Mammalian Viruses and Rickettsiae

Hoyer, Bill H.; Bolton, Ellis T.; Ormsbee, Richard A.; LeBouvier, George; Ritter, Daniel B.; Larson, Carl L.

doi:10.1126/science.127.3303.859

Cited by 65 publications

(13 citation statements)

References 10 publications

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“…onto anion-exchange resins. However, it should be noted that the phenomenon of strong interactions between virus particles and anion-exchange resins has long been known (Puck and Sagik, 1953;Creaser and Taussig, 1957;Hoyer et al, 1958) and has generally been attributed to the presence of negatively charged domains on the surface of the virions rather than to incomplete shielding of the negative charges on the viral DNA. Indeed, "petit" bacteriophage X, which is a hollow precursor of the normal X head, is readily purified on DEAE cellulose, eluting at an even higher ionic strength than that required to elute intact bacteriophage particles (Hendrix and Casjens, 1975).…”

Section: Discussionmentioning

confidence: 98%

A New Method for Purifying Lambda DNA From Phage Lysates

Helms¹,

Graham²,

Dutchik³

et al. 1985

DNA

177

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A new method for preparing small quantities of lambda DNA from phage lysates has been developed. The protocol is based on the concentration and purification of bacteriophage particles from crude lysates using small DEAE-cellulose columns. This chromatographic step gives an absolute separation of the lambda DNA from the cellular nucleic acids and a 20-fold enrichment relative to the major soluble proteins in crude lysates, while effecting a 10-fold concentration of the phage. Final deproteinization and concentration of the lambda DNA is achieved by conventional precipitation steps. The lambda DNA produced by this method is shown to be nondegraded, biologically active, and an excellent substrate for restriction enzymes. A detailed protocol is provided for starting with individual plaques and using the method to obtain purified DNA from large numbers of lambda clones.

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Section: Discussionmentioning

confidence: 98%

A New Method for Purifying Lambda DNA From Phage Lysates

Helms¹,

Graham²,

Dutchik³

et al. 1985

DNA

177

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“…Purified viruses were prepared by ultracentrifugation and column chromatography on diethylaminoethyl cellulose and, where indicated, were labeled with radiophosphorus as described by Hoyer eta/. (12,13).…”

Section: Methodsmentioning

confidence: 99%

“…L strain mouse fibroblast cells were propagated from the strain maintained by Scherer (11).Viruses.--Type 1 poliovirus (Mahoney) was employed as pooled fluid of the 9th and 10thHeLa culture passage of virus received from Connaught Medical Laboratories, Toronto; type 2 poliovirus (MEF-1) was grown in monkey kidney cells or established human cell lines after reception from Connaught Laboratories; Coxsackie B1 (Conn. 5) virus was purchased from The American Type Culture Collection and used after 4 to 5 HeLa passages.Purified viruses were prepared by ultracentrifugation and column chromatography on diethylaminoethyl cellulose and, where indicated, were labeled with radiophosphorus as described by Hoyer eta/. (12,13).RNA Preparation.--Virus for extraction was suspended in 0.02 M phosphate buffer at pH 7.2, supplemented with 5 X 10 -~ ~ ethylenediamine sodium tetraacetate (EDTA) and 10 -2 ~ tris(hydroxymethyl)amino methane (tris) or veronai buffer at pH 7.2. RNA was extracted with phenol at room temperature (1), and stored at 0°C.…”

mentioning

confidence: 99%

Enteroviral Ribonucleic Acid

Holland

Hoyer

McLaren

et al. 1960

The Journal of Experimental Medicine

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Recognition of the infectivity of ribonucleic acid (RNA) of tobacco mosaic (1, 2) and of animal virus (3) initiated investigation of the molecular aspects of viral reproduction. Study of replication of infectious RNA depends upon employment of efficient and reproducible biological assay procedures. Reception of viral genetic material normally depends upon specific interaction of cellular and viral receptors (4--6) while phenol extracted virus has lost this specificity (7). Therefore, assay of infectious RNA may require extracellular environments different from those suitable for assay of whole virus.Plaque formation of infectious RNA was greatly improved by use of 1 sodium chloride solution as described by Alexander et al. (8), but considerable variation among enteroviral RNA titers was noted when different RNA preparations were tested in a number of established human cell strains. The general procedure of Gierer and Schramm (1) consistently yielded infectious poliovirus RNA and minor alterations in the extraction procedure were ineffective. Much of the inefficiency of viral RNA action apparently results from failure to introduce RNA into replicating sites of cells. This paper reports factors affecting the biological activity of enteroviral RNA, and a plaque assay system that has proved sensitive and reproducible. Production of infectious RNA from highly purified poliovirus is reported, in confirmation of the observations of others (9, 10), and evidence is presented that only a small fraction of total extracted virus RNA is adsorbed to cells, and is responsible for infectivity. Material and MethodsCell Cultures and Methods.--Methods and media used for routine cultivation of HeLa cells and monkey kidney, human amnion, and rabbit skin fibroblast cells in primary culture have

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“…labelled virus was purified by ultracentrifugation and DEAE column chromatography ( 7 ) . The preparation used for diffusion contained approximately 10T.O TIDS0 and 100,-000 cpm per 0.1 ml.…”

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confidence: 99%

Radioautographic Studies of Poliovirus Binding by Human Immunoglobulins.

Ainbender

Berger

Hevizy

et al. 1965

Experimental Biology and Medicine

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A previous report from this laboratory demonstrated that radioactive poliovirus could diffuse through agar to combine with electrophoretically separated colostrum and serum antibodies ( 1 ) . The technique as used was relatively insensitive, requiring concentrated, purified immunoglobulins and large volumes of poliovirus. A more sensitive technique, combining immunoelectrophoresis and radioautography with radioactive antigen as one of the reactants, has been described for demonstrating antibody activity. Thus antibodies which bind insulin, thyroglobulins and ragweed have been detected in the IgG,t IgA and IgM classes of immunoglobulins by this method(2-6). The adaptation and use of this radioimmunoelectrophoresis technique to the study of antibody directed against poliovirus is described in this paper.Material and methods. Antigen. P2 labelled type I poliovirus was prepared by growing virus in HeLa cell cultures with citrate-Eagle's medium, 5 % dialysed calf serum and sodium phosphate P2 (0.066 mc/ml). The labelled virus was purified by ultracentrifugation and DEAE column chromatography ( 7 ) . The preparation used for diffusion contained approximately 10T.O TIDS0 and 100,-000 cpm per 0.1 ml.Serum. Serum specimens were obtained from adult human males and females who had not been immunized within the past 2 years. Serum polio neutralizing antibodies were demonstrated by a standard one hour tube-neutralization method, using 100 TIDBo of poliovirus, in HeLa cell cultures(8). The *This research was supponted by U.S.P.H.S. Research Grant AI-05009-03 from Nat. Inst. M t h .t The World Health Organization (Bull. 447, World Health Organ., v30, 1%4) has recommended the following nomenclature for the classes ob immumglobulins: G Immunoglobulin (IgG) for 7s (y2) -globulin ; A Immunoglobulin (IgA) for yla-globulin and M Immunoglobulin (IgM) for y,,,-globulin. Sinai Hospital, New York Citysera were fractionated by a combination of DEAE and Sephadex G-2Q0 column chromatography (9). The type of immunoglobulin in the eluates was determined by radial immunodiffusion( 10). The neutralization titer of each fraction was determined and correlated with the type of immunoglobulin present in the fraction.Radioimmunoelectrophoresis was performed as described by Yagi (2) and Morse(3) using agar consisting of 1% Difco Special Agar Noble in a 25% solution of 0.1 M Verona1 buffer (pH 8.6) on microscope slides. After electrophoresis of the serum, rabbit antiserum directed against human serum was added to the antiserum troughs. As soon as the antiserum had diffused into the agar, the radioactive poliovirus preparation was added to the trough. The plates were incubated 24 hours at room temperature and then washed for 48 hours with a 1% solution of NaCl. The slides were dried and then placed in contact with Kodak Royal Pan film for one week. Exposure was carried out in total darkness. After removal from contact with the film, the slides were stained with amido black. In some experiments the antiserum was allowed to react with the electrophoretic...

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Mammalian Viruses and Rickettsiae

Cited by 65 publications

References 10 publications

A New Method for Purifying Lambda DNA From Phage Lysates

A New Method for Purifying Lambda DNA From Phage Lysates

Enteroviral Ribonucleic Acid

Radioautographic Studies of Poliovirus Binding by Human Immunoglobulins.

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