Translation of Encephalomyocarditis Viral RNA in Oocytes of Xenopus laevis

Abstract

RNA from encephalomyocarditis virus was injected into oocytes of Xenopus laevis. After incubation of the oocytes in [35S]methionine, polyacrylamide gel electrophoresis showed that a new series of polypeptides had been synthesized. They were identical in size to the polypeptides that appeared in ascites cells after infection with this virus. Electrophoretic and chromatographic analysis of the methionine-containing tryptic peptides from three of the induced polypeptides confirmed that they were virus-specific. All of the bands that appeared in ascites cells after infection also appeared in oocytes after injection of RNA from the virus. We conclude that Xenopus oocytes translate a mammalian viral mRNA faithfully and extensively, and perform normal post-translational modifications.Cell organelles or solutions of macromolecules can be injected into living oocytes of Xenopus laevis (1, 2). When messenger RNA for globin or calf-lens crystallin is injected into such oocytes, it is translated (2, 3). Gurdon et al. (2) estimated that each molecule of injected globin mRNA is translated once every 5-10 min for 25 hr. Since the purest sources of natural mRNA currently available are RNA viruses, this paper investigates the ability of oocytes to translate a viral mRNA. Encephalomyocarditis (EMC), a picornavirus, was chosen for this study because the virus-specific proteins synthesized in infected cells have been identified (4), and the activity of the virion RNA as a messenger has been studied in vitro (5-9). EMC RNA has only been partially translated in vitro (7,8), and its translation products in vivo undergo an extensive series of post-translational modifications. MATERIALS AND METHODSAscites Cells and EMC Virus. Strain K2 of EMC virus (10) was grown in Krebs II ascites cells (11) and purified either by the procedure of Burness (12) Lyophilized precipitated proteins were oxidized with performic acid (200,1/50-,ug sample) for 1 hr at 0°, then lyophilized twice. Oxidized proteins were digested with 10 yg of TPCK-treated trypsin (Worthington) in 100jul of triethylamine carbonate (pH 9.0) for 1 hr at 370, followed by addition of a further 10 ,ug of trypsin and 3 hr of incubation. Digests were lyophilized twice to remove triethylamine carbonate, suspended in electrophoresis buffer, and applied to 20 X 40 cm Polygram CEL 300 cellulose-coated thin-layer sheets for electrophoresis at pH 3.5 (5% acetic acid-0.5% pyridine) for 2 hr at 1 kV (25 V/cm). After location of peptides by autoradiography, the cellulose layer was divided into zones, scraped off, and chromatographed on cellulose thin layers in butanol-pyridine-acetic acid-water 5:1:4:4 (18).

A Film Detection Method for Tritium‐Labelled Proteins and Nucleic Acids in Polyacrylamide Gels

“…15 % polyacrylamide slab gels containing 0.09 % bisacrylamide and 0.1 % sodium dodecylsulphate were prepared and electrophoresed using Laemmli's discontinuous buffer system [3] as described previously [4]. When stained gels were required, a solution of 0.…”

Translation of ovalbumin mRNA in Xenopus laevis oocytes. Characterization of the system and effects of estrogen on injected mRNA populations.

“…Xenopus oocytes and eggs have been used as efficient translation systems for mRNAs for a number of proteins including rabbit, mouse and duck hemoglobin (11,12,20), guinea pig a-lactalbumin (21), mouse myeloma immunoglobulin light chain (22)(23)(24), calf lens crystallin protein (25), encephalomyocarditis virus of mouse (26), honeybee promellitin (27), trout testis protamine (28), mouse collagen (27,29), Rana pipiens collagen, (30) and beef thyroglobulin (31,32 It is easily apparent that the Xenopus oocyte is an extremely sensitive translation system: the lowest point on Fig. 8 represents an injection of 0.38 ng mRNAo.…”

The formation of lipid-linked sugars by cell-free preparations of lactating rabbit mammary gland

“…The column eluate was monitored for protein and radioactivity. Samples of the solubilized-protein peak were also examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis on slab gels by using the discontinuous buffer system of Laemmli (1970) as described by Laskey et al (1972). Parallel samples and reference proteins (fi-galactosidase, bovine serum albumin, ovalbumin and lysozyme) were co-electrophoresed.…”