Qualitative changes in DNA indicating differential DNA replication during early embryogenesis of the newt Triturus vulgaris

Development ◽  
1980 ◽  
Vol 57 (1) ◽  
pp. 61-70
Author(s):  
Klaus Lohmann ◽  
Lore Schubert
Keyword(s):  
Density Gradient ◽  
Thermal Denaturation ◽  
Significant Rise ◽  
Regular Shape ◽  
Normal Probability ◽  
Cscl Density Gradient ◽  
Triturus Vulgaris ◽  
Differential Dna Replication ◽  
Early Gastrula ◽  
Qualitative Changes

During gastrulation of the newt Triturus vulgaris considerable changes in the meltingbehaviour and in the CsCl density gradient pattern of DNA occur. The melting curves of DNAs from mid to late gastrulae (stages 11 b-12c) deviate from the regular shape. Whereasthe Tm values are identical in the stages 11 a (early gastrula) and 15 (early neurula), and correspond to the standard DNA (stage 36 = tailbud), a significant rise of Tm (0·8–1·2 °C) has been recorded in the stages 12 a/b (yolk plug). The differences in melting behaviour become visible by the deviation of the curves above Tm. These deviations from the normal sigmoidal shape are caused by the fact that a portion of DNA melts at higher temperatures than usual. Therefore the thermal denaturation of DNA is completed at approximately 3–4 °C later than in standard DNA. Both the derivative curves and the plots on normal probability paper demonstrate a heterogeneity of DNA in the stages 11 b-12c which indicates the presence of an additional GC-rich satellite fraction. These findings are confirmed by CsCl density gradient studies. Thus, in the stages 12a/b a slight shoulder on the heavy side of the gradients occurs, being absent in the other stages. From these facts we have to conclude that there is a stage-dependent multiplication and elimination of GC-rich sequences.

scholarly journals Interactions between different corneal proteoglycans

1978 ◽  
Vol 173 (3) ◽  
pp. 935-939 ◽  
Author(s):  
P Speziale ◽  
M S Speziale ◽  
L Galligani ◽  
C Balduini
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Original Sample ◽  
Chemical Analyses ◽  
Guanidinium Chloride ◽  
Keratan Sulphate ◽  
Cscl Density Gradient ◽  
Two Peaks ◽  
Fraction Density

Proteoglycans were extracted from bovine cornea with 4M-guanidinium chloride and purified by CsCl-density-gradient centrifugation. Under associative conditions two fractions were found: one capable of forming assemblies of high molecular weight and another lacking this property. The heavier fraction (density 1.59 g/ml) was eluted as a single retarded peak from Sepharose 2B, but on DEAE-Sephadex chromatography, gave two peaks: the first (eluted with 0.75 M-NaCl) contained mainly proteochondroitin sulphate and the second (eluted with 1.25 M-NaCl) mainly proteokeratan sulphate. Each of these proteoglycans was more retarded on Sepharose 2B than was the original sample from density-gradient centrifugation. Re-aggregation was obtained by recombination of the two fractions. The lighter fraction (density 1.44 g/ml), containing predominantly keratan sulphate chains, was eluted from DEAE-Sephadex as a single peak with 1.25 M-NaCl and was retarded on Sepharose 2B: this fraction was not able to form aggregates with proteochondroitin sulphate. Chemical analyses of the carbohydrate and protein moieties of the proteoglycans from DEAE-Sephadex confirmed that, in the cornea, different subunits are present with characteristic aggregation properties and hydrodynamic volumes.

scholarly journals Mucins secreted by a transformed cell line derived from human tracheal gland cells1

1997 ◽  
Vol 326 (2) ◽  
pp. 431-437 ◽  
Author(s):  
Jean-Marc LO-GUIDICE ◽  
Marc D. MERTEN ◽  
Geneviève LAMBLIN ◽  
Nicole PORCHET ◽  
Marie-Christine HOUVENAGHEL ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Cell Line ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
High Molecular Mass ◽  
Cscl Density Gradient

High-molecular-mass glycoconjugates are secreted by the continuous cell line MM-39, which has been obtained from cultured human tracheal gland cells transformed by simian virus 40. They were purified on Sepharose® CL-4B and then by two steps of density-gradient centrifugation. High-molecular-mass glycoproteins resistant to digestion by hyaluronidase, chondroitin ABC lyase and heparitinase were obtained, in addition to hyaluronic acid and proteoglycans. They were susceptible to β-elimination. They contained polylactosaminoglycan chains as well as carbohydrate chains with a terminal sialic acid in the NeuAc α2-3 sequence. Most of them have a buoyant density of 1.45 g/ml in CsCl-density-gradient centrifugation, except for MUC1. The MM-39 cells were also characterized by a high expression of MUC1 and MUC4 genes, but they did not express MUC2, MUC3, MUC5B and MUC5AC. Therefore the MM-39 cells synthesized mucin-like glycoproteins as well as lysozyme and mucous proteinase inhibitor [Merten, Kammouni, Renaud, Birg, Mattéi and Figarella (1996) Am. J. Respir. Cell. Mol. Biol. 15, 520–528]; they should be considered as having a mixed, both serous and mucous, phenotype.

Virus purification by CsCl density gradient using general centrifugation

2017 ◽  
Vol 162 (11) ◽  
pp. 3523-3528 ◽  
Author(s):  
Tadahiro Nasukawa ◽  
Jumpei Uchiyama ◽  
Satoshi Taharaguchi ◽  
Sumire Ota ◽  
Takako Ujihara ◽  
...  
Keyword(s):  
Density Gradient ◽  
Virus Purification ◽  
Cscl Density Gradient

scholarly journals Comparison of methods for purification of bacteriophage lysates of gram-negative bacteria for personalized therapy

2021 ◽  
Author(s):  
RB Gorodnichev ◽  
MA Kornienko ◽  
NS Kuptsov ◽  
AD Efimov ◽  
VI Bogdan ◽  
...  
Keyword(s):  
Density Gradient ◽  
Sucrose Gradient ◽  
Sucrose Density Gradient ◽  
Density Gradient Ultracentrifugation ◽  
Phage Lysate ◽  
Purification Method ◽  
Cscl Density Gradient ◽  
Cell Components ◽  
Purification Methods ◽  
Sucrose Gradient Ultracentrifugation

Phage therapy is a promising method of treating antibiotic-resistant infections. To obtain a safe therapeutic formulation, bacterial cell components, including endotoxins, must be removed from the phage lysate. This study was aimed at comparing the efficacy of purification methods for phage lysates intended for therapeutic use. Phages vB_KpnM_Seu621 (Myoviridae) and vB_KpnP_Dlv622 (Autographiviridae) were grown using the KP9068 strain of Klebsiella pneumoniae as a host. The obtained lysates were purified using phage precipitation with polyethylene glycol, CsCl density gradient ultracentrifugation, sucrose density gradient ultracentrifugation, precipitation with 100 kDa centrifugal filter units, and phage concentration on 0.22 µm cellulose filters in the presence of MgSO4. Endotoxin concentrations were determined by LAL testing. The obtained lysates contained 1.25 × 1012 ± 7.46 × 1010 and 2.25 × 1012 ± 1.34 × 1011 PFU/ml of vB_KpnM_Seu621 and vB_KpnP_Dlv622, respectively, and had endotoxin concentrations of 3,806,056 ± 429,410 and 189,456 ± 12,406 EU/ml, respectively. CsCl gradient ultracentrifugation was found to be the optimal conventional purification method in terms of reducing endotoxin concentrations and maintaining phage titers (303 ± 20 — 313 ± 35 EU/ml, 1.5–2.75 × 1012 ± 1.71 × 1011 PFU/ml). Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 were found to be the optimal non-traditional purification methods. A method for phage lysate purification should be selected for each phage preparation individually. Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 hold promise as purification methods that can produce phage preparations suitable for intravenous administration.

Isolation and Structural Studies of “A” Particles from Leydig Cell Tumors

1972 ◽  
Vol 30 ◽  
pp. 78-79
Author(s):  
Nurul H. Sarkar ◽  
Bernhard Kramarsky ◽  
Dan H. Moore
Keyword(s):  
Density Gradient ◽  
Leydig Cell ◽  
High Speed ◽  
Structural Studies ◽  
Low Speed ◽  
Cscl Density Gradient ◽  
Triton X ◽  
Leydig Cell Tumors

The presence of large inclusions of intracytoplasmic A particles (Fig. 1) in Leydig cell tumors has been described (1,2). These A particles were extracted from minced tumors by homogenizing them twice in 5 volumes of SET buffer (0.2M sucrose, 0.001M EDTA and 0.01M Tris HCl, pH 7.2). The extract was clarified by low speed centrifugation and treated with one tenth volume of 5% Triton X-100 in SET followed by one tenth volume of 1M NaCl. The resulting mixture was again clarified by low speed centrifugation and the supernatant was treated with DNAse following high speed centrifugation. The pellet was resuspended in SET, layered over a preformed CsCl density gradient and centrifuged in a Spinco SW 39 rotor.

scholarly journals Deoxyribonucleic acid synthesis in isolated nuclei from baby-hamster kidney cells (BHK-21/C13). Characterization of the system

1979 ◽  
Vol 178 (3) ◽  
pp. 613-620 ◽  
Author(s):  
Julian F. Burke ◽  
Colin K. Pearson
Keyword(s):  
Dna Synthesis ◽  
Density Gradient ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Tween 80 ◽  
Proteinase K ◽  
Baby Hamster Kidney ◽  
Isolated Nuclei ◽  
Brij 58 ◽  
Cscl Density Gradient

A comparative study of some commonly employed laboratory procedures for studying DNA synthesis in isolated nuclei was carried out. Nuclei isolated from baby-hamster kidney (BHK-21/C13) cells synthesize DNA for 30–60min at 37°C in a reaction requiring uni- and bi-valent cations, ATP and all four deoxyribonucleoside 5′-triphosphates. The addition of either ribonucleotides or cytosol from S-phase cells had no effect, but DNA synthesis was stimulated by some dextrans (mol.wt. 5×106). The extent of synthesis was influenced by apparently minor variations in experimental conditions. For example, DNA synthesis by nuclei in Tris/HCl, pH7.5, was only 50% of that observed in Hepes/NaOH, pH7.5; the presence of detergents Triton X-100, Triton N-101, Nonidet P-40, Brij 58 and Tween 80 in the incubation medium altered the amount of synthesis to different extents. Although most detergents inhibited synthesis, a stimulation occurred with Tween 80 (150% of controls). These effects were reversed on washing the nuclei, except that of Brij 58, which inhibited DNA synthesis by 90–95% irreversibly. Anomalous sucrose-density-gradient sedimentation behaviour of the DNA, and of precursor [3H]-dTTP, was observed when nuclei were lysed with solutions of sodium dodecyl sulphate/Mg2+ or with Sarkosyl/Mg2+, but consistent results, showing that the DNA synthesized in vitro sedimented exclusively at about 4S, were obtained when nuclei were lysed with sodium dodecyl sulphate (without Mg2+)/EDTA, digested with proteinase K and heated at 100°C with 11% (v/v) formaldehyde to prevent macromolecular association. These results, coupled with density-labelling studies with bromodeoxyuridine and CsCl-density-gradient analysis, showed that DNA synthesis in these nuclei was replicative and was restricted to a covalent extension of Okazaki pieces previously initiated in vivo. No new initiations were observed, and the DNA was not ligated into larger molecules. The cessation of DNA synthesis after about 60 min was due to the complete utilization of available primer/template DNA.

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