Improved Estimates of Molecular Weight of Plant Virus RNA by Agarose Gel Electrophoresis and Electron Microscopy after Denaturation with Glyoxal

Abstract

SUMMARY The RNA molecules of 13 plant viruses were denatured by heating at 50 °C in 1 m-glyoxal, 50% dimethyl sulphoxide, and their mol. wt. (expressed as the sodium salts) estimated both by electrophoresis in 0.75% agarose gel, and by measuring their lengths in the electron microscope. The two methods gave similar results. Compared with previous estimates obtained by electrophoresis at 60 °C in 2% polyacrylamide gels containing 8 m-urea, the new estimates were similar for molecules of mol. wt. up to about 1.7 × 106 (about 5000 nucleotides) but became increasingly larger as the mol. wt. exceeded this value. The mol. wt. of the RNA-1 molecules of a range of nepoviruses were all found to be about 2.7 × 106 to 2.8 × 106 (about 7800 to 8100 nucleotides) by the glyoxal/agarose method compared with about 2.1 × 106 to 2.2 × 106 (about 6100 to 6400 nucleotides) by the hot urea/polyacrylamide method. The estimated mol. wt. of RNA-1 (B-RNA) of cowpea mosaic virus (CPMV) was 2.39 × 106 (6900 nucleotides), about 20% greater than the commonly used value, those of the single RNA species of apple chlorotic leafspot and heracleum latent viruses were 2.48 × 106 (7170 nucleotides) and 2.52 × 106 (7260 nucleotides) respectively and that of parsnip yellow fleck virus was 3.34 × 106 (9650 nucleotides). Values of 2.37 × 106 (6840 nucleotides) and 0.54 × 106 (1550 nucleotides) were found respectively for RNA-1 and RNA-2 of the CAM strain of tobacco rattle virus (TRV). For the nepoviruses, and for CPMV and TRV, the new mol. wt. estimates were in good agreement with those predicted from the morphology or sedimentation behaviour of the virus particles. Electrophoresis of glyoxylated RNA in agarose gels is preferred to other electrophoretic methods for determining the mol. wt. of RNA molecules larger than about 1.7 × 106 (about 5000 nucleotides), and appears reliable for mol. wt. up to at least about 3.5 × 106 (10000 nucleotides).