A new one-step procedure for the isolation of bacterial RNA, involving lysis by proteinase K in the presence of sodium dodecyl sulfate, is described.Pulse-labeled RNA isolated by this procedure from Bacillus brevis, Bacillus subtiZis, and Escherichia coZi B has been found to contain a substantial fraction (15-40%) of polyadenylated RNA as determined by adsorption to oligo (dT)-cellulose. This contrasts with RNA isolated by procedures involving phenol extraction, a process which appears to lead to the selective loss of polyadenylated RNA. The presence of polyadenylated RNA in E. coZi was confirmed by an independent method which involved hybridization with [3H]polyuridylic acid. Using the proteinase K method for RNA isolation, it was possible to demonstrate the in vitro synthesis of polyadenylated RNA by toluene-

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Sequences were determined of the coding regions of the M-protein genes of the Glasgow and Orsay strains of vesicular stomatitis virus (Indiana serotype) and of two group III (M-protein) mutants derived from each wild type. Synthetic primers were annealed with viral genomic RNA and extended with reverse transcriptase. The resulting high-molecular-weight cDNA was sequenced directly. Both Glasgow and Orsay wild types differed ip 13 bases from a clone of the San Juan strain sequenced by J. K. Rose and C. J. Gallione (J. Virol. 39:519-528, 1981). Six of these base changes caused amino acid changes in each wild type, whereas seven were degenerate. The Orsay aild Glasgow sequences resembled each other more closely than either resembled that of Rose and Gallione, differing in eight nucleotides and four amino acids. Each of the four mutants, however, differed from its pargnt wild type in only one or two point mutations. Every mutation caused a change either from or to a charged amino acid; the change for tsG3l was Lys (position 215) to Glu, the change for ts023 was Gly (position 21) 'to Glu, the change for ts089 was Ala (position 133) to Asp, the changes for tsG33 were Lys (position 204) to Thr and Glu (positioh 214) to Lys. The charge differences predicted from these amino acid changes was confirmed by nonequilibrium pH gradient electrophoresis for tsG31, tsG33, ts023, and the two wild types. These mutations affect residues spanning nearly 85% of the linear sequence, although the mutants possess nearly identical phenotypic properties.

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