Methylation of 45 s ribosomal RNA precursor in HeLa cells

Zimmerman, Ernest F.; Holler, Barbara W.

doi:10.1016/s0022-2836(67)80023-8

Cited by 140 publications

(31 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Under the conditions of electrophoresis shown in Fig. 1 In order to distingush the methylated precursors of ribosomal RNA (rRNA) (24)(25)(26) from the heterodisperse background, labeling with methionine-(methyl) -3H was studied. Fig.…”

Section: Resultsmentioning

confidence: 99%

Kinetics of RNA synthesis in toad bladder epithelium: action of aldosterone during the latent period

Vancura¹,

Sharp²,

Malt³

1971

J. Clin. Invest.

View full text Add to dashboard Cite

A B S T R A C T Processing of RNA in the toad bladder was analyzed by polyacrylamide-gel electrophoresis to determine whether aldosterone causes any changes in the 1 hr before it potentiates transport of sodium ion. No change was found in the quantity or in the specific activity of bulk RNA labeled with uridine-5-3H. In vivo and in vitro with either uridine-5-'H or with methionine-(methyl)-'H as precursors, processing of RNA was extremely slow. Heterodisperse RNA was obvious after 30 min of continuous labeling, but labeling of the 40S precursor of ribosomal RNA was not apparent for 60 min. Labeling of mature 28S and 18S RNA first became apparent after 8 hr. -S RNA was the principal fastmigrating species labeled at 30 min, and 4S RNA was not heavily labeled until 1 hr. Aldosterone (5 x 10-mole/liter) produced no changes. If care were not taken to inhibit metabolism of native bacteria colonizing the bladder, bacterial RNA of high specific activity predominated. We conclude that RNA metabolism in the toad bladder is extraordinarily slow, that a major acceleration of de novo synthesis in response to physiologic doses of aldosterone was not demonstrable, and that some reports to the contrary may have been influenced by artifacts from bacterial RNA metabolism. Earlier evidence for obligatory alterations in RNA metabolism during the latent period is not strong.

show abstract

Section: Resultsmentioning

confidence: 99%

Kinetics of RNA synthesis in toad bladder epithelium: action of aldosterone during the latent period

Vancura¹,

Sharp²,

Malt³

1971

J. Clin. Invest.

View full text Add to dashboard Cite

show abstract

“…The incorporation of '4CH3-methionine into high mol wt RNA allows the selective determination of the synthesis of rRNA (8,17,35). In initial experiments attempting to utilize this specificity, we found 4 to 8 times more '4CH3-methionine radioactivity incorporated into RNA of ribosomes from embryos incubated during the period between 6 and 7 hr, as compared to embryos incubated between 1 and 2 hr.…”

Section: Resltltsmentioning

confidence: 88%

Transcription of Ribosomal and Messenger RNAs in Early Wheat Embryo Germination

Spiegel¹,

Obendorf²,

Marcus³

1975

Plant Physiol.

View full text Add to dashboard Cite

Germinating wheat embryos (Triticum aestivum L). synthesize both ribosomal and messenger RNA at the earliest times after the onset of germination. The rates of synthesis of these two RNAs are determined at various stages in germination by an analysis of newly synthesized radioactive RNA on oligo(dT)-cellulose. The rate of messenger RNA synthesis is essentially constant throughout 18 hours of germination, while that of ribosomal RNA synthesis increases steadily, particularly after the onset of cell expansion (6 hours), reaching at 16 to 18 hours, a rate of synthesis between 5-and 20-fold greater than that observed at the earliest stages. The net effect is a relative decrease in the fraction of transcribed high molecular weight RNA that is mRNA. Throughout the first 7 hours of germination, mRNA is 25 to 30% of the transcribed fraction, whereas by 16 to 18 hours it has declined to a level of 4 to 8%.Protein synthesis has been clearly established as an obligatory early component of seed germination (7,19,32,33 MATERIALS AND METHODSPreparation and Germination of Embryos. Embryos were prepared from wheat seed (Triticwn aestivwn L.) by the procedure of Johnston and Stern (13) using either CCl4-Cyclohexane flotation (20) or sucrose flotation to separate the embryos from the endosperm. The embryos obtained by the sucrose flotation procedure are referred to as "sucrose" embryos. Germination was carried out as previously described (23) Ribosome Isolation. Two 125-mg portions of embryos frozen in dry ice were converted separately into "dry ice powders" (22) and then combined for homogenization in 7 ml of isolation media (0.25 M sucrose, 20 mm KCI, 100 mm tris-HCl [pH 7.6], 5 mM magnesium acetate, 5 mM mercaptoethanol) in a Duall conical glass homogenizer. The homogenate was cleared by centrifugation (23,000g for 10 min) and layered over 2 ml of a cushion of 1.8 M sucrose, 20 mm KCI, 5 mM magnesium acetate, 100 mM tris-HCl (pH 7.6), 5 mm mercaptoethanol, and centrifuged for 90 min at 150,000g in a Spinco Ti5O rotor. The resulting pellet was resuspended in 0.6 ml of 20 mm KCI, 5 mM magnesium acetate, 50 mm tris-HCl (pH 7.6), 1 mm dithiothreitol and clarified by centrifugation for 10 min at 23,000g. The ribosome content was determined by measuring the absorbance at 260 nm of an aliquot (10 A26o = 1 mg of ribosomes). For fractionation, a volume containing 340 ,g of ribosomes was layered on 4.5 ml of a 15 to 38% linear sucrose gradients over 0.5 ml of a 1.5 M sucrose cushion (both gradient and cushion solutions contained 20 mi KCI, 5 mm magnesium acetate, 50 mm tris-HCI [pH 7.6], 1 mM dithiothreitol) and centrifuged for 45 min at 150,00Og in a SW50.1 rotor. In the EDTA release experiment, a volume containing 540 ,ug of ribosomes was layered on 5 ml of a 5 to 21% linear sucrose gradient containing 50 mm KCI, 50 mm tris-HCI (pH 7.6), 10 mm potassium EDTA (pH 7.4), and centrifuged for 80 min at 150,000g in a SW50.1 rotor. The gradients were fractionated with continual monitoring of absorbance at 254 nm; the fractions were ...

show abstract

“…Methylation of RNA with radioactive methionine was carried out according to Zimmerman and Holler (18). Cells were grown for 1 h with 0.5 ,ug of radioactive methionine per ml.…”

Section: Methodsmentioning

confidence: 99%

Amphotericin B and Filipin Effects on L and HeLa Cells: Dose Response

Medoff

Schlessinger³

et al. 1977

Antimicrob Agents Chemother

View full text Add to dashboard Cite

Amphotericin B (AmB) and filipin effects on L and HeLa cells were compared by monitoring drug-induced potassium leakage from cells, changes in radioactive uridine incorporation into cellular ribonucleic acid, protein leakage from cells, and cell viability. L cells were much more susceptible to both AmB and filipin than were HeLa cells, but the overall dose response was similar. For AmB, the various effects were easily separable. Potassium leakage occurred at the lowest concentrations of AmB and was reversible. Inhibition of uridine incorporation and loss of viability occurred at intermediate levels, and protein loss occurred at higher levels. In contrast, filipin was much more potent; its effects on potassium leakage were only minimally reversible, and the separation of the permeabilizing effects from complete cell lysis was possible only over a limited concentration range and for a short time.Amphotericin B (AmB) belongs to the class of polyene antibiotics, which bind to the sterol component of the membranes of all eucaryotic cells (6,13). At sufficiently high concentrations, the polyene antibiotics cause an increase in membrane permeability, with leakage of metabolites and macromolecules from the cell and resultant cell death. Previous studies with fungi and animal cells in culture have shown that at low concentrations AmB can induce nonlethal permneabilizing changes in cell membranes which enlhance uptake of certain small molecules (16) and even macromolecules (9). These properties of AmB have been exploited to achieve the penetration into cells of pharmacologically active compounds (9, 12, 17) that do not penetrate the untreated cell membranes.We have undertaken the present study to compare the nonlethal and lethal effects of AmB on cultured L and HeLa cells, and to determine under which conditions they can be separated and whether the cell damages can be repaired. Similar studies have also been performed with filipin to determine whether permeability changes induced by this polyene antibiotic are separable from lethality. MATERIALS AND METHODSChemicals. AmB in the form of Fungizone was purchased from E. R. Squibb and Sons, Inc., Princeton, N.J. It was dissolved in sterile water before use.The concentrations of the AmB solutions were calculated on the basis of the amounts of AmB in the Fungizone, as stated by the manufacturer. Filipin, in the form of the unfractionated antibiotic complex, which was 86% pure, was donated by The Upjohn Co., Kalamazoo, Mich. The amounts noted refer to the crude product. Filipin was dissolved in methanol before use.[5-3H]uridine (specific activity, 8 Ci/mmol) was purchased from Schwarz/Mann, Orangeburg, N.Y. L-[methyl-3H]methionine (specific activity, 5.0 mCi/ 0.071 mg) was purchased from the New England Nuclear Corp., Boston, Mass. Triton X-100 (Triton) was purchased from Packard Instrumental Co., Downers Grove, Ill. Lithium diluent was purchased from Instrumental Laboratory, Inc., Lexington, Mass.Cell culture. HeLa and L 929 cells were maintained and assayed in monolayer in Ea...

show abstract

Methylation of 45 s ribosomal RNA precursor in HeLa cells

Cited by 140 publications

References 10 publications

Kinetics of RNA synthesis in toad bladder epithelium: action of aldosterone during the latent period

Kinetics of RNA synthesis in toad bladder epithelium: action of aldosterone during the latent period

Transcription of Ribosomal and Messenger RNAs in Early Wheat Embryo Germination

Amphotericin B and Filipin Effects on L and HeLa Cells: Dose Response

Contact Info

Product

Resources

About