Studies on protein and nucleic acid metabolism in virus-infected mammalian cells. 3. Methods for the disruption of Krebs II mouse-ascites-tumour cells

Martin, E. M.; Malec, J; Coote, J. L.; Work, T. S.

doi:10.1042/bj0800606

Cited by 25 publications

(13 citation statements)

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“…), which may be isolated by phenol extraction , and the pure virus may be obtained as a crystalline ribonucleoprotein (Faulkner, Martin, Sved & Work, 1960). It is unlikely that a direct demonstration of the template function of ribonucleic acid could be achieved in intact cells, but the Krebs II cell gave good cellfree preparations capable of protein synthesis (Martin, Malec, Coote & Work, 1961). Since viral protein may be readily assayed or, if necessary, isolated by immunological or haemagglutination techniques, the system seems to offer the possibility of the direct induction of synthesis of a specific protein by a specific ribonucleic acid in a cell-free preparation.…”

Section: Studies On Protein and Nucleic Acid Metabolism Inmentioning

confidence: 99%

Studies on protein and nucleic acid metabolism in virus-infected mammalian cells. 1. Encephalomyocarditis virus in Krebs II mouse-ascites-tumour cells

Martin¹,

Malec²,

Sved³

et al. 1961

Biochemical Journal

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117

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Section: Studies On Protein and Nucleic Acid Metabolism Inmentioning

confidence: 99%

Studies on protein and nucleic acid metabolism in virus-infected mammalian cells. 1. Encephalomyocarditis virus in Krebs II mouse-ascites-tumour cells

Martin¹,

Malec²,

Sved³

et al. 1961

Biochemical Journal

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117

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“…Cells were lysed by double osmotic shock followed by homogenization (Martin, Malec, Coote & Work, 1961). The lysate was centrifuged a t 5008 (max.)…”

Section: Methodsmentioning

confidence: 99%

Intracellular Sites of Synthesis of Encephalomyocarditis Virus Components in Krebs-2 Ascites Tumour Cells

Bellett

Burness

1963

Journal of General Microbiology

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SUMMARYAscites tumour cells were homogenized and fractionated at various times after infection with encephalomyocarditis virus in vitro. Infective ribonucleic acid (RNA) could be obtained from the nuclear fraction soon after infection and the amount of infective RNA recovered from this fraction increased until a maximum was reached a t about 44hr. From 4 h r . onwards the amount of infective RNA associated with the nuclear fraction fell while that from the mitochondrial fraction increased, reaching a maximum at about 53 hr. The amount of haemagglutinin and plaque-forming virus began to increase at about 4 hr. and continued to rise until 8 hr.Most of this virus activity was found in the mitochondrial fraction, although from 6 hr. onwards an increasing amount was found in the microsoma1 fraction.

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“…The rates of valine incorporation into the proteins of nuclei, mitochondria, microsomes and cell sap are in the proportions 0-28:1-0: 1-2:0-67, whereas the rates of orotic acid incorporation into the ribonucleic acids of the same fractions are in the proportions 50:1 0:0-5:3 0. By using the data of Martin et al (1961a) for the distribution of protein and RNA among these fractions, it can be calculated that turnover of RNA in the nucleus accounts for 88 % of the total cell RNA turnover, whereas mitochondria, microsomes and cell sap contribute 24, 33 and 39 % respectively to the total turnover of protein.…”

Section: Experimental and Resultsmentioning

confidence: 99%

“…The flasks were removed, and the cells washed and disrupted by the double-osmotic-shock method. The lysates were fractionated to yield nuclear, mitochondrial and microsome-plus-cell-sap fractions (Martin, Malec, Coote & Work, 1961a). The mitochondrial fraction was analysed for protein and RNA content, and DNA and RNA estimates were carried out on the nuclear fraction.…”

Section: Experimental and Resultsmentioning

confidence: 99%

“…showed that infective RNA could be extracted by cold aqueous phenol from Krebs II ascites-tumour cells that had been infected with encephalomyocarditis virus, although no RNA could be obtained from the virus particles by this treatment. Bellett & Burness (1960) have used the osmotic-shock method of Martin et al (1961a) to localize the formation of infective RNA, and found it to be almost entirely confined to the nucleus during the first 4-5 hr. after infection, thus giving a direct demonstration that the nucleus is the site of infective RNA synthesis in this system.…”

Section: Discussionmentioning

confidence: 99%

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Studies on protein and nucleic acid metabolism in virus-infected mammalian cells. 4. The localization of metabolic changes within subcellular fractions of Krebs II mouse-ascites-tumour cells infected with encephalomyocarditis virus

Martin¹,

Work²

1961

Biochemical Journal

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Martin, Malec, Sved & Work (1961 b) showed that, during a single growth cycle, infection with encephalomyocarditis virus caused no changes in the total deoxyribonucleic acid, ribonucleic acid or protein of the host ascites-tumour cell. However, by the use of [6-14C]orotic acid and [14C]valine, it was shown that there were substantial changes in the rates of turnover of ribonucleic acid and protein at different times during the cycle of virus growth. In particular, about 5 hr. after infection there was a striking increase in the rate of turnover of ribonucleic acid, and this increase coincided in time with the appearance of new virus. However, this stimulation in turnover appeared to be quantitatively greater than could be accounted for by formation of new virus ribonucleic acid.Methods have now been developed for the welldefined separation of disrupted Krebs II ascitestumour cells into nuclei, mitochondria, microsomes and cell sap (Martin, Malec, Coote & Work, 1961 a). By the use of these methods we have been able to investigate the effect of virus infection on the turnover of the ribonucleic acid and protein of the subcellular components of the host cell, and thus to localize more exactly the sites of metabolic change within the infected cell. This paper describes the result of such a study. METHODSThe origin and propagation of both the Krebs II mouseascites-tumour cells and the encephalomyocarditis virus have been described by Martin et al. (1961 b).Conditions of infection. Sufficient virus was added to suspensions of washed ascites-tumour cells in Earle's medium (2-3 x 107 cells/ml.) to infect all cells (approx. 3 plaque-forming units/cell), and the cells were dispensed in 10 ml. portions into incubation flasks. The virus was allowed to adsorb for 30 min. at room temperature, followed by 15 min. at 360. Suspensions of control (uninfected) cells were treated similarly. The cells were then incubated at 36°as described by Martin et al. (1961 b). * Part 3: Martin, Malec, Coote & Work (1961 a).For the study of protein and nucleic acid turnover, 14C-labelled precursors were added at appropriate intervals during the virus growth cycle to flasks containing infected and control cells; 30 min. later the flasks were removed and diluted with an ice-cold solution of the unlabelled precursor in phosphate-buffered saline b), and the cells sedimented by centrifuging at 120g for 5 min. They were then washed twice with buffered saline and stored in an ice bath until required for disruption.Disruption of Kreb8 11 cells andfractionation of subcellular components. Cells were prepared for disruption by washing with calcium-and magnesium-free buffered saline (Martin et al. 1961 b), then with 0-125M-sucrose-0-075M-KCl solution. The cells were disrupted by a combination of double osmotic shock and Potter homogenizer, as described by . Nuclei, mitochondria, microsomes and cell sap were separated from the tumour-coll homogenates by differential centrifuging in 0-25M-sucrose-0-1M-KCI solution . Preparations of isolated nuclei were us...

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Studies on protein and nucleic acid metabolism in virus-infected mammalian cells. 3. Methods for the disruption of Krebs II mouse-ascites-tumour cells

Cited by 25 publications

References 12 publications

Studies on protein and nucleic acid metabolism in virus-infected mammalian cells. 1. Encephalomyocarditis virus in Krebs II mouse-ascites-tumour cells

Studies on protein and nucleic acid metabolism in virus-infected mammalian cells. 1. Encephalomyocarditis virus in Krebs II mouse-ascites-tumour cells

Intracellular Sites of Synthesis of Encephalomyocarditis Virus Components in Krebs-2 Ascites Tumour Cells

Studies on protein and nucleic acid metabolism in virus-infected mammalian cells. 4. The localization of metabolic changes within subcellular fractions of Krebs II mouse-ascites-tumour cells infected with encephalomyocarditis virus

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