Replication of Mengovirus I. Effect on Synthesis of Macromolecules by Host Cell

Plagemann, Peter G.W.; Swim, H. E.

doi:10.1128/jb.91.6.2317-2326.1966

Cited by 109 publications

(60 citation statements)

References 30 publications

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“…The cultivation of Novikoff rat hepatoma cells (subline N1S1-67) in suspension culture in Swim's medium 67 and the method of enumerating cells have been described previously (15,19) . Cultures were frequently tested or contamination with mycoplasma by cultivation in broth or on agar plates, with negative results .…”

Section: Cell Culturementioning

confidence: 99%

Nucleotide Pools in Novikoff Rat Hepatoma Cells Growing in Suspension Culture

Plagemann

1972

The Journal of Cell Biology

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123

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This study was undertaken to measure the absolute levels of nucleoside pools in Novikoff rat hepatoma cells (subline NISJ-67) during growth in suspension culture in the presence of high concentrations of various nucleosides in the medium, and to obtain further evidence for the compartmentalization of the nucleotides in independent cytoplasmic and nuclear pools . The levels of nucleotide pools were measured by growing the cells in medium supplemented with inorganic phosphate-32 P. The nucleotide pool levels (mostly in the form of triphosphates) ranged from about 1 nmole of cytidine nucleotides to 8 nmole of adenosine nucleotides per 10 6 cells . The presence of 1 mm uridine, cytidine, guanosine, or adenosine in the medium resulted in marked increases in the intracellular levels of the corresponding nucleoside phosphates of at least 3-4 nmole/106 cells . These increases were partially compensated for by decreases in the levels of other nucleotides . Evidence is presented to indicate that it is the cytoplasmic pool that expands during incubation with high concentrations of nucleosides in the medium, whereas the nuclear pool remains constant and very small in size . Preincubation of cells with 1 mm uridine-5H for 5 .5 hr, which resulted in a threefold increase in the total intracellular level of uridine nucleotides, had no effect on the subsequent incorporation of uridine-14C into cellular nucleic acids in the nucleus, whether present at a 1 µM or 1 mM concentration in the medium . In contrast, the incorporation of uridine-14 C into cytoplasmic viral-specific RNA by mengovirus-infected Novikoff cells was reduced 60-707 as a result of preincubation of the cells with high concentrations of uridine 3H . Further, within 1-2 min upon addition of 2 .5 or 6 .5 µM 3 H-labeled uridine, cytidine, adenosine, guanosine, or inosine to cultures of Novikoff rat hepatoma cells, the incorporation of label into nucleic acids reached a constant and maximum rate, in spite of the presence of high intracellular concentrations (0.4-3 mM) of the corresponding unlabeled nucleoside triphosphates . Marked differences were also observed in the relative incorporation of the various nucleosides into the different nucleotides of the acid-soluble pool, and of mengovirus RNA and cellular RNA .

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Section: Cell Culturementioning

confidence: 99%

Nucleotide Pools in Novikoff Rat Hepatoma Cells Growing in Suspension Culture

Plagemann

1972

The Journal of Cell Biology

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123

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“…The strain of mengovirus used was that described by Plagemann and Swim (1966). Virus stocks were grown in 17CL.…”

Section: Introductionmentioning

confidence: 99%

The virus-specific intracellular RNA species of two murine coronaviruses: MHV-A59 and MHV-JHM

1981

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Seven virus-specific, polyadenylated RNA species have been identified in mouse cells infected with the murine coronaviruses MHV-A59 (A59V) or MHV-JHM (JHMV). MHVinfected 17CL.l cells were labeled with [zrP]orthophosphate in the presence of actinomycin D and the eytoplasmic RNA was extracted and analyzed by agarose gel electrophoresis. These RNA species range in size from 6.3 X 106 to 6.1 X lo6 daltons. The A59V and JHMV-specific RNAs have identical molecular weights and comigrate in agarose gels. The largest intracellular RNA species is identical to RNA isolated from purified virions, as determined by agarose gel electrophoresis and oligonucleotide fingerprint studies of ribonuclease T1 digests. Oligonucleotide fingerprints of the six subgenomic RNAs show that the sequences they contain are present in virion RNA, confirming their virus-specific nature. The fingerprinting studies also demonstrate that the six subgenomic RNA species make up a nested set. The sequences present in each RNA species are also present in all larger RNA species. These larger RNAs also contain additional sequences consistent with their greater size. The subgenomic RNAs fulfull many of the criteria for mRNAs. Possible mechanisms for generating these RNAs are discussed. 39

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“…Wild-type Novikoff rat hepatoma cells (subline N1 S1-67) and a thymidine kinasedeficient subline thereof [ 171 were propagated in Swim's medium 67 and enumerated as described previously [18,191. Cells were collected by centrifugation at 400g for about 2 min and suspended in a basal medium, BM42B [20], to a concentration of (1-2) X lo7 cells per milliliter for transport measurements or about 2 X lo6 cells per milliliter for uptake studies.…”

Section: Methodsmentioning

confidence: 99%

“…Then 0.2 ml of 0.5 N trichloroacetic acid (TCA) was added to the tube Metabolic Stability of Nucleoside Transport JSS:5 13 and immediately vortexed to disperse the pellet. After 30 min of incubation at 70°C the entire tube and its contents were transferred to a vial containing 8 ml of a modified Bray's solution [18] and were analyzed for radioactivity in a liquid scintillation spectrometer. through oil were determined in parallel runs in which the substrate was replaced by ["C] carboxylinulin in tritiated water [13].…”

Section: Methodsmentioning

confidence: 99%

Metabolic stability of the nucleoside transport system of novikoff rat hepatoma cells

1978

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Rates of transport of uridine and thymidine, estimated with a rapid sampling technique, did not change with culture age. Inhibition of cellular RNA and protein synthesis for periods up to 6 h, did not lead to a loss of nucleoside transport activity. Mild treatment of cell suspensions with trypsin or neuraminidase had no effect on the kinetics of thymidine transport. Thus we conclude, contrary to previous reports, that nucleoside transporters are metabolically stable and that the decreases in nucleoside uptake rates observed with decreased protein synthesis reflect loss of nucleoside kinase activities. These kinases (which have narrow substrate specificity) rather than the membrane-associated, transport apparatus (which has broad substrate specificity) are the most likely sites for regulation of nucleoside uptake.

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Replication of Mengovirus I. Effect on Synthesis of Macromolecules by Host Cell

Cited by 109 publications

References 30 publications

Nucleotide Pools in Novikoff Rat Hepatoma Cells Growing in Suspension Culture

Nucleotide Pools in Novikoff Rat Hepatoma Cells Growing in Suspension Culture

The virus-specific intracellular RNA species of two murine coronaviruses: MHV-A59 and MHV-JHM

Metabolic stability of the nucleoside transport system of novikoff rat hepatoma cells

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