Plasmid detection in a bacteriocinogenic strain of Clostridium perfringens

1980 ◽  
Vol 26 (8) ◽  
pp. 1018-1022 ◽  
Author(s):  
A. W. Li ◽  
P. J. Krell ◽  
D. E. Mahony
Keyword(s):  
Clostridium Perfringens ◽  
Plasmid Dna ◽  
Gel Electrophoresis ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Size Distributions ◽  
Agarose Gel Electrophoresis ◽  
Circular Dna ◽  
Molecular Weights

Bacteriocinogenic Clostridium perfringens, strain 28, harboured plasmid DNA detectable by dye-buoyant density-gradient centrifugation. This plasmid DNA was absent from an ultraviolet light cured variant which had simultaneously lost its immunity and ability to produce bacteriocin. Agarose gel electrophoresis of the plasmid DNA revealed at least six bands but denaturation experiments suggested three plasmids occurring in more than one conformation. Electron microscopy revealed three major size distributions of circular DNA of molecular weights 1,5.6, and 7.1 megadaltons. Some evidence suggests that the 5.6 megadalton plasmid may control bacteriocin 28 production.

Chromosome age and segregation during sporulation of Bacillus megaterium

1978 ◽  
Vol 24 (10) ◽  
pp. 1227-1235 ◽  
Author(s):  
Anthony D. Hitchins
Keyword(s):  
Bacillus Megaterium ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Growth Period ◽  
Circular Dna ◽  
Chromosomal Segregation ◽  
Seven Generations ◽  
Minor Correction

The effect of chromosome age on segregation during sporulation was investigated. Vegetative cells of Bacillus megaterium were labeled with [Me-3H]thymine and then were grown at 30 °C in nonradioactive medium for various times before being allowed to sporulate. The ratio of the amount of label in sporal DNA to that in sporangial DNA, obtained after minor correction for the sporulation frequency, remained essentially constant as the postlabeling growth period was increased from one to seven generations. The spores were preferentially located at the older poles of sporangia, i.e. the poles formed by divisions occurring prior to those forming the sporangia. Therefore, it seems that old (labeled) chromosomes segregate randomly with respect to both the morphological and genealogical polarities of sporangia. Examination of total cell lysates by dye–buoyant density gradient centrifugation revealed the presence of covalently closed circular DNA from cells grown at 37 °C, but none was obtained from cells grown at 30 °C. Thus, possible interference by large amounts of extrachromosomal DNA in the determination of the chromosomal segregation pattern is unlikely.

Study of tobacco mosaic virus in vitro disassembly by sucrose density gradient centrifugation and agarose gel electrophoresis

1984 ◽  
Vol 62 (3) ◽  
pp. 457-462 ◽  
Author(s):  
Richard Hogue ◽  
Alain Asselin
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Density Gradient ◽  
Gel Electrophoresis ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Sucrose Density Gradient Centrifugation

In vitro disassembly of tobacco mosaic virus (TMV) strains U1, U2, U4, U6, and U7 with alkali and urea was studied by sucrose or sucrose–dimethylsulfoxide (DMSO) density gradient centrifugation and by agarose gel electrophoresis. All strains gave similar decapsidation patterns with both agents when partially stripped virus particles (PSVs) were analyzed by sedimentation and electrophoresis. However, U6 was more sensitive to decapsidation than the other strains and U2 exhibited resistance to decapsidation. Agarose gel electrophoresis of TMV decapsidation products allowed the detection of several classes of PSVs in addition to aggregation products involving PSVs and monomer particles. Agarose gel electrophoresis is thus very rapid and useful for analysis of TMV disassembly products especially when aggregation phenomena and kinetic studies with numerous samples are considered.

scholarly journals Covalently closed, circular DNA in kappa endosymbionts ofParamecium

1976 ◽  
Vol 27 (2) ◽  
pp. 161-170 ◽  
Author(s):  
Judith A. Dilts
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Bacterial Chromosome ◽  
Contour Length ◽  
Paramecium Tetraurelia ◽  
Extrachromosomal Dna ◽  
Circular Dna ◽  
Covalently Closed Circular Dna ◽  
Circular Molecule

SUMMARYCaedobacter taeniospiralis(kappa), a bacterial endosymbiont isolated fromParamecium tetraureliastock 51, contains, in addition to the bacterial chromosome, covalently closed circular DNA molecules as shown by isolation on dye-buoyant-density gradients. The closed circular molecule has a contour length of 13·75 ± 0·04 µm with a buoyant density of 1·698 g/cm3. The buoyant density of the bacterial chromosome is 1·700–1·701 g/cm3. Kappa of the 51 group isolated from stock 298 and stock 6g2,P. tetraurelia, also contain the closed circular DNA. Two forms of kappa coexist in paramecia: brights and nonbrights. Examination by density-gradient centrifugation of the DNA of brights and nonbrights shows the extrachromosomal DNA to be associated mainly with brights. It is suggested that the extrachromosomal DNA might be the determinant for the refractile bodies and the helical phage-like structures found in brights.

scholarly journals Proteoglycans of the human intervertebral disc. Electrophoretic heterogeneity of the aggregating proteoglycans of the nucleus pulposus

1988 ◽  
Vol 251 (2) ◽  
pp. 347-356 ◽  
Author(s):  
M R Jahnke ◽  
C A McDevitt
Keyword(s):  
Density Gradient ◽  
Nucleus Pulposus ◽  
Gel Electrophoresis ◽  
Proteinase Inhibitors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Guanidine Hydrochloride ◽  
Buoyant Density ◽  
Hydrodynamic Size ◽  
Keratan Sulphate

Nuclei pulposi were dissected from lumbar discs of radiologically normal human spines of cadavers aged 17, 20 and 21 years. Proteoglycans were extracted with 4 M guanidine hydrochloride (dissociative conditions) with proteinase inhibitors and isolated as A1 fractions by associative density-gradient centrifugation. Aggregating and non-aggregating proteoglycans were separated by Sepharose 2B chromatography. Both aggregating and non-aggregating proteoglycans contained a keratan sulphate-rich region as isolated by chondroitinase/trypsin/chymotrypsin digestion and Sepharose CL-6B chromatography. Agarose/acrylamide-gel electrophoresis of individual fractions of a Bio-Gel A-50m dissociative-column separation of the aggregating proteoglycans revealed two, well-separated bands: S and F, the slower and faster migrating bands respectively. The non-aggregating proteoglycan fractions were eluted under associative conditions (0.5 M-sodium acetate, pH 6.8) and migrated as a single band in the electrophoretic system. The gel-electrophoretic heterogeneity of the aggregating proteoglycans was still evident after hydroxylamine fragmentation and removal of the hyaluronate-binding portion of the molecule. Dissociative density-gradient centrifugation of the aggregating proteoglycans partially separated the Band-S proteoglycans from the Band-F population. Subsequent dissociative chromatography of the high-buoyant-density Band F proteoglycans permitted discrimination of this band into two gel-electrophoresis-distinguishable populations (Bands F-1 and F-2). Enzyme-linked immunosorbent assays with a monoclonal antibody that recognized keratan sulphate demonstrated that the D1 fraction containing the Band F-1 proteoglycans was enriched in keratan sulphate compared with the total aggregating or non-aggregating pool of proteoglycans. The proteoglycans of young adult nucleus pulposus could then be ascribed to one of four structurally and/or electrophoretically distinct populations: (1) the non-aggregating population, which comprised about 70% of the total extractable proteoglycans; (2) the aggregating pool, comprising: (a) Band F-1 proteoglycans, which had a relatively large hydrodynamic size, uronate/protein weight ratio, were enriched in keratan sulphate and had a high buoyant density; (b) Band S proteoglycans, which migrated slower in agarose/acrylamide gels, had a smaller hydrodynamic size, lower buoyant density and a lower uronate/protein ratio than the Band F-1 population; (c) Band F-2 proteoglycans, which were lower in buoyant density, smaller in hydrodynamic size and slightly faster in electrophoretic mobility than the Band F-1 proteoglycans.

scholarly journals Evidence that platelet density depends on the alpha-granule content in platelets

Blood ◽  
1984 ◽  
Vol 63 (2) ◽  
pp. 482-485 ◽  
Author(s):  
BA van Oost ◽  
AP Timmermans ◽  
JJ Sixma
Keyword(s):  
Density Distribution ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Platelet Rich Plasma ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Adenosine Diphosphate ◽  
Density Gradients

Abstract The relation between platelet buoyant density and beta-thromboglobulin (beta-TG), a marker for platelet alpha-granule content, was assessed by three independent approaches. (1) Platelets were separated on iso- osmolar discontinuous Stractan density gradients into five fractions, ranging in density from 1.061 g/ml to 1.091 g/ml (20 degrees C). The beta-TG content (mean +/- SD, n = 17) increased with the platelet density from 27.8 +/- 8.6 micrograms beta-TG/10(9) cells (20% less- dense platelets) up to 65.6 +/- 15.5 micrograms beta-TG/10(9) cells (15% most-dense platelets). (2) Activation of platelets in platelet- rich plasma with thrombin, adenosine diphosphate, collagen, or epinephrine resulted in a decreased density of the platelets. This was only seen when there was simultaneous secretion of beta-TG. (3) The less-dense and the more-dense platelet fractions, after isolation by density gradient centrifugation, were separately treated with thrombin. After complete degranulation, the density distribution of the originally less-dense and more-dense platelets were identical and were much narrower than the density distribution of resting platelets.

The extraction, characterization andin vitrotranslation of RNA from adultSchistosoma mansoni

Parasitology ◽  
1982 ◽  
Vol 84 (2) ◽  
pp. 253-261 ◽  
Author(s):  
M. P. R. Tenniswood ◽  
A. J. G. Simpson
Keyword(s):  
Schistosoma Mansoni ◽  
Lithium Chloride ◽  
Gel Electrophoresis ◽  
Rabbit Reticulocyte Lysate ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Molecular Weights ◽  
Reticulocyte Lysate ◽  
Urea Method

SUMMARYWe have extracted RNA fromSchistosoma mansoniusing the lithium chloride–urea method which gives good yields of undegraded RNA. The results of agarose gel electrophoresis of RNA extracted by this procedure suggest thatS. mansonihas anin vivonick in the large rRNA sub-unit. Translation of the RNA in a rabbit reticulocyte lysate gave significant incorporation of [35S]methionine into synthesized proteins. Immuno-precipitation of these translation products using a hyperimmune monkey serum sedimented between 5 and 8% of the radioactivity, which appeared to be present in approximately 13 proteins of molecular weights 18, 20, 21, 22, 23, 35, 40, 54, 60, 70, 74, 78 and 105 K Daltons.

scholarly journals Physical characterization of the MUC5AC mucin: a highly oligomeric glycoprotein whether isolated from cell culture or in vivo from respiratory mucous secretions

2000 ◽  
Vol 347 (1) ◽  
pp. 37-44 ◽  
Author(s):  
John K. SHEEHAN ◽  
Caroline BRAZEAU ◽  
Saduman KUTAY ◽  
Helena PIGEON ◽  
Sara KIRKHAM ◽  
...  
Keyword(s):  
Cell Culture ◽  
Gel Electrophoresis ◽  
Early Stage ◽  
Average Length ◽  
Buoyant Density ◽  
Gel Chromatography ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Major Band

We have isolated the high-Mr mucins from growth medium of the early stage of an HT-29 cell culture by gel chromatography and isopycnic density gradient centrifugation. The mucins (buoyant density 1.34-1.44 g/ml) were reactive with an anti-peptide antiserum (MAN-5ACI) raised against a sequence from within the MUC5AC mucin. Similar antisera raised against the MUC2 and MUC5B mucins were not reactive. The MUC5AC reduced-mucin subunits exhibited a homogeneous charge distribution on anion-exchange chromatography, but appeared as two bands, one major and one more minor, after agarose gel electrophoresis. The unreduced mucins had an average Mr in excess of 40 MDa and were visualized in the electron microscope as large, fine filamentous threads (many microns in length) that after reduction were greatly reduced in size (number average length 570 nm). Agarose gel electrophoresis of unreduced MUC5AC mucins identified a major band just entering the gel with evidence of a ‘ladder’ of faster-migrating minor bands. Partial reduction of the mucins increased the proportion of the faster bands and at least 16 could be discriminated. Mr measurements showed that these bands differed by single monomer units. The mucins behaved as very stiff extended structures in solution and this characteristic might explain the poor separation of different-sized oligomers in sedimentation-rate experiments. The cell-culture mucin preparation had similar characteristics of charge and buoyant density to MUC5AC mucins from respiratory secretions in vivo. In addition the MUC5AC mucin from respiratory tract secretions exhibited similar behaviour, reduced and unreduced on agarose gel electrophoresis, indicating that the mucin has a similar molecular phenotype in vivo and in vitro.

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.

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