Preparation of an Antiserum against an in vitro Translation Product of Alfalfa Mosaic Virus RNA 3

Abstract

SUMMARYAntisera were raised against P3 (mol. wt. 32 000), the full-length translation product of alfalfa mosaic virus (A1MV) RNA 3. P3 was made by translation in wheat germ extracts, using unfractionated AlMV RNA as message, and the products were then fractionated either by centrifugation through a sucrose gradient, or by phosphocellulose chromatography, each technique being followed by SDS-PAGE. Preparations made by each method were used in sequence to immunize a rabbit. The resulting antisera reacted on immunoblots with P3 synthesized in vitro, at dilutions of about 1 : 10000, but did not react with the translation products of A1MV RNA 1, RNA 2 or RNA 4. Although the antisera contained antibodies against some wheat germ components, most were removed by preabsorbing the antisera with the wheat germ extract. A minor wheat germ translation product of RNA 3, P'3, also reacted with the antisera. Since the mol. wt. of P'3 is apparently 1500 higher than that of P3, it is possible that P'3 is a readthrough product of the P3 cistron. With one antiserum preparation we were able to detect P3 in extracts of tobacco plants infected with AIMV.

A Non-structural Protein of Alfalfa Mosaic Virus in the Walls of Infected Tobacco Cells

“…A second extraction with ESB, at 100 °C, yielded some residual P3 (lane 4). In addition to the major species, lanes 3 and 4 show a minor band (O in lane 3) which reacted specifically with the anti-pep3 serum and migrated between P3 and P'3 (the minor wheat germ translation product of RNA 3 marked with a diamond in lane T; Godefroy-Colburn et al, 1985;Berna et al, 1985). Other, apparently larger species were also visible.…”

“…In a preliminary experiment, A1MV-inoculated plants were kept at 10 °C for 10 days and extracted as described previously (Berna et al, 1985(Berna et al, , 1986. Instead of restricting our search for P3 to the Pe-30, we systematically assayed all the fractions, including the Miracloth residue which had been discarded in the previous study.…”

“…Propagation of AIMV (strain S) in Nicotiana tabacum, preparation of the 1000 g pellet (Pe-1), of the 30000 g pellet (Pe-30) and supematant fractions (S-30) from the leaf extract, fractionation of the S-30 by ammonium sulphate precipitation, preparation of the wheat germ translation products of AIMV RNA and of antisera to C-terminal peptides of P1, P2 and P3 (pepl, pep2, pep3), SDS-PAGE analysis of proteins, immunoblotting with the anti-peptide sera and goat anti-rabbit IgG labelled with peroxidase, and quantification of the immunoreactions by reflection densitometry were all as described by Berna et al (1985Berna et al ( , 1986. Except where otherwise stated, the extractions were done at 20 °C for 15 min and filtration through 'Miracloth' was replaced by centrifugation (3000 r.p.m., 10 min) through an 80-mesh nylon cloth fused to a polyethylene syringe body.…”

Immunodetection of grapevine fanleaf virus satellite RNA-encoded protein in infected Chenopodium quinoa

“…Uncapped transcripts synthesized from 0.4 ilg of EcoRI-linearized plasmid DNA were treated with RNase-free DNase I (Stratagene) and translated in the wheatgerm system in the presence of [3SS]methionine (Amersham) according to GodefroyColburn et al (1985), except that wheatgerm tRNA and RNasin were omitted and that the final concentrations of magnesium acetate and potassium acetate were 2.4 mM and 40 mM, respectively. In some experiments the translation medium was subsequently treated with 10 ~tg/ml RNase in 2 mM-EDTA pH 7-2 for 30 min at 37 °C (Berna et al, 1985) and centrifuged at 12000 g for 30 min to eliminate wheatgerm proteins remaining in the supernatant. One volume of double concentrated ESB was added to all samples before analysis by 10~ SDS-PAGE followed by immunoblotting.…”

Reduction of tobacco mosaic virus accumulation in transgenic plants producing non-functional viral transport proteins

“…A crude cell wall fraction and cytoplasmic membrane fraction sedimenting at 30000g were analysed by SDS-PAGE and immunoblotting with antibodies to an 11 amino acid synthetic oligopeptide corresponding to the carboxy terminus of the TMV UI 30K protein or to a 16 amino acid synthetic oligopeptide corresponding to the carboxy terminus of the BMV 32K protein using peroxidase-conjugated goat anti-rabbit antibodies as the second antibodies. TPs were identified by comparison with an immunoblot of the in vitro translation products visualized with orthodianisidine (Berna et al, 1985).…”