behavioral status differs also in the functional activity of immune cells. Thus, spleen and thymus cells of these animals were characterized by different spontaneous and mitogen-induced proliferative activity, different cytokines synthesis and production. The mechanisms of the influence of immune cells on the formation of behavior status are discussed. Conclusion: The data obtained testify different functional activity of immune system and its cells in mice with aggressive-like and depressive-like types of behavior, which may determine the behavior status of animals.

INVESTIGATING EUCALYPT DISEASES OF UNKNOWN ETIOLOGY: THE MUNDULLA YELLOWS EXPERIENCE

Diseases of eucalypts cause significant losses worldwide. Pathogens include Phytophthora spp., fungi, bacteria and phytoplasmas, but so far no viruses have been isolated or characterised from eucalypts despite observations of mosaic, chlorosis, leaf curl and leaf spot symptoms. A new lethal dieback disease of Australian eucalypts, known as Mundulla Yellows (MY) has emerged as a threat to natural vegetation, ancient iconic trees, natural wildlife habitats and conservation programs. Symptoms are an asymmetric progressive yellowing and slow dieback of the crown. Similar symptoms have been observed in eucalypts growing in Spain, Peru and Syria. No fungi, bacteria or phytoplasmas were found to be associated with MY. Traditional infectivity assays were unsuitable for testing whether MY is a virus-associated disease, so component analysis is being used to seek disease-associated nucleic acids or proteins which may implicate a virus-like agent in the etiology of MY. A number of physically distinct small RNAs were found preferentially in diseased trees, but none had sequences related to those of known viruses. Specific nucleoprotein fractions from MY affected trees contained disease-associated 84 kDa and 116 kDa proteins and quasi-spherical particles 30-40 nm in diameter. Limited sequence analysis of the 84 kDa protein and RNA also associated with the nucleoprotein fraction showed that neither had similarities to known plant viruses. It is concluded that the disease-associated nucleic acids and proteins could either be components of a previously undescribed type of intracellular plant pathogen, or host products resulting from pathogenic changes in affected trees. Targets for future research are the 30-40 nm particles, and the MY associated protein.

C-Reactive Protein: Eighty Years from Discovery to Emergence as a Major Risk Marker for Cardiovascular Disease

C-reactive protein (CRP)1 was discovered in 1930 by William Tillett and Thomas Francis from the Rockefeller University. They described a third serologic fraction, or “fraction C,” that could be isolated from patients infected with pneumococcus that was distinct from previously known capsular polysaccharide and nucleoprotein fractions detectable by specific antibody response (1). A decade later, Oswald Avery and Maclyn McCarty—the research team who originally described the “transforming principle” and the concept that genes are made of DNA—also described CRP as an “acute-phase reactant” that was increased in serum of patients suffering from a spectrum of inflammatory stimuli, including myocarditis and the inflammation associated with rheumatic fever(2)(3)(4). Early clues that this inflammatory biomarker might be linked to atherothrombosis are evident in 2 case reports presented by Gunnar Lofstrom from the State Bacteriologic Laboratory in Stockholm in 1943, in which increases in CRP following acute myocardial infarction are described(5). In the mid 1950s, case series presented by Irving Kroop and others indicated that CRP concentrations consistently increase after coronary ischemia and myocardial necrosis, data that was clinically important, as diagnostic tools for acute coronary syndrome did not yet include creatinine kinase or troponin(6). By the mid 1980s, the work of John Volanakis, Mark Pepys, Irving Kushner, and others had identified CRP as a hepatically derived, nonglycosylated, circulating pentraxin composed of 5 identical subunits arranged with pentameric symmetry that had characteristic calcium-dependent binding to specific ligands, including binding to LDL cholesterol(7)(8)(9)(10)(11)(12)(13). They and other investigators further demonstrated that the bulk of circulating CRP is produced by hepatocytes largely under regulatory control of inflammatory cytokines including interleukin-6 (IL-6) and tumor necrosis factor-α; that the plasma half-life of CRP is approximately 19 h under …

Fractionation by micrococcal nuclease digestion of Drosophila embryo chromatin: isolation of a fraction enriched in two major nonhistone proteins

Limited digestion of Drosophila melanogaster embryo nuclei with mitochondrial nuclease, followed by selective solubilization in 0.1 M NaCl, yields a soluble nucleoprotein fraction (S 3) enriched in two dominant protein bands of apparent molecular weight of 44000 and 48000. The analysis of the nucleosome monomer and multimer peaks, separated on sucrose gradients after slight digestion with micrococcal nuclease, shows that these proteins are associated with chromatin subunits, and that they are principally found in the subnucleosome region of fraction S 3. This doublet is tentatively identified as a member of the noncanonical HMG of Drosophila. The thermal denaturation and the circular dichroism spectra of the fraction soluble in 0.1 M NaCl (S 3) and insoluble fraction (P 3) show that both fractions are also structurally different.

Site directed in-vitro assembly of nucleosomes.

An in-vitro system is described allowing for the assembly of nucleosomes on preselected sites of a cloned tRNA gene. The system consists of a soluble nucleoprotein fraction, a histone source, and circular DNA containing a single stranded stretch (sssDNA). Nucleosomes assemble on the sssDNA, if the three components are incubated as a highly concentrated solution in the presence of the four deoxyribonucleotidetriphosphates and of ATP. The single stranded stretch is rendered double stranded during incubation. The middle axis of one assembled nucleosome always coincides roughly with the midpoint of the original single stranded DNA stretch.

Timing of the synthesis of empty shells and minor nucleoproteins in relation to turnip yellow mosaic virus synthesis in Brassica protoplasts

Following inoculation of Chinese cabbage protoplasts in vitro with turnip yellow mosaic virus, the development of characteristic changes in the chloroplasts was significantly asynchronous when observed by light or electron microscopy. In fully infected protoplasts there were only about 25% as many peripheral vesicles (the site of virus RNA synthesis) as in infected cells in the intact leaf. The yield of virus per infected protoplast was only about 1 10 th that found for an equivalent population of infected palisade cells from inoculated leaves. The production of empty protein shells was not reduced as much. Approximately equal amounts of virus nucleoprotein and empty protein shells were produced at all stages of the virus growth curve. However, if [ 35S]methionine was supplied when virus replication was well established, empty protein shells became labeled at an initial rate almost 10 times that of virus. Thus empty shells and virus must be assembled from differently located “pools” of protein subunits. The minor virus nucleoprotein fraction known to contain a substantial proportion of coat mRNA appears to be assembled earlier than the other minor nucleoprotein components, empty protein shells, or virus.

Behaviour of turnip yellow mosaic virus nucleoproteins under alkaline conditions

A modified CsCl density gradient procedure is described by which purified preparations of turnip yellow mosaic virus (TYMV) can be fractionated to give 12 nucleoprotein components. When the B 1 nucleoprotein is subjected to pH 11.5–11.6 in 1 M KCl (conditions under which artificial empty protein shells are formed), the apparent radius ( r) of the virus increases from about 14.6 to 15.2 nm within 30 sec. The increase in r is most probably due to swelling of the particles. Escape of the RNA from these particles is completed in 3–10 min, while RNA degradation continues for at least 30 min. On return to pH 7.0, r returns to about 14.6 nm. When any of the minor nucleoprotein fractions containing less than the full complement of RNA are subjected to the above conditions, the RNA does not escape from the particle. Experiments with the B 0 components also showed that these particles did not give an increase in r, although the RNA was degraded to about the same extent as that of the B 1 nucleoprotein.

Ultrastructural localization of specific nucleoprotein fractions

Bismuth (Bi) stains nucleoproteins (NPs) by interacting with available amino and primary phosphate groups. These two staining mechanisms are distinguishable by glutaraldehyde crosslinking (Fig. 1,2).Isolated mouse liver nuclei, extracted with salt and acid solutions, fixed in either formaldehyde (form.) or gl utaraldehyde (glut.) and stained with Bi, were viewed to determine the effect of the extractions on Bi stainina. Solubilized NPs were analyzed by SDS-polyacrylamide gel electrophoresis.Extraction with 0.14 M salt does not change the Bi staining characteristics (Fig. 3). 0.34 M salt reduces nucleolar (Nu) staining but has no effect on interchromatinic (IC) staining (Fig. 4). Proteins responsible for Nu and glut.- insensitive IC staining are removed when nuclei are extracted with 0.6 M salt (Fig. 5, 6). Low salt and acid extraction prevents Bi-Nu staining but has no effect on IC staining (Fig. 7). When nuclei are extracted with 0.6 M salt followed by low salt and acid, all Bi-staining components are removed (Fig. 8).

Size distribution and In vitro translation of the RNAs isolated from turnip yellow mosaic virus nucleoproteins

Purified preparations of TYMV contain a number of minor nucleoprotein components distinguishable on the basis of their density in CsCl gradients (R. E. F. Matthews, Virology 12, 521–539, 1960). When analysed by polyacrylamide-gel eletrophoresis, the RNA from each of these various components was found to consist of a characteristic set of molecular-weight species. Full-size RNA (2 × 10 6 daltons) was present only in nucleoproteins B 1 and B 2. Nucleoproteins B 0, B 00, and B 000 contained RNAs ranging in size from approx 1.3 to 0.28 × 10 6 daltons. The 0.28 × 10 6-dalton RNA species was detectable in all nucleoprotein fractions, but was a significant proportion of the RNAs of B 000 and B 00. Translation of the RNA from each of the nucleoproteins in the wheat germ cell-free protein synthesizing system yielded essentially similar patterns of polypeptides which ranged in size from 5000 to 70,000 daltons. The major radioactive product migrated on SDS-polyacrylamide gels to the same position as TYMV coat protein. The data suggest that the 0.28 × 10 6-dalton RNA component is the cistron for coat protein, and that it and other RNAs associated with TYMV infection are encapsidated.

Further studies on the RNA component distribution among the nucleoproteins of cucumber mosaic virus

Abstract Several attempts to obtain a more reliable definition of the RNA component distribution among the nucleoproteins of cucumber mosaic virus (CMV) are described. Preparative isopycnic centrifugation of formaldehyde-stabilized CMV could not be applied because CMV component cross-linking precluded its further dissociation by SDS or Pronase for RNA component analysis. However, in salt stability tests, preparations of CMV-D were found to be incompletely degraded in 2 M RbCl, and the undegraded virus had the same RNA component ratio as untreated CMV-D. The undegraded CMV-D was fractionated following isopycnic centrifugation in RbCl on a preparative scale. RNA 1 (1.07 million daltons) was found predominantly in the heavy nucleoprotein fractions, and RNA 2 (0.95 million daltons) in the light ones. RNA 3 (0.69 million daltons) and RNA 4 (0.33 million daltons) were found in slightly larger proportions in the middle density fractions. RNA 5 (about 0.1 million daltons) was almost evenly distributed among all nucleoprotein fractions. It was concluded that in CMV-D, with its RNA components basically distributed like brome mosaic virus, there are probably other types of nucleoproteins which have RNA 2 and RNA 3 combined with RNA 5, or RNA 5 molecules without the other components.

The selective extraction of histones from rye chromatin.

The selective extraction of histones from rye chromatin was studied using three different methods. Extractions with NaCl-phosphate buffers at pH5.5 gave results similar to those already obtained with other types of chromatin. Histone H1 was selectively extracted with 0.6 M NaCl-0.001 M PO4, while the selectivity of dissociation of the other fractions was reduced at higher NaCl concentrations. The use of phosphate-urea buffers at pH5.5 also revealed that the histones were dissociated at the same concentrations as were calf thymus histones. Histone H1 was extracted with 0.5 M PO4-1 M urea; H1, H2A, and H2B were extracted with 0.8 M PO4-2 M urea; and all histones were removed with 0.8 M PO4-5.3 M urea. It was, however, observed that the dissociated rye histones H2A and H2B were unstable in these buffers. This instability was maximum in the presence of 3 M urea, where both histones were absent from the extracted proteins and the residual nucleoproteins. Finally, a solution of 30% ethanol-0.35 M NaCl-6 M urea produced a rye nucleoprotein fraction containing only histone H1.

Some properties of TYMV nucleoproteins isolated in cesium chloride density gradients

The proportion of the noninfectious nucleoprotein with a normal amount of RNA (B 2 fraction) found in turnip yellow mosaic virus (TYMV) preparations following fractionation in CsCl density gradients, increased from about 15% to 50% as the pH of the CsCl solution was raised from 7.0 to 8.5. The other minor nucleoprotein fractions were not increased in this way. When the pH of the CsCl was controlled in the range 5.0–7.0, and the SW 40 rotor was used, double bands of each of the previously described nucleoprotein components were revealed. Thus ten nucleoprotein bands could be observed, two each for B 000, B 00, B 0, B 1, and B 2. The less dense band ( a band) of each pair was present in greater amounts. Both the bands found in the component previously shown to contain infectivity (B 1) were infectious. Mixing these isolated bands (B 1a and B 1b) did not enhance infectivity. Double bands were formed by some preparations of empty protein shells when these were subjected to centrifugation in CsCl of D = 1.27 g/cm 3. No consistent differences were found in the behaviour of the polypeptide from a and b bands on polyacrylamide gel electrophoresis.

Reactions of procaine amide-induced antinuclear antibodies with fractions derived from calf thymus nuclei.

Antinuclear antibodies induced by procaine amide (PrA) treatment reacted with various nucleoprotein fractions derived from calf thymus nuclei. Individual differences were found between the sera and, in one case, a strong reaction was obtained with a Sm fraction. These differences suggest that PrA-induced antinuclear antibodies are heterogenous.

Influence of Temperature and X-Irradiation on the Cellular Dynamics of the Intestinal Epithelium in Coho Salmon, Oncorhynchus kisutch

The intestinal epithelium of the Coho salmon was used to study the effects of temperature and x-irradiation on the cellular dynamics in a poikilothermic organism. Histological studies showed an absence of villi, and crypts in this system as only primary mucosal folds were observed. Autoradiographic studies following3 H-thymidine (3 H- TdR) labeling indicated that intestinal epithelial cell replication occurs in basal proliferation zones of the intestinal folds and the mucosal cells migrate the entire length of the folds before being sloughed into the lumen. The renewal rates of the intestinal epithelium are temperature dependent and estimated to be 14 ± 1, 24 ± 1, and > 35 days for 18°, 10°, and 5°C, respectively, as evidenced by autoradiographic and biochemical studies of3 H- TdR incorporation and turnover of the nucleoprotein fraction. The uptake of3 H- TdR into the nucleoprotein fraction was temperature dependent. Inhibition of3 H- TdR incorporation into intestinal nucleoprotein by x-rays was time and ...

Studies of immunosuppressive potency of rabbit antimouse antisera raised in response to various murine tissues

Antilymphocytic sera (ALS) capable of delaying rejection of A/J strain skin by C57BL/6J mice were produced in rabbits by the injection of (a) lyophilized thymic tissue from C57BL/6J mice, or (b) certain subcellular fractions of the thymus, including a crude nucleoprotein fraction and a crude insoluble fraction. Serum raised in response to a crude soluble fraction was ineffective. Sera produced by injecting rabbits intravenously with epidermal cells or intraperitoneally with fresh or lyophilized brain tissue from C57BL/6J mice failed to delay rejection of A-strain skin by C57BL/6J mice. All the sera tested were lymphocytotoxic in vitro, whether they were immunosuppressive or not, showing again that cytotoxicity in vitro is not a reliable indication of immunosuppressive action in vivo.

Induction of melanin synthesis in albino mouse skin by DNA from pigmented mice.

Skin cells from albino mouse embryos have been shown to synthesize melanin after exposure to a nucleoprotein fraction of cells from pigmented mice. Although transformed clones were not found in any of 37 control experiments, at least 15 transformations took place in 10 of a series of 49 experiments using undegraded DNA. The active component in the transforming material appears to be DNA because DNase-treated preparations did not induce melanin synthesis.

Contribution to the estimation of nucleic acids in wheat roots

Several methods of NA isolation and estimation were examined in order to determine RNA in the whole root systems of young wheat plants. It was found out that during the hydrolysis of roots with perchloric acid according toOgur andRosen (1950),Spirin (1958) andHeitefuss (1965) a considerable amount of orcine-positive compounds is released which cannot be adequate to the RNA content. Therefore the separate RNA determination in the presence of DNA was excluded even after the NA fractionation by hydrolysis at various temperature and perchloric acid concentration. Besides NA hydrolysates contained a high amount of other compounds absorbing in the UV-region. Compounds interfering with both these methods were present especially in the basal parts of roots. Using the method ofKern (1960) a nucleoprotein fraction was isolated containing about 75 per cent of the total NA content in the tissue. This fraction consisted of more than 90 per cent RNA combined with proteins, namely the ribosomal RNA. After the hydrolysis it could be estimated spectrophotometrically for the hydrolysate did not contain other compounds absorbing in the UV-region. In the root tips the content of this fraction was high in comparison with the basal parts of roots and it changed with the kind of nutrition.

An RNA-histone complex in mammalian cells: the isolation and characterization of a new RNA species.

Many lines of evidence indicate that all specialized cells contain the total genetic information characteristic of the organism, but the mechanisms involved in the selective expression of this total information are unknown. Stedman and Stedman' suggested that the histones, the basic proteins of the nueleus, could serve as the controlling mechanism. Most cells of an organism, however, have the same histones,', I and since the histones of individual organs such as liveer and brain do not change appreciably during development4 or during carcinogenesis,5' it seenms unlikely that the histones alone possess sufficient specificity for the control of gene function. Sypherd and Strauss7 suggested that an associated RNA species complementary to particular sequences of DNA could provide a mechanism for the histones to act as repressors by conferring additional specificity to these proteins. Huang and Bonner8 have recently described an RNA-histone complex in the pea seedling. We have concurrently carried out experiments on the nucleoprotein fraction isolated from normal rat liver nuclei and have found an RNA species with a high adenine and uridine content which is intimately associated with the nucleoprotein fraction and is resistant to ribonuclease. After extraction from the basic protein with phenol and detergent, this RNA species is RNase-sensitive and is heterogeneous by sucrose density gradient analysis. Materials and Methods.-Male Wistar rats weighing 100-120 gm from the Columbia University Colony were used in these experiments. They were fed rat pellets (A. E. Staley Manufacturing Co., Rockland Ill.) ad libitum. Labeled nucleoprotein: In the experiments to be reported, either the P32-labeled RNA-nucleoprotein complex or the nucleoprotein labeled with C14-lysine was used. For each preparation. 2-5 animals were injected intraperitoneally with neutral isotonic solutions containing either 2 mc of carrier-free p32 (Radiochemical Centre, Amersham, England) or 50 .Ac lysine-UL-C14 (NuclearChicago Corp., Des Plaines, Ill.) per 100-gm rat. The animals were allowed free access to food and water at all times. Three hours following p32 administration and 4 hr after the injection of lysine, liver nuclei were prepared and purified by a modification of the method of Maggio et al.9 With a Potter-Elvehjem homogenizer 9-12 gm of liver were homogenized in 3 vol of ice-cold buffer, pH 7.5, which consisted of 5 X 10-3 M1 Tris, 0.25 M sucrose, 5 mM MgC2, 1 mM aCl2, and 30 mM KCl. The homogenate was filtered through cheesecloth and centrifuged at 800 X g at 00 for 10 min. The crude nuclear pellet was washed two times with buffer and was repelleted after each wash at 800 X g for 10 min. After washing, the pellet was resuspended in a small volume of similar buffer containing 2 M sucrose and then layered over additional hypertonic buffer in a centrifuge tube. The contents of the upper third of the tube were stirred to distribute the nuclear sispension, which was then centrifuged for 1 hr in an SW 25:1 head at 25,000 rpm in a model L2 ultracentrifuge (Beckman Instruments, Inc., Palo Alto, Calif.) at 50. The supernatant was discarded, and the walls of the tube were cleansed of debris. The nuclear pellet was resuspended in buffer containing 0.25 M sucrose and then pelleted at 800 X g for 10 min. Following an additional wash and centrifugationi, the pellet was washed twice with 0.15 M NaCl, 0.01 M sodium citrate, pH 6.5.

Binding of Ca and Mg by lipoprotein and nucleoprotein subfractions of rat liver cell microsomes.

AbstractRat liver cell microsomes were subfractionated in D2O‐sucrose solutions of various densities into three subfractions with densities > 1.23, 1.18–1.23 and 1.12–1.18. There was an inverse correlation between the nucleic acid and lipid contents of these subfractions; the highest density subfraction had a relatively high nucleic acid (20.9%) and low lipid (15.1%) content and the lowest density subfraction had a low nucleic acid (3.4%) and a high lipid (45.2%) content. Whole microsomes were determined to be composed of 55.0% protein, 8.6% nucleic acid and 36.4% lipid. This composition of microsomes was calculated to reflect a nucleoprotein and lipoprotein content of 28% and 72%, respectively. Investigation of Ca and Mg binding by the three microsome subfractions and by the whole microsome fraction after equilibration for 12 hours in a medium of pH 6.7 containing 8–9 meq Ca or Mg/liter indicated that all fractions retained considerable amounts of the divalent cations (1.5–2.6 meq/g N). The nucleoprotein fraction was calculated to bind 0.35–0.40 meq Ca or Mg/g dry weight of nucleoprotein and the lipoprotein fraction 0.12 meq Ca or Mg/g dry weight of lipoprotein. Nevertheless, the lipoprotein fraction of microsomes is responsible for 45% of the total binding of Ca or Mg by the whole microsome fraction. These observations support the contention that cellular membrane structures, composed essentially of lipoprotein, can bind considerable quantities of the divalent cations.