CsCl Density Gradient Centrifugation Research Articles

Glycogen in thermoacidophilic archaebacteria of the genera Sulfolobus, Thermoproteus, Desulfurococcus and Thermococcus

Glycogen has been found in thermoacidophilic archaebacteria of the genera Sulfolobus, Thermoproteus, Desulfurococcus and Thermococcus. Thermoplasma acidophilum yielded a related, though less defined compound. Glycogen was identified by elementary analysis, infrared spectroscopy, the nature of the hydrolysis products, the iodine reaction, and the nature of the products of periodate oxydation and reduction. The average chain length was 7. From crude extracts of Sulfolobus and Thermoproteus complexes of glycogen with 4 respectively 2 proteins have been isolated by CsCl density gradient centrifugation. In either case, one of the proteins was identified as glucosyl transferase. The glucosyl transferase of Sulfolobus acidocaldarius strain B 12 utilizes UDP-glucose as well as ADP-glucose as substrates, with K m values of 0.42 and 0.2 mM respectively and turnover numbers of 4.6 and 5.2 per second respectively. In electron micrographs the isolated glycogen protein complex appears as scale like aggregates, whereas in cell sections amorphous bodies fill large portions of the cells.

The composition and polymeric structure of mucus glycoprotein from human gallbladder bile

1. Human gallbladder bile was obtained from three patients with mixed-type gallstones undergoing cholecystectomy, and each patient sample was analysed individually. 2. Mucus glycoprotein was isolated by Sepharose 4B gel filtration followed by equilibrium density gradient centrifugation in CsCl. The purified glycoprotein was free from contaminant non-covalently bound protein as demonstrated by SDS-polyacrylamide gel electrophoresis. 3. The mucus glycoprotein was present in human bile at concentrations of 0.2–0.5 mg/ml. 4. Amino acid analysis of the purified glycoprotein was similar to that of other mucus glycoproteins in being rich in serine, threonine and proline, which accounted for 39% w/w of the total amino acids. 5. The hexosamine content of the glycoprotein depended on the blood group; patients with blood group O had a molar ratio of glucosamine:galactosamine of 4.0:1, and those with blood group A a molar ratio of 2.1:1. 6. Analysis of the mucus glycoprotein by gel filtration on Sepharose 2B showed that the major glycoprotein component was excluded. On treatment with 0.2 M mercaptoethanol or proteolytic enzymes, but not with 4 M guanidinium chloride, this excluded peak was broken down to a lower molecular weight glycoprotein component which eluted as a single included peak on gel filtration. 7. A protein of 70 000 molecular weight, identified by SDS-polyacrylanide gel electrophoresis, is released following reduction of the glycoprotein. 8. These results show (1) that human gallbladder mucus glycoproteins, in common with several other gastrointestinal mucus glycoproteins, consist of a polymeric structure of lower molecular weight glycoprotein units joined by disulphide bridges, and (2) that parts of the protein core are accessible to proteolysis, while other parts are glycosylated and protected from proteolysis, presumably by the carhohydrate side-chains. 9. Mucus glycoproteins from pig gallbladder bile were also shown to consist of large molecular weight polymers maintained by disulphide bridges and broken down by proteolysis.

Circular forms of viral DNA in Marek's disease virus-transformed lymphoblastoid cells.

The state of viral DNA in three Marek's disease virus (MDV)-transformed chicken cell lines (MDCC-MSB-1, MDCC-HP-2, MDCC-RP-1) was investigated by CsCl-density gradient centrifugation, and sedimentation analysis in neutral glycerol gradients. Each cell line contained MDV DNA integrated into the host cell genome. Additionally, free viral DNA could be identified in MDCC-MSB-1 and MDCC-HP-2 cells, sedimenting at about 100 S, and banding at the position of circular DNA in CsCl-ethidiumbromide gradients. Thus, MDV DNA with properties of circular plasmid DNA could be demonstrated in 2 virus-transformed cell lines. The significance of circular plasmid forms of viral DNA remains yet to be clarified, since these are apparently not regularly found in MDV-transformed cells.

Satellite DNA of Drosophila nasuta nasuta and D. n. albomicana: localization in polytene and metaphase chromosomes.

The DNA from the two Drosophila nasuta races, D. n. nasuta and D. n. albomicana was investigated by CsCl density gradient centrifugation. D. n. nasuta has one major AT-rich satellite DNA sequence with a density of 1.664 g/cm3, while D. n. albomicana has at least three satellites with densities of 1.674 g/cm3, 1.665 g/cm3 and 1.661 g/cm3. The isolated satellite sequences hybridize in situ to all heterochromatic regions of all metaphase chromosomes of both races. In polytene chromosomes the satellite sequences hybridize exclusively to the chromocenter. All chromosomal regions hybridizing with the satellites show also bright quinacrine fluorescence.

Characterization of DNA of the algaChlamydomonas geitleri Ettl

Preparations of total DNA isolated from algal cells ofChlamydomonas geitleri were characterized by routine methods (UV-spectra, thermal denaturation, sedimentation analysis, centrifugation in CsCl density gradients). In this way, the fundamental physicochemical characteristics, undetermined for this alga up to now, have been attained.The comparison of the characteristics of the preparations of DNA showed that during isolation it was more convenient to prefer procedures that use the lysis of cells. A mechanical disintegration may sometimes lead to the degradation of the macromolecules of DNA.The ascertained characteristics of DNA in the alga under study are compared here with the corresponding values of DNA known for the most widely studied volvocal algaChlamydomonas reinhardii as well as for other species of green algae. The greatest difference between the two species comparcd was in the contents of bases G + C. The determined values inChlamydomonas geitleri from 43.4 to 45.4 mol.% are much lower than the values known inChlamydomonas reinhardii (about 65.5 mol.%) or those published for some volvocal algae (60 to 68 mol.%).

Guanine-plus-cytosine content, hybridization percentages, and EcoRI restriction enzyme profiles of spiroplasmal DNA.

The guanine-plus-cytosine (G + C) content of spiroplasmal DNA was calculated from the melting temperature determined spectrophotometrically and the buoyant density determined by equilibrium density gradient centrifugation in CsCl. Only two ranges of G + C values were found: 25-27 mol% and 29-32 mol%. The DNA of the following spiroplasmas has 25-27 mol% G + C: Spiroplasma citri (serogroup I-1); the spiroplasmas pathogenic to the honeybee (KC3, BC3, and B63; serogroup I-2); the corn stunt strain (E275; serogroup I-3); the tick strain 277F (serogroup I-4); the drosophila strain (serogroup II); and one group of flower spiroplasmas (serogroup III). The DNA of a second group of flower spiroplasmas (serogroup IV) and the SMCA strain (serogroup V) has a G + C content of 29-31 mol/. The classification of flower spiroplasmas into two groups on the basis of G + C content agrees well with the groupings based on serologic and protein analysis. Spiroplasmas isolated from honeybees in Morocco (B13) or froghoppers in Corsica (L89) have 29-31 mol% G + C, a value that corroborates the relatedness of these strains and the flower spiroplasmas of serogroup IV found by serologic analysis. Reannealing experiments between the vivo-labeled DNA of S. citri and unlabeled DNA of other spiroplasmas gave the following percentages of hybridization: 64% with honeybee spiroplasma DNA, 49% with corn stunt spiroplasma DNA, and 19% with tick spiroplasma 277F DNA; no significant hybridization was observed with DNA of any other spiroplasma. The taxonomic position of the tick spiroplasma 277F within serogroup I was confirmed by hybridization experiments involving [3H]DNA of this strain. The value of polyacrylamide gel analysis of DNA fragments produced by the action of EcoRI restriction enzyme on DNAs from various spiroplasmas is also discussed.

Purification and stability of epizootic hemorrhagic disease virus

Purification of epizootic hemorrhagic disease virus (EHDV) from sonicated cell culture supernatant was effected using polyethylene glycol (PEG) precipitation and two CsCl density gradient centrifugations. During purification procedures the pH was maintained at values close to pH 8.0 as pH stability for EHDV was maximal between pH 7.2 and pH 9.O. During CsCl isopycnic centrifugation at pH 8.0, a 0.1 M or greater buffer was necessary to preserve maximum viral infectivity. This method of purification resulted in the recovery of 75% or more of the infectious virus contained in infected cell culture supernatants. For the extraction of infected cell-associated virus, sonication effectively liberated infectious virus, while freeze-thawing resulted in a significant loss of viral infectivity. The use of Freon allowed for complete recovery of infectious virus while ether or chloroform resulted in lower recoveries of infectious virus.

Transfection of Escherichia coli spheroplasts with a bacteriophage Mu DNA-protein complex.

We disrupted bacteriophage Mu particles by freeze-thaw treatment and recovered the DNA by CsCl density gradient centrifugation. This CsCl-purified DNA had a buoyant density which was indistinguishable from that of phenol-extracted Mu DNA. It was, however, 10(3) times more infective than phenol-extracted DNA for spheroplasts of exoV endI Escherichia coli. Infectivity was destroyed by proteinase K as well as by pancreatic DNase, indicating that the infective form was a DNA-protein complex. The infective properties of the complex demonstrated that the protein protects. Mu DNA against degradation by exonuclease V and that it serves at least one other function in bacteriophage Mu infection. The infectivity of the CsCl-purified DNA was due to a small class of highly infective molecules which sedimented 1.2. times faster than phenol-extracted Mu DNA on neutral sucrose gradients. This change in sedimentation rate is best explained by the formation of protein-linked circular monomers or linear dimers of Mu DNA. In vitro labeling of the DNA-protein complex, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed that the CsCl-purified DNA contained a noncovalently associated 65,000-dalton polypeptide. A 65,000-dalton protein was also found to be a minor component of the bacteriophage Mu particle. No protein was found in phenol-extracted Mu DNA. These results suggest that the 65,000-dalton protein is necessary for successful phage infection and is normally injected into the host cell with the Mu genome.

Indications for Changes in DNA Composition Correlated with Early Embryonic Differentiation (Triturus vulgaris, Urodela)*: (amphibia-Triturus vulgaris/embryogenesis/cell differentiation/DNA differential replication/gene amplification).

During early embryogenesis of the newt Triturus vulgaris (early gastrula to early neurula) the DNA was characterized by various methods of genome analysis. By preparative CsCl density gradient centrifugation an AT-rich satellite fraction (about 10% of total DNA) was found in all developmental stages studied. An additional GC-rich fraction, with a portion of about 3% of the genome, could be visualized only in the yolk plug stages. Filter hybridization experiments with labelled ribosomal RNA indicate that the number of rRNA cistrons is by a factor of 1.69±0.17 higher in the DNA of the mid-gastrula than of the tailbud stage. In ethidium bromide-CsCl gradients this additional (possibly amplified) rDNA bands at the same density as linear DNA. The analytical cleavage of DNA with ten restriction endonucleases reveals an extreme heterogeneity of the Triturus genome. The methylation pattern of DNA, studied with the aid of the isoschizomers Hpa II and Msp I, remains constant during early development. The reassociation kinetics of DNA, recorded spectrophotometrically, show that the portion of DNA that reassociates until Cot 10 increases significantly from 20% in the gastrula stages to 30% in the early neurula stage.

Fractionation of the liver membrane lipoprotein (LSP) and characterisation of its antigenic determinants by autoantibodies and a heterologous anti-serum.

Sera from 50 patients with chronic active liver disease were investigated by radioimmunoprecipitation test for autoantibodies against human and rabbit liver membrane lipoprotein (LSP), and a human kidney equivalent protein. Twenty-one of 50 chronic active liver disease sera were positive for autoantibodies against human liver membrane lipoprotein. Eleven of 50 sera reacted with rabbit liver membrane lipoprotein and four of 50 sera with human kidney equivalent protein as well. I order to identify the antigenic determinants of these autoantibodies 125I-labelled human and rabbit liver membrane lipoprotein and human kidney equivalent protein were fractionated by CsCl density gradient centrifugation. Distribution of labelled antigen fractions in density gradients revealed two peaks with maximum radioactivity at 1.34 and 1.11 g/ml. Density gradient fractions were assayed for antigen activity by radioimmunoprecipitation test using autoantibody positive sera of patients with chronic active liver disease. Anti-human and anti-rabbit liver membrane lipoprotein and anti-human kidney equivalent protein positive human sera all localised their corresponding determinants in the low density fractions (1.08-1.10 g/ml). An antiserum against human liver membrane lipoprotein, raised in rabbits after short-term immunisation, recognised the low density 125I-human liver membrane lipoprotein subfraction. This serum had previously been absorbed with human kidney homogenate, plasma, and blood cells. Thus, organ-specific and non-organ specific determinants were both localised in the low density liver membrane lipoprotein subfraction. They could not be separated by the described fractionation procedure. It is to be supposed that organ-specific determinants of a low density membrane protein of hepatocytes are targets circulating autoantibodies in chronic active liver disease.

The biosynthesis of glycoproteins by cultured bovine tendon fibroblasts.

Confluent bovine fetal tendon fibroblasts maintained in a chemically defined medium incorporated L-[6-3H]fucose and L-[5-3H]proline in a linear manner into non-diffusible macromolecules for up to 48 hrs. Equilibrium CsCl density gradient centrifugation indicated that [3H]fucose-labelled macromolecules released into the medium were predominantly glycoproteins. The [3H]fucose-labelled glycoproteins in the culture medium were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. This technique demonstrated the presence of a number of high mol. wt. fucosylated components, the most notable of which was a glycoprotein of apparent mol. wt. 150,000. Immunological procedures allowed the tentative identification of four glycoproteins including fibronectin which was found in the cell medium and in extracts of the cell layer. Two of the glycoproteins (mol. wts. 150,000 and 270,000) released into the incubation medium were shown to be related to the microfibrillar components of elastic tissue. One or more of the newly synthesized [3H]fucose labelled molecules was shown to be immunologically related to a glycoprotein (mol. wt. 60,000) extracted from bovine Achilles tendon. These studies represent the first demonstration of the synthesis of microfibril-related and tendon glycoprotein-related macromolecules by tendon fibroblasts in culture.

Heparan sulfate proteoglycan of cultured cells: Demonstration of a lipid- and a matrix-associated form

Two different forms of cell-associated [ 35S]-heparan sulfate proteoglycans were identified in prelabeled cultured cells, including glial cells, endothelial cells and fibroblasts. One of them migrated characteristically in the excluded volume fraction in Sepharose CL-2B chromatography and flotated in CsCl density gradient centrifugation. Further, it showed affinity for a hydrophobic gel, Octyl-Sepharose. The molecular size was markedly reduced and the density elevated by treatment with detergent or lipid solvents. These findings indicate an admixture of lipid in this proteoglycan and suggest a location for the molecule in the plasma membrane. This proteoglycan was found in all cell species examined. - The other type of heparan sulfate proteoglycan had a larger molecular size than most previously described heparan sulfate proteoglycans and had a buoyant density around 1.32 g/ml, probably due to an unusually high ratio of protein to carbohydrate. This heparan sulfate proteoglycan was found only in extracts of cells capable of forming a fibrillar extracellular matrix, but not in extracts of cells devoid of matrix. It was retained in cell-free preparations of extracellular matrix, indicating that it may be a specific product of this compartment.

Isolation and characterization of a proteodermatan sulfate from calf skin.

A proteodermatan sulfate was extracted from calf skin with 3 M MgCl2 at 4 degrees C in the presence of protease inhibitors and purified by DEAE-cellulose chromatography followed by CsCl density gradient centrifugation. The molecular weight of the proteoglycan was estimated to be approximately 115,000, based on the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteoglycan contains neutral sugars such as galactose, xylose, mannose, fucose, and glucose, but its amino acid composition is similar to that of proteodermatan sulfates from bovine heart valve, tendon, sclera, and pig and rat skin. The core protein is a glycoprotein with a molecular weight of 56,000 and seems to be monodisperse. However, the dermatan sulfate component, which is the only glycosaminoglycan constituent composed of 95% 4-sulfated disaccharide unit and 5% 6-sulfated disaccharide unit, seems to be polydisperse with a number average molecular weight of 17,000, as judged from both gel chromatography and chemical analysis. Analysis by beta-elimination and reduction of the dermatan sulfate-peptide(s) indicated that the glycan components are linked to the core protein through an O-glycosidic linkage between xylose and serine residues. From these results, we propose a model for the structure of calf skin proteodermatan sulfate that is composed of 3--4 chains of dermatan sulfate components which are covalently linked to a core (glyco-) protein via O-glycosidic linkages.

Deletion mutants of adenovirus 2: Isolation and initial characterization of virus carrying mutations near the right end of the viral genome

A series of eight deletion mutants of human adenovirus 2 have been isolated by a procedure which employs temperature-sensitive adenovirus mutants as helper viruses. Seven of these deletions are located in the right end of the viral genome and cover portions of early regions 3 and/or 4. The mutant with the largest deletion (807) has been separated from its helper by CsCl density gradient centrifugation. Measurements of the synthesis of viral DNA and late viral protein in 807-infected cells show that the mutant synthesizes DNA normally, and. that it produces reduced amounts of some, but not all, late proteins. These observations suggest that while the region 82.5–95 on the adenoviral genome contains no gene essential for DNA replication, it does include a gene or site whose function affects the synthesis of late proteins encoded by widely separated regions of the viral genome.

A 70000-molecular-weight protein isolated from purified pig gastric mucus glycoprotein by reduction of disulphide bridges and its implication in the polymeric structure.

The glycoprotein of pig gastric mucus has been isolated free of non-covalently bound protein as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and equilibrium density-gradient centrifugation. After reduction with 0.2 M-mercaptoethanol, protein was released from the glycoprotein, which consisted of a major 70000-mol.wt. component and a minor 60000-mol.wt. component. The 70000-mol.wt. protein fraction was separated from the reduced glycoprotein by either density-gradient centrifugation in CsCl or by gel filtration. Analysis of the 70000-mol.wt. protein fraction showed that, within the limits of the analysis, it was non-glycosylated, and its amino acid analysis was quite different from that of the reduced glycoprotein, which is high in serine, threonine and proline. There was a ratio of one 70000-mol.wt. protein per native glycoprotein molecule of 2 X 10(6) mol.wt. Dissociation of the native glycoprotein into glycoprotein subunits (5 X 10(5) mol.wt.) by reduction or proteolysis results in the release or hydrolysis respectively of the 70000-mol.wt. protein. A similar 70000-mol.wt. protein is demonstrated in human gastric mucus glycoprotein. A structural role for the proteins in these mucus glycoproteins is proposed.

Studies on human migration inhibitory factor: characterization of three molecular species.

Human migration inhibitory factor (MIF), obtained from supernatants of peripheral blood mononuclear cells stimulated with concanavalin A, was analyzed by gel filtration, isoelectrofocusing, and CsCl density gradient centrifugation. A distinct pattern of heterogeneity was determined on the basis of its harvesting time and biochemical criteria. Supernatants from cells cultured for 1 day contained a single peak of MIF activity with an isoelectric point of 4.3 to 5.2, an apparent m.w. of 23,000, and a density of 1.314 g/ml, the same as the density of the marker protein, 125I-HSA (1st day pH 5-MIF). Furthermore, this species of human MIF was sensitive to treatment with trypsin, strongly suggesting its being a protein, but not to treatment with neuraminidase and corresponds therefore to guinea pig pH 5-MIF. However, when 2nd day supernatants were analyzed under the same conditions, 2 MIF species were found. One species with an isoelectric point of 2.4 to 3.3 had an apparent m.w. of 65,000 (2nd day 3-MIF). The other species with an isoelectric point of 4.3 to 5.6 had an apparent m.w. of 23-43,000 (2nd day pH 5-MIF). Upon centrifugation in CsCl density gradients, the density (rho 25 of 1.314 to 1.414 g/ml) of both species was found to be greater than that of the pure protein, 125I-HSA. In addition, both species were chymotrypsin and neuraminidase sensitive but not trypsin sensitive, further suggesting their glycoprotein nature.

Purification and partial characterization of virus-like particles from Schneider line 2 Drosophila cells

A procedure has been developed for the purification of virus-like particles (VLPs) from Schneider line 2 Drosophila cells. The VLPs were precipitated with polyethylene glycol from the cytoplasmic fraction of lysed cells and further purified by equilibrium centrifugation in CsCl density gradients, in which they band at a density of 1.366 g/ml. Electron micrographs of these preparations revealed polyhedral particles with a diameter of 310–330 Å. We have also found particles of this size in thin sections of the intact cells. Sedimentation of the VLPs through 10–70% sucrose gradients yields a sedimentation coefficient of 235 S. Preliminary studies show that the VLPs contain double-stranded RNA species of 10 S, 14.5 S, 16 S, and 18 S.

Shedding of the major plasma membrane sialoglycoprotein from the surface of 13762 rat ascites mammary adenocarcinoma cells

ASGP-1 (ascites Sialoglycoprotein 1) the major sialoglycoprotein of 13762 rat ascites mammary adenocarcinoma cells, is shed from MAT-B1 (nonxenotransplantable) and MAT-C1 (xenotransplantable) sublines when incubated in vitro after labeling in vivo with [ 3H]glucosamine. The rates of shedding of label in both particulate and soluble form are similar for the two sublines, but the turnover of label in the cells is 80% greater for MAT-C1 cells ( t 1 2 2.4 days) than for MAT-B1 cells ( t 1 2 4.1 days). Shed soluble ASGP-1 was smaller than ASGP-1 in the particulate fraction by gel filtration in dodecyl sulfate. By CsCl density gradient centrifugation, gel filtration, and sucrose density gradient centrifugation, all in 4 m guanidine hydrochloride, the shed soluble ASGP-1 was found to be slightly more dense and smaller than ASGP-1 purified from membranes. No differences in sialic acid or oligosaccharides released by alkaline borohydride treatment were found between the shed soluble ASGP-1 and purified ASGP-1. These results suggest that the shed soluble ASGP-1 is released from the membrane by a proteolytic cleavage. This mechanism is supported by the inhibition of the release of soluble shed ASGP-1 by aprotinin, a protease inhibitor. Soluble ASGP-1 in ascites fluid is also smaller by gel filtration, but is more heterogeneous, suggesting a similar release mechanism in vivo followed by more extensive degradation in the ascites fluid.

Molecular Origin of Transducing DNA in Bacteriophage SPP1

Transducing particles produced by bacteriophage SPP1 infection of Bacillus subtilis were separated from plaque-forming units by CsCl density-gradient centrifugation. The density in CsCl of DNA isolated from such purified transducing particles was similar to that of bacterial DNA, indicating that the transducing particles probably contain DNA of exclusively bacterial origin. Bacterial DNA synthesized after infection of the donor culture was also encapsulated in the transducing particles. The number of transducing particles was at least 10 times higher than that of the transductants.

Isolation of the complementary strands of vaccinia virus DNA.

Separation of the complementary strands of vaccinia virus DNA by density centrifugation was attempted. Only non-cross-linked DNA molecules were used in these experiments. The best results were obtained using centrifugation in alkaline Cs2SO4 density gradients after denaturation of the DNA, followed by recycling of the isolated strands. Under these conditions, the heavy strand banded at a density of 1.480 g/cm3 and the light strand at 1.465 g/cm3. Incubation of denatured vaccinia virus DNA with ribopolymers, followed by centrifugation in CsCl density gradients, did not result in strand separation. Annealing experiments indicated that the strands isolated by alkaline Cs2SO4 density gradients were complementary.