scholarly journals DNA Extraction from 0.22 μM Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

10.3791/1352 ◽  
2009 ◽  
Author(s):  
Jody J. Wright ◽  
Sangwon Lee ◽  
Elena Zaikova ◽  
David A. Walsh ◽  
Steven J. Hallam
Keyword(s):  
Density Gradient ◽  
Dna Extraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Cesium Chloride

Studies on the Cytotoxicity of Myleran and Dimethyl Myleran

1972 ◽  
Vol 50 (10) ◽  
pp. 1074-1081 ◽  
Author(s):  
M. P. Mitchell ◽  
I. G. Walker
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Alkylating Agents ◽  
Gradient Centrifugation ◽  
Cesium Chloride ◽  
Cross Linking ◽  
Alkaline Denaturation ◽  
Dna Strands ◽  
Cross Links

Myleran and dimethyl Myleran (DMM) are two potentially bifunctional alkylating agents which might be expected to form cross-links between guanine residues on the same or opposite strands of native DNA. When L cells were treated with these agents DMM was considerably more toxic than Myleran. When DNA, RNA, and protein were subsequently isolated and analyzed it was found that both agents reacted with DNA to the same extent, the degree of labelling being linear with the concentration of the agents. RNA and protein were labelled to a rather greater extent. When DNA was analyzed chromatographically, no evidence was found for the formation of alkylated guanine residues after treatment with DMM. With Myleran, a product corresponding to the N-7 alkylated monoguaninyl derivative was formed, but no diguaninyl product was detected. Similar results were obtained when DNA was treated in vitro. No evidence of cross-linking by either agent was found when DNA treated in vitro was subjected to alkaline denaturation and subsequent renaturation, and then analyzed by cesium chloride density gradient centrifugation. It is concluded that neither Myleran nor DMM causes cross-linking of DNA strands, nor do they form links between adjacent guanine residues in one strand of DNA.

scholarly journals Proof-of-concept for the yadokari nature: a capsidless replicase-encoding but replication-dependent (+)ssRNA virus hosted by an unrelated dsRNA virus

2021 ◽  
Author(s):  
Subha Das ◽  
Md Mahfuz Alam ◽  
Rui Zhang ◽  
Sakae Hisano ◽  
Nobuhiro Suzuki
Keyword(s):  
Density Gradient ◽  
Reverse Genetics ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Expression System ◽  
Cesium Chloride ◽  
Dsrna Virus ◽  
Equilibrium Density ◽  
Proof Of Concept ◽  
Ssrna Virus

We have previously proposed a new virus lifestyle or yadokari/yadonushi nature exhibited by a positive-sense ssRNA virus, yadokari virus 1 (YkV1), and an unrelated dsRNA virus, yadonushi virus 1 (YnV1) in a phytopathogenic ascomycete, Rosellinia necatrix . We have proposed that YkV1 diverts the YnV1 capsid to trans-encapsidate YkV1 RNA and RNA-dependent RNA polymerase (RdRp) and replicate in the heterocapsid. However, it remains uncertain whether YkV1 replicates using its own RdRp, and whether YnV1 capsid co-packages both YkV1 and YnV1 components. To address these questions, we first took advantage of the reverse genetics tools available for YkV1. Mutations in the GDD RdRp motif, one of the two identifiable functional motifs on the YkV1 polyprotein, abolished its replication competency. Mutations were also introduced in the conserved 2A-like peptide motif, hypothesized to cleave the YkV1 polyprotein co-translationally. Interestingly, the replication proficiency of YkV1 mutants in the host fungus agreed with the cleavage activity of the 2A-like peptide tested using a baculovirus expression system. Cesium chloride equilibrium density gradient centrifugation allowed for the separation of particles, with a subset of YnV1 capsid solely packaging YkV1 dsRNA and RdRp. These results provide proof-of-concept that a capsidless (+)ssRNA virus is hosted by an unrelated dsRNA virus. Importance Viruses typically encode their own capsids that encase their genomes. However, a capsidless (+)ssRNA virus, YkV1, depends on an unrelated dsRNA virus, YnV1, for encapsidation and replication. We have previously shown that YkV1 highjacks the capsid of YnV1 for trans-encapsidation of its own RNA and RdRp. YkV1 was hypothesized to divert the hetero-capsid as the replication site, as is commonly observed for dsRNA viruses. Herein, mutational analyses showed that the RdRp and 2A-like domains on the YkV1 polyprotein are important for its replication. The active RdRp must be cleaved by a 2A-like peptide from the C-proximal protein. Cesium chloride equilibrium density gradient centrifugation allowed for the separation of particles, with YnV1 capsid solely packaging YkV1 dsRNA and RdRp. This study provides proof-of-concept of a virus neo-lifestyle where a (+)ssRNA virus snatches capsids from an unrelated dsRNA virus to replicate with its own RdRp, thereby mimicking the typical dsRNA virus lifestyle.

Uranyl Acetate and Rotary Shadowing to Increase the Contrast of Small Aggregated Virus Particles

1969 ◽  
Vol 27 ◽  
pp. 424-425
Author(s):  
Lee F. Ellis ◽  
Richard M. Van Frank ◽  
Walter J. Kleinschmidt
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Uranyl Acetate ◽  
Interferon Inducer ◽  
Virus Particles ◽  
Staining Methods ◽  
Rotary Shadowing

The extract from Penicillum stoliniferum, known as statolon, has been purified by density gradient centrifugation. These centrifuge fractions contained virus particles that are an interferon inducer in mice or in tissue culture. Highly purified preparations of these particles are difficult to enumerate by electron microscopy because of aggregation. Therefore a study of staining methods was undertaken.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

Sign in / Sign up

Export Citation Format

Share Document