Purification of viruses via CsCl gradient and ultracentrifugation v1

protocols.io ◽  
2015 ◽  
Author(s):  
Jozef Nissimov
Keyword(s):  
Cscl Gradient

scholarly journals A simplified CsCL protocol for lambda DNA purification: no enzymatic treatment/one phenol extraction

2000 ◽  
Vol 23 (1) ◽  
pp. 65-66 ◽  
Author(s):  
Roberto V. Santelli ◽  
Luci Deise Navarro-Cattapan
Keyword(s):  
Gradient Centrifugation ◽  
Enzymatic Treatment ◽  
Dna Purification ◽  
Phenol Extraction ◽  
Cscl Gradient ◽  
Centrifugation Method ◽  
Lambda Dna ◽  
Tedious Process

A modification of the CsCl gradient centrifugation method for DNA phage purification is presented. It avoids the enzymatic steps as well the need for a preliminary phage titration, a tedious process proposed in the majority of the protocols in use. The quality of the DNA obtained makes it amenable for additional manipulations like digestions, ligations, labelling, subcloning, etc.

scholarly journals Hepatitis B viral core proteins with an N-terminal extension can assemble into core-like particles but cannot be enveloped

1999 ◽  
Vol 80 (10) ◽  
pp. 2647-2659 ◽  
Author(s):  
Eric Ka-Wai Hui ◽  
Yong Shyang Yi ◽  
Szecheng J. Lo
Keyword(s):  
Hepatitis B ◽  
Kinase Activity ◽  
Core Protein ◽  
Surface Antigens ◽  
Human Hepatoma Cells ◽  
The Core ◽  
Core Proteins ◽  
B Virus ◽  
Transfection Medium ◽  
Cscl Gradient

The structure of hepatitis B virus (HBV) nucleocapsids has been revealed in great detail by cryoelectron microscopy. How nucleocapsids interact with surface antigens to form enveloped virions remains unknown. In this study, core mutants with N-terminal additions were created to address two questions: (1) can these mutant core proteins still form nucleocapsids and (2) if so, can the mutant nucleocapsids interact with surface antigens to form virion-like particles. One plasmid encoding an extra stretch of 23 aa, including six histidine residues, fused to the N terminus of the core protein (designated HisC183) was expressed in Escherichia coli and detected by Western blot. CsCl gradient and electron microscopy analyses indicated that HisC183 could self-assemble into nucleocapsids. When HisC183 or another similar N-terminal fusion core protein (designated FlagC183) was co-expressed with a core-negative plasmid in human hepatoma cells, both mutant core proteins self-assembled into nucleocapsids. These particles also retained kinase activity. Using an endogenous polymerase assay, a fill-in HBV DNA labelled with isotope was obtained from intracellular nucleocapsids formed by mutant cores. In contrast, no such signal was detected from the transfection medium, which was consistent with PCR and Southern blot analyses. Results indicate that core mutants with N-terminal extensions can form nucleocapsids, but are blocked during the envelopment process and cannot form secreted virions. The mutant nucleocapsids generated from this work should facilitate further study on how nucleocapsids interact with surface antigens.

scholarly journals Unstable association in vitro between donor and recipient DNA in Bacillus subtilis

1982 ◽  
Vol 152 (1) ◽  
pp. 275-283
Author(s):  
J Van Randen ◽  
K Wiersma ◽  
G Venema
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Gradient Centrifugation ◽  
Cross Linking ◽  
Dna Fragments ◽  
Dna Complexes ◽  
Cscl Gradient ◽  
Donor Moiety

In addition to stable donor-recipient DNA complexes, unstable complexes between donor and recipient DNA were formed in vitro with Bacillus subtilis. Whereas the stable complexes survived CsCl gradient centrifugation at pH 11.2 and phenol plus sodium p-aminosalicylate extraction with 0.17 M NaCl, the unstable complexes dissociated during these manipulations. The donor moiety from the unstable complexes remained associated with the recipient DNA during phenol plus sodium p-aminosalicylate treatment at 0.85 M NaCl. The unstable complexes could be stabilized artificially by cross-linking with 4,5',8-trimethylpsoralen. Dissociation of the complexes during CsCl gradient centrifugation could be prevented by centrifuging at pH 10. Heterologous DNA fragments derived from phage H1 DNA appeared to be unable to form complexes with the recipient B. subtilis DNA. Unstable complexes were also formed with Escherichia coli DNA, although under all conditions tested, more complex was detectable by using homologous B. subtilis DNA.

Isolation and characterization of a mitochondrial RNA polymerase from Drosophila melanogaster

1987 ◽  
Vol 65 (2) ◽  
pp. 173-182 ◽  
Author(s):  
Michael Goldenthal ◽  
James T. Nishiura
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Polymerase ◽  
Nuclear Dna ◽  
Gradient Centrifugation ◽  
Mitochondrial Rna ◽  
Mitochondrial Rna Polymerase ◽  
Isolation And Characterization ◽  
Cscl Gradient ◽  
Nuclear Rna

A DNA-dependent RNA polymerase was solubilized from sucrose gradient isolated, DNase-treated mitochrondria of Drosophila melanogaster. The isolated mitochondria were not detectably contaminated with nuclear DNA as shown by CsCl gradient centrifugation and polylysine Kieselguhr chromatography. The detergent-solubilized RNA polymerase was sensitive to rifampicin, resistant to α-amanitin, had an apparent molecular mass of about 60 kilodaltons, and displayed a tendency to aggregate, both in crude extracts or when purified. The mitochondrial RNA polymerase could be distinguished from nuclear RNA polymerases on the basis of size, salt optima, rifampicin sensitivity, and α-amanitin resistance.

scholarly journals Occurrence of Cryptosporidium and Giardia in Wild Ducks along the Rio Grande River Valley in Southern New Mexico

2002 ◽  
Vol 68 (1) ◽  
pp. 161-165 ◽  
Author(s):  
Ryan C. Kuhn ◽  
Channah M. Rock ◽  
Kevin H. Oshima
Keyword(s):  
New Mexico ◽  
Standard Deviation ◽  
Detection Limit ◽  
Giardia Lamblia ◽  
Fluorescent Antibody ◽  
Gradient Centrifugation ◽  
Rio Grande ◽  
Cesium Chloride ◽  
Cscl Gradient ◽  
Rio Grande River

ABSTRACT Fecal samples were taken from wild ducks on the lower Rio Grande River around Las Cruces, N. Mex., from September 2000 to January 2001. Giardia cysts and Cryptosporidium oocysts were purified from 69 samples by sucrose enrichment followed by cesium chloride (CsCl) gradient centrifugation and were viewed via fluorescent-antibody (FA) staining. For some samples, recovered cysts and oocysts were further screened via PCR to determine the presence of Giardia lamblia and Crytosporidium parvum. The results of this study indicate that 49% of the ducks were carriers of Cryptosporidium, and the Cryptosporidium oocyst concentrations ranged from 0 to 2,182 oocysts per g of feces (mean ± standard deviation, 47.53 ± 270.3 oocysts per g); also, 28% of the ducks were positive for Giardia, and the Giardia cyst concentrations ranged from 0 to 29,293 cysts per g of feces (mean ± standard deviation, 436 ± 3,525.4 cysts per g). Of the 69 samples, only 14 had (oo)cyst concentrations that were above the PCR detection limit. Samples did test positive for Cryptosporidium sp. However, C. parvum and G. lamblia were not detected in any of the 14 samples tested by PCR. Ducks on their southern migration through southern New Mexico were positive for Cryptosporidium and Giardia as determined by FA staining, but C. parvum and G. lamblia were not detected.

scholarly journals The proteoglycans of the human intervertebral disc

1975 ◽  
Vol 145 (3) ◽  
pp. 549-556 ◽  
Author(s):  
J H Emes ◽  
R H Pearce
Keyword(s):  
Intervertebral Disc ◽  
Analytical Ultracentrifugation ◽  
Salt Solutions ◽  
Heavy Component ◽  
Guanidinium Chloride ◽  
Nasal Cartilage ◽  
Cscl Gradient ◽  
Ultracentrifugal Analysis ◽  
Low Shear

The methods of Hascall & Sajdera (1969) were used to compare the proteoglycans of human intervertebral disc with those of bovine nasal cartilage. In contrast with cartilage, most of the hexuronate of disc could be extracted at low shear with water or dilute salt solutions. Extracts of disc with 4M-guanidinium chloride were centrifugated in 0.4M-guanidinium chloride in a CsCl gradient. Analytical ultracentrifugation of the hexuronate-containing heavy component revealed two fractions. both more polydisperse than those of cartilage. Also the more rapidly sediminting component was a much smaller fraction of the total. After prior extraction with 0.4M-guanidinium chloride, 4M-guanidinium chloride extracts of disc were found, by ultracentrifugal analysis, to be enriched in components resembling the proteoglycan monomer and aggregating factors of cartilage.

scholarly journals Efficacy and Pharmacokinetics of Bacteriophage Therapy in Treatment of Subclinical Staphylococcus aureus Mastitis in Lactating Dairy Cattle

2006 ◽  
Vol 50 (9) ◽  
pp. 2912-2918 ◽  
Author(s):  
J. J. Gill ◽  
J. C. Pacan ◽  
M. E. Carson ◽  
K. E. Leslie ◽  
M. W. Griffiths ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Bovine Mastitis ◽  
Treated Group ◽  
Microbial Infection ◽  
Cure Rate ◽  
Bacteriophage Therapy ◽  
Lactating Cows ◽  
Cscl Gradient ◽  
Serial Samples ◽  
Lactating Dairy Cattle

ABSTRACT Bovine mastitis is an inflammation of the udder caused by microbial infection. Mastitis caused by Staphylococcus aureus is a major concern to the dairy industry due to its resistance to antibiotic treatment and its propensity to recur chronically. Growing concerns surrounding antibiotic resistance have spurred research into alternative treatment methods. The ability of lytic S. aureus bacteriophage K to eliminate bovine S. aureus intramammary infection during lactation was evaluated in a placebo-controlled, multisite trial. Twenty-four lactating Holstein cows with preexisting subclinical S. aureus mastitis were treated. Treatment consisted of 10-ml intramammary infusions of either 1.25 × 1011 PFU of phage K or saline, administered once per day for 5 days. The cure rate was established by the assessment of four serial samples collected following treatment. The cure rate was 3 of 18 quarters (16.7%) in the phage-treated group, while none of the 20 saline-treated quarters were cured. This difference was not statistically significant. The effects of phage intramammary infusion on the bovine mammary gland were also studied. In healthy lactating cows, a single infusion of either filter-sterilized broth lysate or a CsCl gradient-purified phage preparation elicited a large increase in the milk somatic cell count. This response was not observed when phage was infused into quarters which were already infected with S. aureus. Phage-infused healthy quarters continued to shed viable bacteriophage into the milk for up to 36 h postinfusion. The phage concentration in the milk suggested that there was significant degradation or inactivation of the infused phage within the gland.

scholarly journals A Novel Open and Infectious Form of Echovirus 1

2016 ◽  
Vol 90 (15) ◽  
pp. 6759-6770 ◽  
Author(s):  
Mira Myllynen ◽  
Artur Kazmertsuk ◽  
Varpu Marjomäki
Keyword(s):  
Sucrose Gradient ◽  
Structural Changes ◽  
Dense Particle ◽  
Intermediate Particle ◽  
Receptor Interactions ◽  
Cscl Gradient ◽  
Gradient Separation ◽  
Domain I ◽  
Infectious Form ◽  
In Cells

ABSTRACTOne of the hallmarks of enterovirus genome delivery is the formation of an uncoating intermediate particle. Based on previous studies of mostly heated picornavirus particles, intermediate particles were shown to have externalized the innermost capsid protein (VP4) and exposed the N terminus of VP1 and to have reduced infectivity. Here, in addition to the native and intact particle type, we have identified another type of infectious echovirus 1 (E1) particle population during infection. Our results show that E1 is slightly altered during entry, which leads to the broadening of the major virion peak in the sucrose gradient. In contrast, CsCl gradient separation revealed that in addition to the light intact and empty particles, a dense particle peak appeared during infection in cells. When the broad peak from the sucrose gradient was subjected to a CsCl gradient, it revealed light and dense particles, further suggesting that the shoulder represents the dense particle. The dense particle was permeable to SYBR green II, it still contained most of its VP4, and it was able to bind to its receptor α2β1integrin and showed high infectivity. A thermal assay further showed that the α2β1integrin binding domain (I-domain) stabilized the virus particle. Finally, heating E1 particles to superphysiological temperatures produced more fragile particles with aberrant ultrastructural appearances, suggesting that they are distinct from the dense E1 particles. These results describe a more open and highly infectious E1 particle that is naturally produced during infection and may represent a novel form of an uncoating intermediate.IMPORTANCEIn this paper, we have characterized a possible uncoating intermediate particle of E1 that is produced in cells during infection. Before releasing their genome into the host cytosol, enteroviruses go through structural changes in their capsid, forming an uncoating intermediate particle. It was shown previously that structural changes can be induced by receptor interactions and, in addition, by heating the native virion to superphysiological temperatures. Here, we demonstrate that an altered, still infectious E1 particle is found during infection. This particle has a more open structure, and it cannot be formed by heating. It still contains the VP4 protein and is able to bind to its receptor and cause infection. Moreover, we show that in contrast to some other enteroviruses, the receptor-virion interaction has a stabilizing effect on E1. This paper highlights the differences between enterovirus species and further increases our understanding of various uncoating forms of enteroviruses.

scholarly journals Biosynthesis of respiratory-tract mucins. Incorporation of radioactive precursors into glycoproteins by canine tracheal explants in vitro

1973 ◽  
Vol 136 (4) ◽  
pp. 837-844 ◽  
Author(s):  
Daniel B. Ellis ◽  
Glenn H. Stahl
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sialic Acids ◽  
Equilibrium Density ◽  
Agarose Gels ◽  
Cscl Gradient ◽  
Tracheal Explants

1. Canine tracheal explants, cultured in medium 199, actively incorporated radioactive precursors into secreted macromolecules in vitro. 2. Puromycin, 6-diazo-5-oxo-l-norleucine and ouabain markedly inhibited the incorporation of these precursors. 3. Exogenous glucosamine at concentrations above 20mm caused a greater than 50% inhibition of the incorporation of l-[G-3H]fucose and l-[U-14C]serine. 4. Carbohydrate content of the purified secretions was approximately 50% and consisted principally of galactose, N-acetylglucosamine, N-acetylgalactosamine, fucose and sialic acids. 5. Chromatography on DEAE-cellulose and Bio-Gel A-150m and equilibrium density-gradient centrifugation in a CsCl gradient confirmed the presence of mucous glycoproteins. 6. Electrophoresis on 1% agarose gels gave profiles that were identical with canine respiratory mucus obtained in vivo. 7. These results support the utility of the explant system for studies of respiratory secretions.

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