Isolation and preliminary characterization of herpes Channel Catfish virus DNA

1980 ◽  
Vol 26 (2) ◽  
pp. 130-134 ◽  
Author(s):  
Jean Robin ◽  
Alice Rodrigue
Keyword(s):  
Channel Catfish ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Sedimentation Coefficient ◽  
Sodium Deoxycholate ◽  
Cesium Chloride ◽  
Equilibrium Density ◽  
Channel Catfish Virus ◽  
Phenol Extraction ◽  
Infected Cells

The DNA of Channel Catfish virus (CCV) was selectively extracted from infected cells with a 5% solution of sodium deoxycholate, deproteinized using sodium sarcosinate and pronase, and purified by phenol extraction followed by equilibrium density gradient centrifugation in a cesium chloride solution. CCV DNA displays a buoyant density of 1.715 g/cm3 in such a solution, as would be expected from a duplex DNA containing 56.1% of guanine plus cytosine. As estimated from both its sedimentation coefficient and length in the electron microscope, CCV DNA is a linear duplex molecule of approximately 85 × 106 daltons.

Purification and structure of Semliki Forest virus isolated from mouse brain

1968 ◽  
Vol 14 (2) ◽  
pp. 153-159 ◽  
Author(s):  
P. Faulkner ◽  
S. M. McGee-Russell
Keyword(s):  
Semliki Forest Virus ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Cesium Chloride ◽  
Equilibrium Density ◽  
Thin Sections ◽  
Virus Particles ◽  
Fundamental Symmetry ◽  
The Core ◽  
Intact Virus

Semliki Forest virus from brains of infected suckling mouse was separated by cesium chloride equilibrium density gradient centrifugation into two fractions, both able to agglutinate red blood cells of goose. The heavy fraction (HF density 1.24) had more infectivity, had a sedimentation coefficient of 285 S, and contained intact virus particles, diameter about 75 mμ. The light fraction (LF density 1.205) of equivalent HA (hemagglutinating activity), had less than 1% of the infectivity of the HF and did not contain intact virus particles. It consisted of "empty" virus particles and fragments of the fragile envelope material. The complete virus particle consists of an envelope of complex character surrounding a well-defined core. Shadow-cast preparations of HF show pointed and truncated shadows which suggest cubical symmetry, and the core of full particles, in thin sections, shows an angular, often pentagonal or hexagonal configuration. This suggests that the cubical symmetry of the full particle depends upon the fundamental symmetry of the core, which appears to be icosahedral.

Triton WR-1339 injected into rats enters renal renin granules

1988 ◽  
Vol 255 (3) ◽  
pp. E353-E356
Author(s):  
B. J. Morris
Keyword(s):  
Plasma Proteins ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Extracellular Fluid ◽  
Sucrose Density Gradient ◽  
Equilibrium Density ◽  
Juxtaglomerular Cells ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton Wr 1339 ◽  
Extracellular Substances

To test directly the possibility that substances in extracellular fluid can gain access to renin storage granules in renal juxtaglomerular cells, rats were injected with Triton WR-1339, which binds to plasma proteins. A heavy granule fraction was prepared, and isopycnic sucrose density gradient centrifugation was performed. The renin granule peak was found to be altered from a mean equilibrium density of 1.202 g/ml in control rats to 1.196 g/ml for rats injected with Triton WR-1339 (P less than 0.005). The distribution of angiotensinogen, which is bound in kidney granules having a different buoyant density, was also examined and found to be unaltered. After injection, Triton WR-1339 binds to circulating plasma proteins. The results for renin support the possibility of pinocytotic uptake of protein-Triton WR-1339 complexes by the juxtaglomerular cells with subsequent fusion of the endocytotic lysosomal vacuoles with renin granules accounting for the translocation of ingested substances into the granule matrix. If so, the potential would therefore exist for interaction(s) of ingested extracellular substances with renin or other components in the granules. The present study has therefore demonstrated directly that endogenous extracellular substances may enter renin granules.

A NON-NUCLEOTIDE POLYMER FOUND IN THE DNA OF THE SAND DOLLAR, ECHINARACHNIUS PARMA: II. PRELIMINARY CHARACTERIZATION

1967 ◽  
Vol 45 (2) ◽  
pp. 281-287 ◽  
Author(s):  
Herbert S. Rosenkranz
Keyword(s):  
Buoyant Density ◽  
Sedimentation Coefficient ◽  
Negative Electrode ◽  
Cesium Chloride ◽  
Amino Sugar ◽  
Electrophoretic Analysis ◽  
Positive Test ◽  
Preliminary Characterization ◽  
Acid Hydrolysate

A preliminary characterization of the non-nucleotidic component present in the DNA of Echinarachnius parma was undertaken. This material has an extremely high sedimentation coefficient (907 S). It contains no deoxyribose and presumably no ribose. After acid hydrolysis it was strongly ninhydrin-positive and also gave positive tests for reducing sugars as well as a slightly positive test for amino sugars. Upon electrophoretic analysis of an acid hydrolysate, three ninhydrinpositive spots were detected. One of these migrated to the negative electrode with a mobility identical with that of galactosamine, the other migrated to the positive electrode, and the third was neutral at pH 6.3. The spot with a mobility identical with that of galactosamine also gave a positive test for amino sugar. The material was not attacked by α-amylase. However, digestion with a crude trypsin preparation resulted in loss of the banding property in gradients of cesium chloride. Exposure to purified trypsin did not completely digest it, but caused an increase in buoyant density.

Die Bildung von RNS-Doppelstrang zur Vermehrung eines RNS enthaltenden Bakteriophagen

1964 ◽  
Vol 19 (7) ◽  
pp. 593-604 ◽  
Author(s):  
Hans Christian Kaerner ◽  
Hartmut Hoffmann-Berling
Keyword(s):  
Molecular Weight ◽  
Buoyant Density ◽  
Sedimentation Coefficient ◽  
Radioactive Material ◽  
Double Stranded Rna ◽  
Infected Cells ◽  
Rna Phage ◽  
Denaturation Profile ◽  
Molecular Weight Form ◽  
Latent Phase

The RNA phage fr induces in Escherichia coli cells the production of double stranded RNA, which is identified by its thermal denaturation profile ( Tm 101 °C in 0,2-m. Na⊕ ), by its nonreactivity with formaldehyde and by its buoyant density in Cs2SO4 (1,609 g cm-3 , compared to that of fr-RNA = 1,634 g cm-3 ). Unless denatured the double strand is resistant to RNase. In its high molecular weight form the double stranded R N A has twice the weight of fr-RNA, as estimated from the sedimentation coefficient (s20 = 14,5). The base ratios are those expected for a double stranded replicative from of fr-RNA. By melting and annealing one of the strands of the non-radioactive material can be exchanged for 32P-fr-RNA from phage particles. Infectiosity of the doublestranded RNAhas not yet been shown. Extracts from infected cells contain double strand bound to the 30 - 50 s fraction; there is also double strand in the supernatant, apparently in the form of relatively low molecular weight fragments. The double stranded RNA, isolated at the height of infection, accounts for 3 - 8% of the cellular RNA. Cells infected with 32P-fr show a surprisingly large part of the infecting RNA bound to ribosomes quite late in the latent phase. The meaning of this result is discussed.

Association of deoxyribonuclease-sensitive material with adenovirus penton aggregates after treatment of infected cell cultures with sodium deoxycholate

1971 ◽  
Vol 17 (8) ◽  
pp. 1009-1013 ◽  
Author(s):  
R. G. Marusyk ◽  
E. Norrby
Keyword(s):  
Infected Cell ◽  
Cell Cultures ◽  
Buoyant Density ◽  
Sodium Deoxycholate ◽  
High Density ◽  
Monomeric Form ◽  
Sensitive Material ◽  
Dna Labeling ◽  
Infected Cells ◽  
Simian Adenovirus

Sodium deoxycholate treatment of adenovirus-infected cell cultures was found to release rapidly sedimenting penton components with a buoyant density in CsCl of about 1.34 g/ml. The high-density penton material derived from human and simian adenovirus-infected cells occurred in monomeric form, while that derived from canine adenovirus-infected cells was in the form of non-symmetrical groupings of pentons aggregated at the vertex capsomere. Deoxyribonuclease treatment and 3H-thymidine-DNA labeling experiments revealed that the high-density of the pentons so obtained was due to a small associated segment of nuclease-sensitive material. A possible explanation of the nature of the nuclease-sensitive material is given.

Subunit composition of goblet-shaped particles from the cell wall of Flexibacter polymorphus

1983 ◽  
Vol 29 (12) ◽  
pp. 1689-1693 ◽  
Author(s):  
H. F. Ridgway ◽  
R. A. Lewin
Keyword(s):  
Cell Envelope ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Cesium Chloride ◽  
Protein Subunits ◽  
Nonionic Detergent ◽  
Gliding Bacterium ◽  
Triton X

Submicroscopic goblet-shaped particles (goblets) were released from the cell envelope of the marine gliding bacterium Flexibacter polymorphus when treated with the nonionic detergent Triton X-100 followed by sonication. The goblets were purified by cesium chloride density gradient centrifugation and exhibited an equilibrium buoyant density of 1.30 g/mL at 23 °C. They were composed of protein and a small amount of carbohydrate (approximately 3.4% by weight). Aqueous suspensions exhibited an absorption maximum in the ultraviolet at a wavelength of 276 nm and a smaller shoulder at 281 nm. Phospholipids were not detected in purified preparations of goblets, though they are known to be prominent constituents of the intact membranes of this microbe. Polyacrylamide gel electrophoresis of goblets solubilized in sodium dodecyl sulfate and 2-mercaptoethanol indicated four major polypeptide components ranging in molecular weight from 13 000 to 80 000. This number of different protein subunits corroborates earlier ultrastructural observations indicating a multisubunit composition.

scholarly journals Replication of kinetoplast DNA of Crithidia acanthocephali. I. Density shift experiments using deuterium oxide.

1976 ◽  
Vol 70 (2) ◽  
pp. 406-418 ◽  
Author(s):  
J E Manning ◽  
D R Wolstenholme
Keyword(s):  
Nuclear Dna ◽  
Deuterium Oxide ◽  
Gradient Centrifugation ◽  
Unit Length ◽  
Buoyant Density ◽  
Cesium Chloride ◽  
Kinetoplast Dna ◽  
Open Circular ◽  
Linear Molecules ◽  
Topological Interlocking

The protozoan Crithidia acanthocephali contains, within a modified region of a mitochondrion, a mass of DNA known as kinetoplast DNA (kDNA). This DNA consists mainly of an association of approximately 27,000 covalently closed 0.8-mum circular molecules which are apparently held together in a definite ordered manner by topological interlocking. After culturing of C. acanthocephali cells for 25 generations in medium containing 75% deuterium oxide, both nuclear DNA (rhonative, nondeuterated=1.717 g/cm3) and kDNA (rhonative, nondeuterated=1.702 g/cm3) increased in buoyant density by 0.012 g/cm3. The replication of the two DNAs was studied by cesium chloride buoyant density analysis of DNAs from exponentially growing cells taken at 1.0, 1.4, 2.0, 3.0, and 4.0 cell doublings after transfer of cells from D2O-containing medium into medium containing only normal water. The results obtained from analysis of both native and denatured nuclear DNAs indicate that this DNA replicates semiconservatively. From an analysis of intact associations of kDNA, it appears that this DNA doubles once per generation and that the newly synthesized DNA does not segregate from parental DNA. Fractions of covalently closed single circular molecules and of open circular and unit length linear molecules were obtained from associations of kDNA by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium gradient centrifugation. Buoyant density profiles obtained from these fractions indicate that: (a) doubling of the kDNA results from the replication of each circular molecule rather than from repeated replication of a small fraction of the circular molecules; (b) replication of kDNA is semiconservative rather than conservative, but there is recombination between the circles at an undefined time during the cell cycle.

scholarly journals Isolation and biochemical characterization of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density

1982 ◽  
Vol 203 (3) ◽  
pp. 683-689 ◽  
Author(s):  
H D Keiser ◽  
J B Adlersberg ◽  
H M Steinman
Keyword(s):  
Biochemical Characterization ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Exchange Chromatography ◽  
Equilibrium Density ◽  
Nasal Cartilage ◽  
Cartilage Proteoglycan ◽  
Sepharose 4B

Relatively homogeneous fractions of proteoglycan fragments were prepared from tryptic digests of the 4M-guanidinium chloride extract of bovine nasal cartilage. Glycosaminoglycan-containing fragments were separated from non-proteoglycan contaminants by ion-exchange chromatography and fractionated by equilibrium density-gradient centrifugation under dissociative conditions. The fractions of highest buoyant density were chromatographed on a column of Sepharose 4B, digested with chondroitinase ABC and chromatographed on a column of Sepharose 6B, yielding two distinct fractions: fraction B/6B-4 contained fragments from the chondroitin sulphate-bearing region of the proteoglycan monomer, and fraction B/6B-2 fragments from the keratan sulphate-rich region, most probably including a chondroitin sulphate-bearing monomer segment. By dansyl chloride analysis, fraction B/6B-2 had alanine and leucine as sole and fraction B/6B-4 had isoleucine and leucine as greatly predominant N-terminal amino acids, indicative of the relative homogeneity of these preparations of cartilage proteoglycan monomer fragments.

scholarly journals Identification of the Raji cell membrane-derived C1q inhibitor as a receptor for human C1q. Purification and immunochemical characterization.

1984 ◽  
Vol 160 (5) ◽  
pp. 1375-1389 ◽  
Author(s):  
B Ghebrehiwet ◽  
L Silvestri ◽  
C McDevitt
Keyword(s):  
Disulfide Bonds ◽  
Gradient Centrifugation ◽  
Raji Cell ◽  
Buoyant Density ◽  
Equilibrium Density ◽  
Macromolecular Complex ◽  
Density Fraction ◽  
Dependent Cell ◽  
Low Ionic Strength ◽  
Sepharose 4B

We have shown previously that an activity which is capable of precipitating purified C1q and inhibiting some of the C1q-dependent biologic reactions could be solubilized from the membranes of both normal human peripheral B lymphocytes and a B cell-derived lymphoblastoid cell line (Raji), both of which are known to possess receptors for human C1q. In this report we present evidence that this membrane-associated C1q inhibitor is a chondroitinase-insensitive macromolecule and is the receptor for human C1q. The receptor was solubilized from membranes of Raji cells with Nonidet P-40 and purified to homogeneity using C1q-Sepharose 4B affinity chromatography. Equilibrium density gradient centrifugation analysis revealed that the complex could be resolved into a protein-rich, low density fraction and a carbohydrate-rich, high density fraction. The large hydrodynamic size, coupled with the high buoyant density, suggests that a proteoglycan is a constituent of the complex and indicates that the receptor might be a macromolecular complex of a proteoglycan portion noncovalently linked to a 60-70 kD glycoprotein. The glycoprotein moiety, in turn, consists of two or more identical (70,000 mol wt) polypeptide chains held together by disulfide bonds and constitutes the C1q receptor (C1qR). Sucrose density ultracentrifugation analysis showed that the isolated receptor sediments with an apparent rate of 4.2 S. Immunochemical analyses demonstrated that a typical preparation of the C1qR complex consists of approximately 23% uronic acid and approximately 21% galactosamine with a galactosamine-to-glucosamine ratio of 3.2. Binding of C1q to the receptor was found to be optimal at low ionic strength and neutral or near-neutral pH (7-7.4). The isolated receptor was found to inhibit C1q hemolytic function, abrogate C1q-dependent rosette formation, and block the C1q-dependent, cell-mediated cytotoxicity, all of which are activities mediated by the receptor.

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