Current status of viroid taxonomy.

Viroids are the smallest autonomous infectious nucleic acids known so far. With a small circular RNA genome of about 250-400 nt, which apparently does not code for any protein, viroids replicate and move systemically in host plants. Since the discovery of the first viroid almost forty-five years ago, many different viroids have been isolated, characterized and, frequently, identified as the causal agents of plant diseases. The first viroid classification scheme was proposed in the early 1990s and adopted by the International Committee on Taxonomy of Viruses (ICTV) a few years later. Here, the current viroid taxonomy scheme and the criteria for viroid species demarcation are discussed, highlighting the main taxonomic questions currently under consideration by the ICTV Viroid Study Group. The impact of correct taxonomic annotation of viroid sequence variants is also addressed, taking into consideration the increasing application of next-generation sequencing and bioinformatics for known and previously unrecognized viroids.

[Molecular properties of potato spindle tuber viroid (PSTVd) isolates from the collection of the Russian Research Institute of Phytopathology].

The complete nucleotide sequences of more than 100 isolates of PSTVd collected from locations in the territory of Russia and the former USSR have been determined. These sequences represent 42 individual sequence variants, each containing 1-10 mutations with respect to the "intermediate" or type strain of PSTVd (GenBank Acc. No. v01465). Isolates containing 2-5 mutations were the most common, and 24 sequence variants are described here for the first time. Twenty one isolates contained a mutation found only in Russian and Ukrainian isolates of PSTVd up till now; i.e., replacement of the adenine at position 121 with cytosine (A121C). Many of these isolates contained two mutations--deletion of one of the three adenine residues occupying positions 118-120 plus replacement of the adenine at position 121 with either uracil or cytosine (A120, A121U/C). Both combinations of mutations were phenotypically neutral, i.e. symptom expression in Rutgers tomato was unaffected. Phylogenetic analysis of the sequences of different PSTVd isolates presented in work together with sequences of other naturally-occurring isolates obtained from Internet databases suggesting that known PSTVd isolates may be divided into four groups: i) a group of isolates from potato, tomato and solanaceous ornamentals where the type strain of PSTVd (PSTVd.018) may be considered to represent the ancestral sequence, ii) a group of isolates from potato, tomato and solanaceous ornamentals where PSTVd.123 play the same role as PSTVd.018 for the first group, and iii) a group of potato isolates where PSTVd.125 is a possible ancestral sequence. The fourth and most divergent group of PSTVd isolates differs significantly from these first three groups. The majority of isolates in this group originate from New Zealand and Australia and infect different solanaceous hosts (tomato, pepper, cape gooseberry, potato, and others).

Mexican papita viroid and tomato planta macho viroid belong to a single species in the genus Pospiviroid.

Tomato planta macho viroid (TPMVd) and Mexican papita viroid (MPVd) are two closely related (>90% sequence identity) members of the genus Pospiviroid. Their current status as members of separate species is based upon the reported ability of TPMVd to replicate in Gomphrena globosa and the inability of this viroid to evoke flower break in N. glutinosa. Characterization of a viroid recently isolated from diseased tomato plants grown in Mexico (identical to GenBank accession GQ131573) casts doubt on this earlier report and indicates that these viroids should be classified as members of a single species. Giving priority to the older name, we propose including both of these viroids in the current species Tomato planta macho viroid.

Sensitive and rapid diagnosis of potato spindle tuber viroid disease by nucleic Acid hybridization.

A sensitive and reliable new method for the detection of potato spindle tuber viroid in potato tubers has been developed. The method is based on hybridization of highly radioactive recombinant DNA to viroid RNA that has been attached to a solid support. The method can be automated and permits the rapid testing of large numbers of tubers.

Molecular Detection and Identification of Group 16SrI and 16SrXII Phytoplasmas Associated with Diseased Potatoes in Russia.

Phytoplasmal diseases have long been suspected to occur in several potato-growing regions in Russia on the basis of symptoms and the presence of insect vectors. Symptoms resembling stolbur are most prevalent, but round leaf disease, potato witches'-broom, and potato purple top wilt also occur (1). The phytoplasma etiologies of these diseases have never been verified by molecular means. During the summer of 2006, 33 potato plants exhibiting various symptoms including purple top, round leaves, and stolbur-like symptoms characterized by purple top, stunting, bud proliferation, and formation of aerial tubers were randomly collected from the Volga River Region, Central Region, and Northern Caucasian Region in Russia. DNA extracts were prepared from 1.0 g of petioles and leaf mid veins according to a modified procedure with the Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA) as previously described (2). A nested PCR with primer pair P1/P7 in the first amplification followed by R16F2n/R16R2n in the second amplification was performed to detect phytoplasmas in infected potato samples (4). Potato plants maintained in the greenhouse were used as healthy controls. A negative control devoid of DNA templates was included in all PCR assays. One microliter of diluted (1:30) PCR product from the first amplification was used as the template in the nested PCR. Eight of 33 potato samples tested positive in the first PCR. Twelve of 33 potato samples tested positive in nested PCR. Nine of the 12 potato samples that tested positive for phytoplasma exhibited stolbur-like symptoms; the other three samples exhibited round leaves, stunting, or proliferation. The remaining symptomatic samples that exhibited nonspecific purple or yellow discoloration of terminal leaves, without other specific stolbur-like symptoms, may be infected by other pathogens. Restriction fragment length polymorphism (RFLP) analysis of nested PCR products (R16F2n/R16R2n amplicons, 1.2 kb) was performed. PCR products (6 µl) were digested singly with the restriction enzymes AluI, HaeIII, HhaI, HpaII, KpnI, MseI, RsaI, and Tsp509I. Comparison of RFLP profiles with published profiles (3) was used for identification of the putative phytoplasmas detected. Among the 12 PCR positive potato samples, 10 showed very similar or identical RFLP profiles to stolbur phytoplasma, a strain belonging to stolbur phytoplasma group (16Sr XII), subgroup 16SrXII-A and closely related strains, and two showed RFLP profiles similar to those of aster yellows phytoplasma group (16SrI). Nucleotide sequence analysis of cloned 16S rDNA (GenBank Accession Nos. EU344884-EU344890 and EU333396-EU333400) confirmed the results of the RFLP analyses and also indicated that the two samples showing 16SrI profiles were simultaneously infected with two phytoplasma strains belonging to subgroups 16SrI-A and 16SrI-B. To our knowledge, this is the first confirmation by molecular procedures that stolbur phytoplasma (16SrXII-A) is prevalent in several potato-growing regions and is the first report of 16SrI-A and 16SrI-B phytoplasmas associated with potatoes in Russia. References: (1) D. Z. Bogoutdinov. Potato Phytoplasmas and Methods of Their Study. Samara State Agricultural University, Samara, 2000. (2) M. J. Green et al. Plant Dis. 83:482, 1999. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) I.-M. Lee et al. Plant Dis. 90:989, 2006.

Russian isolates of potato spindle tuber viroid.

Forty PSTVd isolates collected from five regions of Russia (North-western, Central, Volga region, Northern Caucasus and the Far East) were sequenced during 2006-2008. All isolates lacked the adenine residue present at position 123 of the type strain; i.e., PSTVd-intermediate (GenBank V01465). Nineteen Russian isolates also contained an adenosine --> cytosine substitution at position 120. Twenty two additional unique changes were also observed in one or more of the isolates sequenced.

Identification of phytoplasma species associated with potato diseases in Russia.

Four out of six known potato diseases attributed to phytoplasma infection were previously reported to occur in Russia based on a combination of biological properties such as symptomatology and/or vector relationships and electron microscopy of infected phloem tissue. In 2007, the first molecular identification of potato diseases causing symptoms including purple top, round leaves, stunting, bud proliferation and formation of aerial tubers was carried out using PCR methods. A nested PCR using primer pair P1/P7 in the first amplification followed by R16F2n/R16R2n in the second amplification was performed to detect phytoplasma in infected potato samples. PCR products were digested singly with several restriction enzymes. Comparison of RFLP profiles with published profiles was used for identification of the putative phytoplasma detected. The majority of 49 PCR positive potato samples showed RFLP profiles of 16S rDNA sequences very similar or identical to stolbur phytoplasma, a strain belonging to stolbur phytoplasma group (16Sr XII), subgroup 16SrXII-A, and only two showed RFLP profiles similar to those of aster yellow phytoplasma strains ('Candidatus Phytoplasma asteris') belonging to aster yellows phytoplasma group (16SrI), subgroup 16SrI-A and 16SrI-B. The results demonstrated that stolbur phytoplasma is prevalent in several potato growing regions of Russia.

Dioxin-dependent activation of murine Cyp1a-1 gene transcription requires protein kinase C-dependent phosphorylation.

Transcriptional activation of the murine Cyp1a-1 (cytochrome P(1)450) gene by inducers such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (dioxin) requires the aromatic hydrocarbon (Ah) receptor and the interaction of an inducer-receptor complex with one or more of the Ah-responsive elements (AhREs) located about 1 kb upstream from the transcriptional initiation site. We find that treatment of mouse hepatoma Hepa-1 cells with 2-aminopurine, an inhibitor of protein kinase activity, inhibits CYP1A1 mRNA induction by TCDD as well as the concomitant increase in CYP1A1 enzyme activity. Formation of DNA-protein complexes between the Ah receptor and its AhRE target is also inhibited by 2-aminopurine, as determined by gel mobility shift assays. Phosphorylation is required for the formation of Ah receptor-specific complexes, since in vitro dephosphorylation of nuclear extracts from TCDD-treated Hepa-1 cells abolishes the capacity of the Ah receptor to form specific complexes with its cognate AhRE sequences. To determine whether any one of several known protein kinases was involved in the transcriptional regulation of the Cyp1a-1 gene, we treated Hepa-1 cells with nine other protein kinase inhibitors prior to induction with TCDD; nuclear extracts from these cells were analyzed for their capacity to form specific DNA-protein complexes. Only extracts from cells treated with staurosporine, a protein kinase C inhibitor, were unable to form these complexes. In addition, staurosporine completely inhibited CYP1A1 mRNA induction by TCDD. Depletion of protein kinase C by prolonged treatment with phorbol ester led to the complete suppression of CYP1A1 mRNA induction by TCDD. We conclude that (i) phosphorylation is necessary for the formation of a transcriptional complex and for transcriptional activation of the Cyp1a-1 gene; (ii) the phosphorylation site(s) exists on at least one of the proteins constituting the transcriptional complex, possibly the Ah receptor itself; and (iii) the enzyme responsible for the phosphorylation is likely to be protein kinase C.

Recovery of Four Novel Potato spindle tuber viroid Sequence Variants from Russian Seed Potatoes.

First described in the early 1930s, the limited distribution of potato "gothic" disease made it of little economic significance in European Russia until the early 1970s when meristem-tip culture was widely adopted throughout the former USSR to increase production of virus-free seed potatoes. Shortly thereafter, the yield and quality of Russian seed potatoes began a dramatic decline. Symptoms of potato "gothic" resemble those of Potato spindle tuber viroid (PSTVd) (3), and initial suspicions that in vitro plantlets and seed potatoes might be viroid-infected were later proved correct when Kastalyeva et al. (2) showed that approximately 50 to 70% of in vitro plantlets and tubers collected from different regions of Russia as well as the in vitro germplasm collection maintained by the All-Russian Potato Research Institute (ARPRI) were infected with PSTVd. Measures have since been taken to reduce the incidence of PSTVd infection, and numerous PSTVd isolates were collected from territories of the former USSR; however, none of these isolates have been characterized at the molecular level. Overlapping reverse transcription (RT)-PCR products (1) were generated from four PSTVd isolates maintained in field-grown tubers at the VNIIF using two pairs of primers; PSTVd180F (5'-TCACCCTTCCTTTCTTCGGGTGTC-3') + PSTVd179R (5'-AAACCCTGTTTCGGCGGGAATTAC-3') and PSTVd112F (5'-ACT GGCAAAAAAGGACGGTGGGGA-3') + PSTVd359R (5'-AGGAACC AACTGCGGTTCCAAGGG-3'). Automated sequence analysis of the resulting uncloned PCR products revealed the presence of four previously unknown PSTVd variants (GenBank Accession Nos. EF044302-EF044305). All four tubers were also infected with Potato virus M and Potato virus Y and one tuber also contained Potato virus S. ELISA tests for Potato leaf roll virus were negative. Each isolate appeared to contain only a single 358-359 nt variant differing from PSTVd-intermediate strain (GenBank Accession No. V01465) at 2-5 positions. The three closely related variants originating from Leningradskaya Province (Northwest Russia) contain two to three changes in the variable domain and central conserved region and induced intermediate symptoms in Rutgers tomato. The fourth variant originating from Samarskaya Province (Volga River Region) contains additional changes in the pathogenicity domain and induced mild symptoms. Minor differences among the Leningradskaya variants may represent sequence drift during extended (9 to 11 year) tuber passage. The presence of additional sequence changes in the variant from Samarskaya is consistent with independent origin and/or prolonged separation. Additional studies with a wider range of Russian isolates of PSTVd are currently underway to develop diagnostic methods suitable for future large-scale screening programs. References: (1) Y. Hu et al. Virology 219:45, 1997. (2) T. B. Kastalyeva et al. Vestn. RASKHN 3:22, 1992. (3) Y. A. Leontyeva. Potato spindle tuber ('gothic') as one of the most important diseases in the Volga region. (In Russian.) Ph.D. thesis. Agricultural University of Leningrad, Pushkin, 1971.

Human CYP1A2: sequence, gene structure, comparison with the mouse and rat orthologous gene, and differences in liver 1A2 mRNA expression.

We have sequenced the human CYP1A2 (cytochrome P(3)450) gene, 1,906 basepairs (bp) of the 5' flanking region, and 113 bp of the 3' flanking region. The gene spans almost 7.8 kilobases, comprising seven exons and six introns. The transcriptional start site was determined by both primer extension and S1 mapping. Including the first noncoding exon of 55 bp, the entire mRNA is 3,121 bp in length, and the open reading frame, starting with nucleotide 10 of exon 2, encodes 515 amino acids (mol wt = 58,294). Between the human CYP1A2 and CYP1A1 (cytochrome P(1)450) genes, exons 2, 4, 6, and especially 5 are strikingly conserved in both nucleotide similarity and total number of bases. Alignment of the upstream sequences and exon 1 of human CYP1A2 with that of mouse or rat CYP1A2 revealed two possibly significant regions of similarity: 1) 68% in the approximately 150 bases immediately 5' from the mRNA cap site and 2) 80% identify between the human -841 to -758 segment and the mouse -1,529 to -1,439 segment. The canonical 5-bp box (CACGC), found upstream of all mammalian CYP1A1 genes to date and believed to interact with the inducer.aromatic hydrocarbon receptor complex, was not found on either strand in the 1,906 bp of the 5' flanking region of human CYP1A2. In contrast, alignment of the upstream sequences, exon 1, and intron 1 of human CYP1A1 with that of mouse or rat CYP1A1 revealed large, highly conserved regions. Conserved regions were found in intron 1 of the human, mouse, and rat CYP1A2 gene. These data suggest that the regulatory elements controlling the CYP1A2 gene might differ in location from those controlling the CYP1A1 gene. Among 12 human liver samples, striking differences (greater than 15-fold) in the 3.3-kilobase 1A2 mRNA levels were seen. This result may reflect significant genetic differences in constitutive and/or inducible CYP1A2 gene expression that could play an important role in individual risk of environmental toxicity or cancer.

Mutational analysis of viroid pathogenicity: tomato apical stunt viroid.

A series of nucleotide substitutions within the pathogenicity domain of tomato apical stunt viroid have been evaluated for their effects upon infectivity and symptom expression. None of the 12 A----G substitutions and one C----U substitution that were examined abolished infectivity in a whole plant bioassay, and the resulting progeny were characterized by nucleotide sequence analysis of cDNAs amplified by the polymerase chain reaction. Four of the 13 substitutions gave rise to altered progeny, but the patterns of sequence changes observed were unexpectedly complex. Mutations that did not rapidly revert to the wild-type sequence are located near the right border of the pathogenicity domain, a region which shows considerable natural sequence variability. None had a detectable effect upon symptom expression. The ability to observe viroid sequence evolution in vivo may provide insight into the molecular interactions responsible for viroid host range and symptom formation.

Possible involvement of the phloem lectin in long-distance viroid movement.

Incubation with cucumber phloem exudate in vitro results in a dramatic decrease in the electrophoretic mobility of Hop stunt viroid. UV cross-linking and a combination of size exclusion and ion exchange chromatography indicate that this phenomenon reflects a previously unsuspected ability of phloem protein 2, a dimeric lectin and the most abundant component of phloem exudate, to interact with RNA. In light of its demonstrated ability to move from cell to cell via plasmodesmata as well as long distances in the phloem, our results suggest that phloem protein 2 may facilitate the systemic movement of viroids and, possibly, other RNAs in vivo.

Total shoulder arthroplasty.

Surgeons perform total shoulder arthroplasty (TSA) procedures to reduce patients' intractable arthritic pain and to repair humeral head fractures. Total shoulder arthroplasty has undergone remarkable advances--not only in prosthetic improvements and refinements--but in patient outcomes. Advantages to TSA procedures include decreased pain and patients' increased ability to perform activities of daily living.

In vitro synthesis and characterization of DNA complementary to potato spindle tuber viroid.

When DNase-digested calf thymus DNA is used as primer, the RNA-directed DNA polymerase of avian myeloblastosis virus will accept potato spindle tuber viroid (PSTV) as a template for DNA synthesis. The DNA obtained hybridizes specifically to PSTV and low molecular weight RNA isolated from PSTV-infected plants; no hybridization to unrelated viral RNAs or tow molecular weight RNA isolated from uninoculated host plants was detectable. Purified PSTV cDNA is heterogeneous in length, but the mean chain length is approximately one-fourth that of PSTV. The kinetics of PSTV PSTV cDNA hybridization and the thermal denaturation profile of the resulting hybrids are consistent with the known molecular weight (1.27 x 10(5)) and G + C content (54.7%) of PSTV.

The Rep68 protein of adeno-associated virus type 2 stimulates expression of the platelet-derived growth factor B c-sis proto-oncogene.

Rep68 protein, encoded by adeno-associated virus type 2 (AAV), has been previously shown to bind to specific sequences within the viral genome and in human chromosome 19. The effect of AAV Rep protein on human cellular genes is of interest because AAV is being developed as a gene therapy vector. We have identified sequences related to the Rep recognition sequence in the AAV P5 promoter in or near the c-sis proto-oncogene and the genes coding for a hepatocyte glucose transporter, alpha-A-crystallin, and carcinoma marker GA733-1. The ability of Rep68 to bind to these sites was established by gel shift assays, and the effect of Rep68 on the expression of these genes was tested by semiquantitative reverse transcriptase PCR. Rep68 enhances the expression of the c-sis proto-oncogene, which codes for the B polypeptide of platelet-derived growth factor, a multifunctional growth factor that is involved in embryonic development, tissue regeneration, osteogenesis, fibrosis, atherosclerosis, and neoplasia.

Glutathione: a protective agent in Salmonella typhimurium and Escherichia coli as measured by mutagenicity and by growth delay assays.

Cultures of some aerobically grown strains of Salmonella typhimurium and Escherichia coli contain up to 24 microM extracellular glutathione (GSH) [Owens RO, Hartman PE (1985): Environ Mutagen 7(Suppl 3): 47] in addition to having intracellular GSH concentrations in the millimolar range. The addition of 26 microM GSH to cultures of Salmonella typhimurium strain TA1534 partially protected the bacteria from the toxic effects causing growth delay by 54 microM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). When MNNG was preincubated with equimolar GSH, the mutagenicity of the MNNG was neutralized. The addition of micromolar GSH to cultures of an Escherichia coli GSH- strain protected the cells from growth inhibition by micromolar concentrations of mercuric chloride, methyl mercuric chloride, silver nitrate, cisplatin, cadmium chloride, cadmium sulfate, and iodoacetamide. In the cases of mercuric chloride, cisplatin, MNNG, silver nitrate, and iodoacetamide, reaction products with GSH were detected by paper chromatography. In contrast to reduced GSH, micromolar concentrations of oxidized glutathione (GSSG) provided little or no protection and formed no detectable reaction products. Export of GSH by enteric bacteria may provide an important defense mechanism against exogenous toxic agents otherwise active in the micromolar range.

Export of glutathione by some widely used Salmonella typhimurium and Escherichia coli strains.

Significant levels of extracellular glutathione (GSH) were detected in aerobically grown cultures of some strains of Salmonella typhimurium LT-2 and in Escherichia coli K-12, B, and B/r but not in cultures of nine freshly isolated clinical isolates of E. coli. Cultures of S. typhimurium generally contained less total GSH (intracellular plus external) than did E. coli cultures. S. typhimurium TA1534 contained about 2 mM intracellular GSH and exported about 30% of its total GSH. The external GSH concentration increased logarithmically during exponential growth and peaked at about 24 microM in early-stationary-phase cultures. External accumulation of GSH was inhibited by 30 mM NaN3. GSH was predominantly exported in the reduced form. Two-dimensional paper chromatography of supernatants from cultures labeled with Na2(35)SO4 confirmed the presence of GSH and revealed five other sulfur-containing compounds in the media of S. typhimurium and E. coli cultures. The five unidentified compounds were not derivatives of GSH.

Mutational analysis of potato spindle tuber viroid reveals complex relationships between structure and infectivity.

Viroids are single-stranded, covalently closed circular RNA pathogens that can be isolated from certain higher plants afflicted with specific diseases. Their small size (246-375 nucleotides; M(r) 0.8-1.3 x 10(5)) and ability to replicate autonomously make viroids a unique model system in which to study the relationships between the structure of an RNA and its biological function. The demonstrated infectivity of certain cloned viroid cDNAs allows the use of site-specific mutagenesis techniques to probe structure-function relationships suggested by comparative sequence analysis. Several site-specific mutations that disrupt base pairing in either the native structure or secondary hairpin I destroyed the ability of potato spindle tuber viroid cDNA to initiate infection. Alterations in the terminal loops of the native structure also abolished cDNA infectivity. One pseudorevertant, a mutant cDNA containing compensating changes that restore base pairing in the native structure, was marginally infectious; a second pseudorevertant in which base pairing was restored within the stem of secondary hairpin I was not infectious. The behavior of these mutants dramatically demonstrates the effect of remarkably small structural changes on viroid infectivity and emphasizes the importance of the conserved rod-like native structure for viroid function.