Viroids are uncoated infectious RNA molecules pathogenic to certain higher plants. Four different highly purified viroids were studied. By ultracentrifugation, thermal denaturation, electron microscopy, and end group analysis the following features were established: (i) the molecular weight of cucumber pale fruit viroid from tomato is 110,000, of citrus exocortis viroid from Gynura 119,000, of citrus exocortis viroid from tomato 119,000, and of potato spindle tuber viroid from tomato 127,000. (ii) Viroids are single-stranded molecules. (iii) Viroids exhibit high thermal stability, cooperativity, and selfcomplementarity resulting in a rod-like native structure. (iv) Viroids are covalently closed circular RNA molecules. Viroids exert biological functions like viruses since they are infectious and pathogenic. In contrast to viruses, however, all known viroids seem to exist only as uncoated RNA molecules and their genetic information is not sufficient to code for a protein with a molecular weight larger than 10,000. They are pathogens of certain higher plants. Little is understood, as yet, about their mechanisms of replication and pathogenicity. Moreover, the available information is too limited to lead to a concise structural model of viroids (1). Previous data on molecular weights of viroids were obtained by indirect methods and the determinations were based on particular assumptions about their unknown conformation. Analysis in sucrose gradients, in polyacrylamide gels, and in the electron microscope resulted in molecular weight values ranging between 25,000 and 150,000 for both the viroid of the spindle tuber disease of potato (PSTV) (e.g. refs. 2-4) and the viroid of the exocortis disease of citrus (CEV) (e.g., refs. 5-7). The sedimentation coefficients reported range from 4 S to 25 S (2, 6, 7). A considerable number of structural models have been proposed (e.g. refs. 4, 7, and 8); single strands with hairpins or double strands with incomplete base pairing have been considered as the most probable structure of viroids.The availability of highly purified viroid RNA in sufficient quantities enabled us to investigate the structure in detail.

No abstract

Double mutations in the quinolone resistance determining region of the gyrase A gene (gyrA) have recently been reported to be associated with high-level resistance to fluoroquinolones in clinical isolates of Escherichia coli. We examined the type and frequency of such mutations in a large number of clinical isolates that were obtained from ten different geographical locations and had been genotypically characterized by pulsed field gel electrophoresis (PFGE) of chromosomal DNA digests. Of 36 isolates with ofloxacin MICs > or = 4 mg/L that represented at least 24 distinct genotypes, 35 had double mutations at amino acid codons 83 and 87 of gyrA, while two isolates with ofloxacin MICs of 0.5 and 4 mg/L, respectively, each had a single mutation at codon 83. Mutations at codon Ser-83 were uniform, resulting in substitution by Leu. The additional mutations at amino acid codon 87 in the 35 double-mutants were diverse, resulting in Asp-87 substitutions by residues Asn (23 isolates), Gly (7 isolates), Tyr (4 isolates), or His (1 isolate) without a discernable correlation with fluoroquinolone MICs or with phenotypic resistance to chemically unrelated antibacterial agents. Maximal differences between MICs of double-mutants with the same amino acid substitution were eight-fold. The changes of amino acid residues at codon Asp-87 differed between individual patient isolates with the same genotype (and similar MICs), suggesting that the amino acid codon 87 mutations (and possibly the development of high-level fluoroquinolone resistance) might have occurred after the transmission and sharing of a precursor strain carrying the Ser-83-->Leu mutation.

On purification of human adenovirus type 12 (Adl2) by equilibrium sedimentation in CsCl density gradients, two bands of particles, are

The binding and replication of purified potato spindle tuber viroid (PSTV) by DNA-dependent RNA polymerase II from wheat germ was studied in analytical ultracentrifugation experiments and in vitro transcription assays. The equilibrium association constant for the viroid-polymerase interaction is 1.9 X 10(7) M-1. Both ultraviolet and fluorescent monitoring during the sedimentation experiments showed two distinguishable viroid-polymerase complexes. These are interpreted as resulting from a 1:1 and 2:1 enzyme-to-viroid binding stoichiometry. A265/A280 ratios across the sedimenting boundaries, the sedimentation velocity of the complexes, as well as electron microscopic data support this interpretation. The role of viroid secondary structure in enzyme binding and polymerization is discussed in the light of these results and compared with binding and polymerization data for virusoid RNA, single- and double-stranded RNA, and double-stranded DNA.