Hugh D. Robertson

Experimental data concerning viroid-specific nucleic acids accumulating in tomato plants establish, together with earlier studies, the major features of a replication cycle for viroid RNA in plant cells. Many features of this pathway, which involves multimeric strands of both polarities, may be shared by other small infectious RNA's including certain satellite RNA's and "virusoid" RNA's which replicate in conjunction with conventional plant viruses. The presence, in host plans, of an elaborate machinery for replicating these disease agents suggests a role for endogenous small RNA's in cellular development.

Abstract A nuclease with specificity for double-stranded RNA (RNase III) has been found in extracts of Escherichia coli. It remains within osmotically shocked cells attached to the ribosomes. It sediments with the ribosomes in less than 0.20 m NH4Cl, but is detached at higher concentrations. A purification is described which utilizes this property. The free enzyme, after diethylaminoethyl Sephadex and carboxymethyl Sephadex chromatography, shows an absolute specificity for polymers containing double helical polyribonucleotide regions—other polymers (single- and double-stranded DNAs, single-stranded RNAs) are not digested, nor do they inhibit the digestion of double-stranded RNAs when present in excess. RNase III shows an absolute requirement for divalent cations, including Mg++ and Mn++, and for monovalent cations, including NH4+, K+, and Na+. The optimal pH range for the reaction is from pH 7.6 to pH 9.75. RNase III is inactivated by exposure to many substances at monovalent cation concentrations below 0.2 m. The enzyme is not a factor required for protein synthesis in vitro. Its mode of action appears to be endonucleolytic.

Abstract Precursor molecules of Escherichia coli wild type and mutant tyrosine tRNA's contain at both their 5' and 3' termini extra nucleotides in addition to those of the mature tRNA molecule. The early steps of processing these precursor molecules must involve specific ribonuclease cleavage. We report the isolation from E. coli extracts of the specific endonucleolytic RNase which cleaves only a single phosphodiester bond of the 129 nucleotide tyrosine tRNA precursor molecule. This cleavage removes all extra nucleotides present at the 5' terminus of the precursor as a 41 nucleotide fragment, exposing the 5' end of the mature tRNA. After sufficient purification, this activity has no effect upon the extra nucleotides at the 3' end of the tRNA precursor. Therefore processing of the two ends of this molecule must be carried out by different enzymatic activities. This novel RNase activity, which we have called RNase P, has been purified by washing ribosomes with 0.2 m NH4Cl, followed by ammonium sulfate fractionation and chromatography on DEAE-Sephadex and phosphocellulose. At this stage it shows no evidence of other E. coli RNase activities. RNase P requires both monovalent and divalent cations for optimal activity, and has a pH optimum of 8.0. In the course of purifying RNase P, we have discovered in other subcellular fractions of E. coli RNase activities potentially responsible for additional steps of precursor tRNA processing.

Nucleic acids isolated from uninfected and potato spindle tuber viroid-infected Rutgers tomato plants were fractionated on agarose gels under two different sets of denaturing conditions and hybridized to (125)I-labeled viroid in a series of blot hybridization experiments. Complementary strand nucleic acids detected in extracts of infected plants were heterogeneous in size, with four discrete bands containing molecules approximately 700, 1050, 1500, and 1800 nucleotides long. Enzymatic studies indicated that these viroid minus strands are composed exclusively of RNA and, as extracted, are present in complexes containing extensive double-stranded regions. After treatment with several RNases under conditions favoring digestion of single-stranded regions, the high molecular weight minus strands can no longer be detected and roughly unit-length minus strands appear. A model for the structure of the viroid replication intermediate is proposed.