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assembly of flexuous plant viruses and their proteins.recent experiments on the disassembly and assembly of some flexuous plant viruses and their proteins are described. the properties of reconstituted potato virus x and those of assembled potato virus y protein are considered as well as the suitability of other flexuous viruses for reconstitution studies.197613429
antiphytoviral activity of 2,4 dioxo hexahydro triazine.three treatments with 2,4 dioxo hexahydro triazine (dht) significantly reduced the concentration of potato virus x (pvx) in systemically infected tobacco plants. in hypersensitive plants dht caused a reduction in the number of local lesions produced by pvx. in systemic and hypersensitive hosts, treatment with dht resulted in a more or less marked reduction in the concentration of, and in the number of local lesions caused by, potato virus y (pvy), potato virus a (pva), tobacco mosaic virus (tmv) ...197942301
partial purification and characterization of the potato virus y helper component. 1977860406
[anomalous structures in the cytoplasm of cells infected with potato virus y]. 1977895578
[purification of pvy, pvx, tmv and spherical virus of pinks using polyethylene glycol and gel filtration]. 19761012092
characterization of the protein and nucleic acid of potato virus y strains isolated from pepper. 19751114695
some characteristics of infectious rna from potato virus y. 19751162905
[electron microscopic study of double infection induced by potato virus x and potato virus y in nicotiana glutinosa]. 19751214639
evidence for sense rna-mediated protection to pvyn in tobacco plants transformed with the viral coat protein cistron.the coat protein (cp) cistron of the tobacco veinal necrosis strain of potato virus y (pvyn), supplemented with translational start signals, was cloned into an agrobacterium tumefaciens ti transformation vector. transformation of tobacco leaf discs resulted in 99 transgenic lines which were subsequently analysed for the presence and expression, at both the transcriptional and translational level, of the cp-gene. although cp-specific rna transcripts were produced in all plants no cp could be dete ...19921280473
establishment of hybridoma cell line secreting specific monoclonal antibodies against turnip mosaic virus and analysis of properties of the mcab.monoclonal antibodies (mcab) of hybridomas derived from the fusing of the mouse splenocytes immunized by tumv with balb/c mouse myeloma cells (sp2/0-ag14). five kinds of hybridoma cell line were produced by indirect-elisa screening and cloning three times with limiting dilution. four kinds of hybridoma produced antibodies respectively reactive to tumv c1, c3, c4 and c5. one kind was reactive to all five strains of tumv. in indirect-elisa and sandwich-elisa tests, tumv specific monoclonal antibod ...19921343828
factors influencing immune electron microscopy of flexuous potato viruses.effects of ph of extraction buffers, ph and titer of trapping antisera and their combinations, virus acquisition time and virus host on the trapping efficiency of flexuous potato viruses x, s and y (pvx, pvs and pvy) in immune electron microscopy were evaluated. addition of ethylene-diamine-tetraacetic acid to the extraction buffer improved trapping of pvy, adversely affected pvs but not pvx. combinations of antisera had differential adverse effect on trapping which was maximum with the mixture ...19921364019
engineering resistance to mixed virus infection in a commercial potato cultivar: resistance to potato virus x and potato virus y in transgenic russet burbank.potato virus x (pvx) and potato virus y (pvy) infection in potato may result in the loss of certification of seed potatoes and affect quality and yield of potatoes in commercial production. we transformed a major commercial cultivar of potato, russet burbank, with the coat protein genes of pvx and pvy. transgenic plants that expressed both cp genes were resistant to infection by pvx and pvy by mechanical inoculation. one line was also resistant when pvy was inoculated with viruliferous green pea ...19901366358
protection against detrimental effects of potyvirus infection in transgenic tobacco plants expressing the papaya ringspot virus coat protein gene.we obtained transgenic tobacco plants expressing the papaya ringspot virus (prv) coat protein (cp) gene by transformation via agrobacterium tumefaciens. expression was effectively monitored by enzyme-linked immunosorbent assays (elisa) of crude tissue extracts. subcloned plants derived from eight original ro transformants were inoculated with potyviruses: tobacco etch (tev), potato virus y (pvy), and pepper mottle (pemv). plants that accumulated detectable levels of the prv cp showed significant ...19911367635
the complete nucleotide sequence of pepper mottle virus genomic rna: comparison of the encoded polyprotein with those of other sequenced potyviruses.the complete nucleotide sequence of a pepper mottle virus isolate from california (pepmov c) has been determined from cloned viral cdnas. the pepmov c genomic rna is 9640 nucleotides excluding the poly(a) tail and contains a long open reading frame starting at nucleotide 168 and potentially encoding a polyprotein of 3068 amino acids. comparison of the pepmov c presumptive polyprotein with those of other sequenced members of the potyvirus group, including tobacco etch virus (tev), tobacco vein mo ...19921413501
a comparison of pepper mottle virus with potato virus y and evidence for their distinction.pepper mottle virus (pepmov) was identified as a distinct potyvirus infecting peppers in arizona and florida in the 1970's. the distinction of pepmov from potato virus y (pvy) has recently been challenged on the basis of sequence comparisons of the coat proteins and of the 3' nontranslated regions of the viral rnas. we summarize the biological, cytological, serological, and in vitro translational studies which compare the apparent differences, and also similarities, between pepmov and pvy. we co ...19921450758
is pepper mottle virus a strain of potato virus y?on the basis of serological properties and host plant reactions pepper mottle virus (pepmov) has been classified as a potyvirus related to, but distinct from, other pepper-infecting potyviruses, potato virus y (pvy) and tobacco etch virus (tev). recent amino acid and nucleotide sequence data show that pepmov is more closely related to pvy than previously assumed. pepmov shows a high degree of homology to various pvy strains in both the coat protein and the 3' non-translated sequences, while unre ...19921450759
evidence that pepper mottle virus and potato virus y are distinct viruses: analyses of the coat protein and 3' untranslated sequence of a california isolate of pepper mottle virus.pepper mottle virus (pepmov) is a member of the large and complex genus potyvirus, and is classically distinguished from other members of the genus by differential host range and cytopathology as well as serology of the coat protein and cytoplasmic inclusion body proteins. here we report the deduced amino acid sequence of the coat protein of a california potyvirus identified by a variety of classical methods as pepmov (pepmov c). comparison of the 3' untranslated nucleic acid sequence and the de ...19921450760
production and characteristics of strain common antibodies against a synthetic polypeptide corresponding to the c-terminal region of potato virus y coat protein.comparing the predicted amino acid sequence between two japanese potato virus y (pvy) strains, necrotic strain and ordinary strain, it was found that the c-terminal regions (h2n-httedvspsmhtllgvknm-cooh) of the coat proteins in the two strains were completely conserved. the conserved amino acid sequence was also found in the c-terminal region coat protein of pvy-36, a strain which did not react with monoclonal antibodies specific to the necrotic and the ordinary strain respectively. antibodies w ...19921474133
coat protein properties suggest that azuki bean mosaic virus, blackeye cowpea mosaic virus, peanut stripe virus, and three isolates from soybean are all strains of the same potyvirus.the interrelationship of a number of potyviruses infecting legumes has been investigated by comparing molecular properties of their coat proteins. comparison of the coat proteins by the techniques of amino acid analysis and page was inadequate to distinguish strains from distinct potyviruses. however, high-performance liquid chromatographic peptide profiles of tryptic digests of coat proteins of these legume-infecting potyviruses enabled such assignments to be made. these data indicate that amin ...19921500273
effects of antisense oligodeoxynucleotide hybridization on in vitro translation of potato virus y rna.potato virus y (pvy), a potyvirus, has an rna genome containing 9704 nucleotides of which 185 belong to the 5' nontranslated region (ntr). contrary to most eukaryotic mrnas that have a cap structure, the potyvirus rna has a genome-linked protein (vpg). in order to understand the mechanisms of pvy rna translation initiation, hybrid-arrest translation was used to localize sequences involved in binding of proteins and/or ribosomes. the 5' ntr was fused to the beta-glucuronidase (gus) reporter gene. ...19921549909
nucleotide sequence of the 3'-terminal region of potato virus a rna.the sequence of the 3'-terminal region of the genome of the potato virus a (pva) was obtained from two independent cdna clones. this sequence is 1383 nucleotides long and contains an open reading frame of 1178 nucleotides, ending with the translation termination codon taa and followed by untranslated region of 205 nucleotides. since the n-terminal amino acid of the coat protein of pva was blocked, the position of the putative coat protein cleavage site has been deduced by searching for consensus ...19921604933
cloning and sequencing of the 3' half of a potato virus y (o strain) genome encoding the 5k protein, protease, polymerase and coat protein. 19921630927
comparison and differentiation of potyvirus isolates and identification of strain-, virus-, subgroup-specific and potyvirus group-common epitopes using monoclonal antibodies.a panel of monoclonal antibodies (mabs) generated against an admixture of 12 potyvirus isolates was used to compare and differentiate diverse potyviruses. both native and denatured virions of strains of bean yellow mosaic (bymv), potato virus y, tobacco etch, pea seed-borne mosaic, iris severe mosaic, iris mild mosaic and asparagus virus-1 potyviruses were used as immunogen and as antigen for screening of the hybridoma cell lines. thirty cell lines secreting potyvirus-specific antibodies reactiv ...19911703561
the use of peptide-mediated electrofusion to select monoclonal antibodies directed against specific and homologous regions of the potyvirus coat protein.whilst monoclonal antibodies (mab) to potyviruses have been generated, it has not been possible to produce molecules with high specificity or broad reactivity to defined conserved amino acid sequences. in the current study, peptide-mediated electrofusion was used to select for high efficiency antibody-secreting hybridomas after mice were immunized with highly immunogenic viral coat protein. mice were immunized with coat protein from either one potyvirus (potato virus y, pvy-d) or a mixture of fi ...19911719098
[the antigenic characteristics of the capsid proteins in strains of the potato virus y].the antigenic properties of capsid proteins of potato virus y (pvy) strains have been studied, the most wide antigenic specificity of necrotic group strains has been marked. several antigen-active strains of necrotic group strains (pvy-n far east, pvy-n leningrad, pvy-n moscow) and common group (pvy-o-3 moscow, pvy-o far east) have been revealed. antisera against these strains reacted with any pvy strain. virus specific surface epitope, corresponding to position 198-208 of polypeptide chain has ...19911722712
the complete nucleotide sequence of pea seed-borne mosaic virus rna.the complete nucleotide sequence of the rna genome of pea seed-borne mosaic virus (psbmv) was determined from cloned cdna and by direct sequencing of viral rna. the psbmv genomic sequence was determined to be 9924 nucleotides in length excluding the poly(a) tract. the rna contained an open reading frame (orf) of 9618 nucleotides with the potential to encode a polyprotein with a calculated mr of 364000 (364k). the orf was flanked by a 5' untranslated leader sequence of 143 nucleotides and a 3' un ...19911940858
cloning and mapping of the potato virus y genome and its in vitro expression.full-length cdna of genomic rna of potato virus y (pvy) was cloned in one piece into a lambda vector. the order of the ecori and sali fragments of the inserted cdna was determined. this is the first report of the cloning of a long, expressible, potyvirus genome. the availability of such a clone is a prerequisite for any further study of the molecular biology of this group of viruses, as they are expressed into a self-processed primary polyprotein.19901980175
resistance in solanum brevidens to both potato virus y and potato virus x may be associated with slow cell-to-cell spread.a series of experiments was carried out to investigate the nature of the resistance of the wild potato species, solanum brevidens, to potato virus x (pvx) and potato virus y (pvy). in vitro inoculation of leaf protoplasts of s. brevidens and the virus-susceptible dihaploid s. tuberosum genotype pdh40 with pvx or pvy using polyethylene glycol showed that protoplasts of both species were similar in susceptibility. however, examination of protoplasts prepared from the leaves of s. tuberosum and s. ...19911993865
development and application of a nonradioactive nucleic acid hybridization system for simultaneous detection of four potato pathogens.cdna clones of potato virus x (pvxcp strain), potato virus y (pvyo strain), potato leaf roll virus (plrv) and potato spindle tuber viroid (pstv) were used separately or combined for the detection of the corresponding rnas in extracts of infected plants. a general method for the rapid preparation of rna extracts without use of organic solvents (i.e. phenol) was developed for this purpose. plant extracts from a range of field, artificially inoculated germplasm genotypes, micro-propagated and proto ...19912016393
replication of potato virus x rna is altered in coinfections with potato virus y.potato virus x (pvx) and potato virus y (pvy) may coinfect tobacco to cause a classic synergistic disease. in the acute stage the disease is characterized by a dramatic increase in the accumulation of infectious pvx particles, with no corresponding increase or decrease in the accumulation of pvy. the accumulation of pvx genomic rna and coat protein has been examined in doubly versus singly infected tobacco leaves. these experiments indicate that the levels of both viral components increase in do ...19912024486
the establishment of rat hybridoma cell lines secreting mcab against strains of potato virus y and analysis of its stability.the rat splenocytes immunized with potato virus y (pvyn) and ratmyeloma (ir983) were fused by peg (m. w.1450). three kinds of stable hybridoma cell lines secreting specific monoclonal antibodies (mcabs) were derived. one kind of the cell lines producing mcabs reacts to pvyn specifically. another reacts to pvyo specifically. the third one reacts to both of the two strains. tested by the methods of sandwich-elisa and indirect-elisa, all kinds of mcabs did not react to seven plant viruses: tobacco ...19902104212
comparative sequence of the helper component (hc) region of potato virus y and a hc-defective strain, potato virus c.potato virus c (pvc), a non-aphid transmissible strain of potato virus y (pvy), was found to code for a protein (pvc-hc) which is similar in molecular weight and immunological reactivity to the helper component protein of pvy (pvy-hc). pvc-hc, however, was inactive with respect to its ability to effect aphid transmission of either pvc or pvy. the 5'-terminal 2.7-kb regions of pvc and pvy were sequenced. within the hc region there was 92% nucleotide homology between the two strains; comparison of ...19902219708
potato virus y helper component protein is associated with amorphous inclusions.the distribution of the helper component (hc) protein of potato virus y in tissues and cells of infected plants was studied by immunoblotting and immunogold labelling techniques. this hc protein was found in leaf blade and vein tissue but not in the petiole of leaves. in infected cells, the protein was localized in rod-shaped cytoplasmic inclusions known as amorphous inclusions.19902230744
nucleotide sequence of an altered virulence potato virus y coat protein gene (pvyh strain). 19902251149
polymerase chain-reaction-mediated cloning and expression of the coat protein gene of potato virus y in escherichia coli.complementary dnas (cdnas) to the rna genome of a necrotic strain of potato virus y in japan (hokkaido univ. isolate of pvy-t:pvy-th) were synthesized and cloned into a plasmid pbr322. about 4.3 kbp of the cdna sequence containing the 3'-poly(a) tract of pvy-th was inserted into a recombinant plasmid pbryt88. the coat protein coding region (cp gene) in pbryt88 was amplified using the polymerase chain reaction (pcr) and subcloned into a plasmid puc119. the nucleotide sequence of the cp gene was d ...19902288070
nucleotide sequence of the coat protein gene of pea seed-borne mosaic potyvirus.the nucleotide sequence of a 1355 bp cdna representing the 3'-terminal sequences of pea seed-borne mosaic virus (psbmv) was determined. this sequence contained a single long open reading frame (orf) of 1189 bp ending with a single taa termination codon. downstream from the orf was an untranslatable region of 189 bp followed by eight bp of polyadenylate. the probable location of the psbmv coat protein codons within the long orf was determined by comparing the inferred amino acid sequence with oth ...19902391503
localization of virus-specific and group-specific epitopes of plant potyviruses by systematic immunochemical analysis of overlapping peptide fragments.virus-specific or group-specific antibody probes to potyviruses can be produced by targeting the immune response to the virus-specific, n-terminal region of the capsid protein (29-95 amino acids depending on the virus) or to the conserved core region (216 amino acids) of the capsid protein, respectively. immunochemical analysis of overlapping, synthetic octapeptides covering the capsid protein of the johnsongrass strain of johnsongrass mosaic virus (jgmv-jg) has delineated the peptide sequences ...19892479020
the use of peroxidase anti-peroxidase (pap) complexes in the detection of plant viruses by elisa.an elisa test system for detection of plant viruses in field samples is described, based on the unlabelled antibody method using a peroxidase-antiperoxidase (pap) complex. novel features of the system include the use of acid-treated naked bacteria as combined carrier-adjuvants for the production of rabbit antiviral antibodies, and the use of acid-treated chicken antibodies (igy) for antigen trapping in the elisa. systems for detection of potato virus y (pvy), potato leafroll virus (plrv), grapev ...19892685004
nucleotide sequence of the 3'-terminal region of potato virus yn rna.the sequence of the 3'-terminal 1611 nucleotides of the genome of the tobacco veinal necrosis strain of potato virus y (pvyn) was determined. the sequence revealed an open reading frame of 1285 nucleotides, of which the start was not identified, and an untranslated region of 316 nucleotides upstream of a poly(a) tract. comparison of the open reading frame with the amino-terminal sequence of the viral coat protein enabled mapping of the start of the coat protein at amino acid -267, and indicated ...19892732687
nucleotide sequence of potato virus y (n strain) genomic rna.the complete nucleotide sequence of the genomic rna of the potyvirus potato virus y strain n (pvyn) was obtained from cloned cdnas. this sequence is 9704 nucleotides long and can encode a polyprotein of 3063 amino acids. the positions of the cleavage sites at the n terminus of the capsid and cytoplasmic inclusion proteins have been determined. other putative protein cleavage sites have been deduced by searching for consensus sequences and by analogy with the polyprotein of the tobacco vein mottl ...19892732709
molecular cloning of a potato virus y genome: nucleotide sequence homology in non-coding regions of potyviruses.an aphid-transmissible field isolate of potato virus y, strain mm, was purified from a pepper host and cloned. all but 18 terminal nucleotides of the 9.7 kb genome are apparently contained in two overlapping cdna clones. the library of cdna clones is likely to be representative of the viral rna population present in infected plants because restriction endonuclease maps derived from cloned and uncloned cdna are colinear, each region of the genome is represented by several independent clones, and ...19892769227
nucleotide sequence of the coat protein gene of a necrotic strain of potato virus y from new zealand.the sequence of the 3'-terminal 1,134 nucleotides of the genome of a new zealand isolate of a necrotic strain of potato virus y (pvyn) has been determined. this sequence contains one large open reading frame of 796 nucleotides, the start of which was not identified, which is capable of encoding a protein of 264 amino acid residues with a molecular weight of 29,631. comparison of the amino acid sequence with a published coat protein sequence of another strain, pvy-d, and with the amino acid seque ...19892802998
cloning of the potato virus y genes encoding the capsid protein cp and the nuclear inclusion protein nib.dna was synthesized complementary to the rna genome of potato virus y (pvy; strain go 16) and cloned into lambda vectors. the size of pvy-specific eco ri-restricted cdna ranged from 0.3 to approximately 22kb. two of the cdna clones each of which contained some 4kb of cdna sequence starting from the 3'-polyadenylated terminus were characterized by sequence analysis. presence of a single open reading frame suggests that pvy-specific proteins are synthesized as a polyprotein precursor. as with othe ...19892802999
on the inhibition of plant virus multiplication by ribavirin.the inhibition of the replication of potato virus x (pvx), belladonna mottle virus, tobacco mosaic virus, potato virus y (pvy), and tobacco necrosis virus by ribavirin and pyrazofurin is described with emphasis on the inhibition of pvx by ribavirin. ribavirin inhibits an early step of pvx replication. the inhibition is reversed to different degrees by all ribo- and deoxyribonucleosides, most strongly by thymidine. in tobacco leaves, nucleosides compete with ribavirin for phosphorylation to monop ...19872821895
coat protein of potyviruses. 4. comparison of biological properties, serological relationships, and coat protein amino acid sequences of four strains of potato virus y.four strains of potato virus y, pvy-d, pvy-10, pvy-18, and pvy-43, obtained from different australian sources were compared on the basis of their biological, serological and coat protein structural properties. each of the strains could be distinguished on the basis of their reactions on selected test plant species. two of the pvy strains, pvy-d and pvy-10, induced symptoms similar to those produced by the pvyo strain group. the reactions of pvy-18 and pvy-43, although comparable to pvyn in some ...19883144263
biotinylated nucleic acid hybridization probes for potato virus detection.cdna libraries, representative of potato viruses x (pvxc strain) and y (pvy degrees strain) genomes were obtained. a pvx cdna cloned fragment was sequenced and biotinylated to be used as hybridization probe for the detection of purified virus or nucleic acid extracts of infected plants. dot hybridization assay was sensitive to detect 4 ng of viral particles, corresponding to about 200 pg of viral rna. the level of detection in infected plant extracts was as effective as that obtained with the el ...19883214274
nucleotide sequence of the capsid protein gene of potato virus y (pvy).the nucleotide sequence of the 3' terminal region of potato virus y (pvy) was determined. starting with a poly(a) tail of 18 residues a non-coding region of 335 nucleotides precedes the region encoding for the virus coat protein (cp) 801 nucleotides long ending with a tga. this region was located by comparing the predicted amino acid sequence with the one determined for the pvy capsid protein by shukla et al. (1). both sequences contained 267 amino acids sharing about 94% homology. they differ, ...19883245130
partial purification of inclusions induced by tobacco etch virus and potato virus y. 19714107552
a virus-induced component of plant sap needed when aphids acquire potato virus y from purified preparations. 19744214349
location of the factor for resistance to potato virus y in tobacco. 19734782667
potato virus y particles in plasmodesmata of tobacco leaf cells. 19744813267
evidence that a component other than the virus particle is needed for aphid transmission of potato virus y. 19744818682
enhancement by potato virus y of potato virus x synthesis in doubly infected tobacco depends on the timing of invasion by the viruses. 19744821699
independent assembly of virions in tobacco doubly infected by potato virus x and potato virus y or tobacco mosaic virus. 19744826211
on the size of the protein subunits in potato virus y. 19725034362
cross protection test with four strains of potato virus y in nicotiana tabacum l. cv. samsun. 19695396065
increased hypersensitivity induced in tobacco by systemic infection by potato virus y. 19685651028
intranuclear crystals and cytoplasmic membranous inclusions, associated with infection by two brazilian strains of potato virus y. 19685698672
effect of ph and other conditions on immunosorbent electron microscopy of several plant viruses.the efficiency of trapping, using immunosorbent electron microscopy, of particles of cucumber mosaic virus, lily symptomless virus, potato virus y, and carnation mottle virus, depended markedly on the ph of the virus extract. varying the ph of the serum had a smaller, though still significant effect. with potato virus y, the optimal ph of the virus extract varied with the host plant. it appears that for each virus and host a specific testing procedure is required to find the optimal trapping con ...19826752161
the ultrastructure of nicotiana tabacum cells infected with potato virus x and potato virus y. 19827143535
distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants.tobacco etch potyvirus engineered to express the reporter protein beta-glucuronidase (tev-gus) was used for direct observation and quantitation of virus translocation in plants. four tev-gus mutants were generated containing capsid proteins (cps) with single amino acid substitutions (r154d and d198r), a double substitution (dr), or a deletion of part of the n-terminal domain (delta n). each modified virus replicated as well as the parental virus in protoplasts, but was defective in cell-to-cell ...19947511101
resistance against multiple plant viruses in plants mediated by a double stranded-rna specific ribonuclease.many plant viruses have single-stranded rnas as their genomes. during the course of replication, genomic rnas and their complementary template rnas are likely to form double-stranded (ds) rna at least transiently. to attack replication intermediates of many plant rna viruses specifically, we introduced a yeast-derived ds-rna specific rnase gene called pac 1 into plants. the transformed plants showed a decrease in lesion numbers when they were challenged with tomato mosaic virus, and a delay in t ...19957556661
transmission by aphids of a naturally non-transmissible plum pox virus isolate with the aid of potato virus y helper component.two spanish plum pox virus (ppv) isolates, 5.15 and 3.3, were used in transmission experiments involving the aphid vector myzus persicae, with woody and herbaceous host plants. these isolates differ in the size of their coat protein (cp) and sequence analysis revealed that isolate 3.3 has a 15 amino acid deletion near the n terminus of the cp, affecting the same positions as in a previously reported non-aphid-transmissible ppv isolate from germany. aphid transmission experiments showed that isol ...19957561767
characterization of antigenic epitopes of potato virus y.immunochemical analysis of overlapping synthetic hexapeptides covering the entire length of the coat protein of potato virus y (pvy) revealed immunodominant regions both at the n-terminal and at the c-terminal end of the coat protein. immunization of rabbits with synthetic peptides representing n- and c-terminal regions of the coat protein resulted in production of antibodies that reacted with pvy. antigenicity of pvy peptides was found to correlate with predicted beta turns, with hydrophilicity ...19937680576
improvement in the sensitivity of pvyn detection by increasing the cdna probe size.cloned cdna to a north american isolate of tobacco veinal necrotic strain of potato virus y (pvyn) rna was used for the detection of pvyn in diseased samples. digoxigenin-labelled cdna probes were prepared from approximately 0.56 kb, approximately 1.2 kb, approximately 2.5 kb, and approximately 3.25 kb overlapping clones representing the 3'-terminus of the pvyn genome. it was shown that the probe sizes, as determined by alkaline gel-electrophoresis, generally corresponded to the size of the temp ...19947714042
digoxigenin-labelled molecular probe for the simultaneous detection of three potato pathogens: potato spindle tuber viroid (pstvd), potato virus y (pvy), and potato leafroll virus (plrv).a molecular probe, p3pot, was constructed of pstvd, pvy, plrv cdna fragments introduced into puc18 vector. sequencing of the inserts revealed that cloned fragments covered conservative parts of pathogenic genomes. dot-blot hybridization of digoxigenin-labelled construct to crude extracts from plants infected with different potato viruses proved high sensitivity and specificity of the p3pot probe. this makes p3pot probe an useful tool for the routine testing, and selection of virus-free potatoes.19947732766
cleavage of potato virus y polyprotein in escherichia coli depending on the proteolytic activity of viral protease.we have cloned a 5-kb cdna corresponding to the 3'-half of genomic rna of potato virus y (pvy), the type member of plant potyvirus. based on its nucleotide sequence, the cdna was presumed to encode pvy protease responsible for the self-processing of polyprotein precursor expressed from pvy genome. to test its proteolytic activity, the cdna was modified so as to express the protease-polymerase-coat protein (cp) portion of pvy polyprotein in escherichia coli. by western blotting analysis of the ce ...19937763774
mutational analysis of the amino acid residues essential for the cis and trans cleavage activity of the potato virus y 50-kda protease.to examine the proteolytic activities of various truncated derivatives of the potato virus y (pvy) 50-kda protease, the derivatives were expressed in escherichia coli in polyprotein forms fused with coat protein (cp). for the intermolecular cleavage reaction, the truncated proteases were expressed together with the substrate protein containing the polymerase-cp junction. the activity was evaluated by the amount of the mature cp released from the precursor by the intra- and intermolecular cleavag ...19937764224
digoxigenin-labelled cdna probes for the detection of potato virus y in dormant potato tubers.an isolate (y139) of the potato virus y strain pvyo, common in the potato-growing regions of eastern canada, was cloned and segments of about 1800 bases from the 3'-end of the pvy-genome were sequenced. these clones were used to prepare molecular detection probes by labelling with digoxigenin. five methods reported to yield nucleic acid preparations from tuber tissue were compared. two of the methods failed to yield nucleic acids from tubers suitable for use in dot-blot hybridization. the other ...19957769026
5' proximal potyviral sequences mediate potato virus x/potyviral synergistic disease in transgenic tobacco.the interaction of potato virus x (pvx) and potato virus y (pvy) in tobacco causes a synergistic disease characterized by a dramatic increase in symptom severity, a change in the regulation of pvx rna replication, and an increase in accumulation of pvx. in this study we demonstrate that pvx also interacts synergistically with three other members of the potyvirus group of plant viruses, tobacco vein mottling virus (tvmv), tobacco etch virus (tev), and pepper mottle virus. these synergisms resembl ...19957831814
small-scale field tests with transgenic potato, cv. bintje, to test resistance to primary and secondary infections with potato virus y.the coat protein (cp) gene of the potato virus y (pvy) strain n605 has been cloned into a plant binary expression vector and introduced into the potato variety bintje. the transformed lines, bt6, that contained two copies of the cp gene showed complete resistance to the homologous strain pvy-n605 and a good resistance to the related strain pvy-o803 in the greenhouse. the good resistance of bt6 to primary and secondary infections by pvy was confirmed in two successive field tests where the virus ...19947919216
characterization and strain identification of a potato virus y isolate non-reactive with monoclonal antibodies specific to the ordinary and necrotic strains.an isolate of potato virus y, named pvy-36, reacted with polyclonal antibody against pvy-o (an ordinary strain), but not with any of eight monoclonal antibodies (mabs) specific to pvy-o or with two mabs specific to pvy-t (a necrotic strain). from its host range and symptomatology, pvy-36 belongs to the pvyo group. the nucleotide sequence of the coat protein (cp) coding region of the pvy-36 genome was determined and the amino acid sequence was predicted. based on the cp amino acid sequence, pvy-3 ...19947928283
molecular cloning and sequencing of the capsid and the nuclear inclusion protein genes of a north american pvyn isolate.the sequence of the 3'-terminal 3258 nucleotides of a tobacco veinal necrosis strain of a potato virus y (pvyn) isolate from north america was determined. the sequence revealed an open reading frame of 2931 nucleotides, of which the start codon was not identified. the nontranslated region contains 327 nucleotides upstream of a poly(a) tract. the open reading frame encodes a large polyprotein containing 976 amino acids. the data indicate that the coat protein (cp) and the nuclear inclusion protei ...19947954114
[the effect of the 5'-leader of potato x-virus on expression of the gene for the potato y virus membrane protein in transgenic solanum tuberosum].the 5'-leader of potato virus x (pvx) genomic rna was successfully used to generate potato plants expressing the coat protein of the potato virus yn (russian isolate) (pvy cp). two expression cassettes were constructed that carried the pvy cp coding sequence along with the pvy 3'untranslated region under the control of the camv 35s promoter and poly(a) signal. the cassettes contained different sequences between the 35s promoter and the cp artificial aug initiating codon of the coat protein: eith ...19947990802
transgenic plant virus resistance mediated by untranslatable sense rnas: expression, regulation, and fate of nonessential rnas.haploid leaf tissue of tobacco cultivars k326 and k149 was transformed with several transgenes containing cdna of the potato virus y (pvy) coat protein (cp) open reading frame (orf). the various transgenes containing the pvy cp orf sequence produced (1) the expected mrna and cp product, (2) an mrna rendered untranslatable by introduction of a stop codon immediately after the initiation codon, or (3) an antisense rna that was untranslatable as a result of the incorrect orientation of the pvy cp o ...19947994177
principles and background for the construction of transgenic plants displaying multiple virus resistance.we investigated the possibility of reconstructing the 2'-5' oligoadenylate (2-5a) pathway into the plant kingdom to achieve multiple virus resistance. differently phosphorylated 2-5a trimers and tetramers inhibited tmv rna translation in cell-free systems. in wheat germ extracts the most potent inhibitors were nonphosphorylated forms of 2-5a. triphosphorylated forms of 2-5a were deposphorylated and hydrolysed in plant extracts. since we could not detect homologous dna to mammalian 2-5a synthetas ...19948032271
the 5' untranslated region of pvy rna, even located in an internal position, enables initiation of translation.potato virus y (pvy) is the type member of the potyvirus group. potyviruses, like picorna-, como-, and nepoviruses, belong to the picornavirus-like supergroup. all these viral rnas have a vpg at their 5' end, and for four picornaviruses and one comovirus internal initiation of translation has been reported. to know if such a translational mechanism holds true for potyviral rnas, the 5' nontranslated region (ntr) of pvy rna was placed in an internal position, either by adding 91 bases upstream of ...19938122396
replicase-mediated resistance to potato virus y in transgenic tobacco plants.nicotiana tabacum 'turkish samsun nn' plants were transformed with nuclear inclusion b (nib) gene sequences of potato virus y, o strain (pvyo). the full-length construct included an additional in-frame initiation codon contiguous to the putative n-terminal amino acid codon and a stop codon replacing the c-terminal amino acid codon. of 13 independently transformed lines, four yielded 37 (out of 100) plants in the r1 generation that were resistant to pvyo infection. progeny of 13 out of 15 of r1 p ...19948167367
a pcr-microplate hybridization method for plant virus detection.a sensitive nonradioactive method for detection of plant viruses was evaluated. a cdna fragment from the coat protein coding region of the potato virus y (pvy) rna genome amplified by reverse transcription (rt) followed by polymerase chain reaction (pcr), was directly adsorbed onto polystyrene microplate wells after heat denaturation. the adsorbed cdna was hybridized with a digoxigenin (dig)-labelled cdna probe without a prehybridization step. the probe was also prepared by pcr using the same pa ...19948188816
mutations in the helper component protease gene of zucchini yellow mosaic virus affect its ability to mediate aphid transmissibility.the nucleotide sequence of the helper component protease (hc-pro) genes of three zucchini yellow mosaic virus (zymv) strains has been compared with that of a helper-deficient strain of zymv-hc. the comparisons revealed three unique deduced amino acid differences. two of these mutations were located in regions which are conserved in other potyviruses. the role of these mutations in aphid transmissibility was examined by exchanging dna fragments of part of the deficient hc-pro gene with the respec ...19948207404
efficient pathogen-derived resistance induced by integrated potato virus y coat protein gene in tobacco.the coat protein (cp) gene from potato virus y (hungarian isolate, pvy-h) was engineered into agrobacterium tumefaciens binary vector for expression in different tobacco lines. three different nicotiana tabacum breeding lines were transformed and the integration of the cp gene was confirmed by pcr technique using genomic dna preparations. the transcription and expression of the integrated cp gene was detected by northern and western blots. pathogen-derived resistance was demonstrated by inoculat ...19938268262
mutations in zucchini yellow mosaic virus helper component protein associated with loss of aphid transmissibility.zucchini yellow mosaic virus (zymv) is a potyvirus transmitted by aphids in a non-persistent manner. isolates having partially or totally lost their ability to be transmitted by aphids have been isolated and found to be affected in their helper component activities. we have sequenced the helper component coding region of poorly aphid-transmissible (pat) variants of two strains of zymv, e15 and r5a. mutations have been identified at the nucleotide level leading to two amino acid changes in the e1 ...19938277279
coat protein gene-mediated resistance to potato virus y in tobacco: examination of the resistance mechanisms--is the transgenic coat protein required for protection?we transformed sr1 tobacco with a cdna fragment of the potato virus y strain n (pvyn) 605 genome which contained the 3' end of the nib polymerase gene, the coat protein (cp) coding sequence, and most of the viral 3' untranslated region. complete resistance to several pvy strains was obtained in plants of the r2 generation. the resistance to pvyn and pvyo strains was maintained when the plants were inoculated dually with the potato potyviruses v or a (pvv or pva). only partial resistance to pvyn ...19938324247
genetic engineering of potyvirus resistance using constructs derived from the zucchini yellow mosaic virus coat protein gene.three versions of the zucchini yellow mosaic virus (zymv) coat protein gene were engineered for expression in plants: the full-length coat protein sequence, the conserved core portion of the gene, and an antisense version. these constructs were introduced into muskmelon (cucumis melo) and tobacco plants (nicotiana tabacum) via agrobacterium tumefaciens-mediated transformation; gene expression was verified by northern and western analysis. transgenic r0 and r1 muskmelon plants expressing the full ...19938324251
signal for potyvirus-dependent aphid transmission of potato aucuba mosaic virus and the effect of its transfer to potato virus x.a british isolate of potato aucuba mosaic potexvirus (pamv) was transmitted by aphids (myzus persicae) which had fed previously on a source of potato y potyvirus (pvy). nucleotide sequence analysis of the pamv coat protein gene indicated that amino acid residues 14 to 16 from the n terminus of the coat protein have the sequence dag, which is also found in the coat proteins of potyviruses and is required for their aphid transmissibility. a recombinant virus isolate (txpa7) was produced in which a ...19938336116
taxonomic relationships between distinct potato virus y isolates based on detailed comparisons of the viral coat proteins and 3'-nontranslated regions.detailed comparisons were made of the sequences of the coat protein (cp) cistrons and 3'-nontranslated regions (3'-ntr) of 21 (geographically) distinct isolates of potato virus y (pvy) and a virus isolate initially described as pepper mottle virus (pepmov). multiple sequence alignments and phylogenetic relationships based on these alignments resulted into a subgrouping of virus isolates which largely corresponded with the historical strain differentiation based on biological criteria as host ran ...19938347079
nucleotide sequence of the capsid protein cistrons from six potato virus y (pvy) isolates infecting tobacco.complementary dna libraries representing the capsid protein cistron of the potato virus y (pvy) isolate 'chilean', 'hungarian', msnr, nsnr, o, and 'potato us' were synthesized and used as template for polymerase chain reaction (pcr) amplification. an aug codon for initiating a discrete capsid protein (cp) open reading frame was embedded upstream of the first codon of the cp cistrons. pcr-amplified products of the expected size of 0.8 kilo bases were cloned into the transcription vector pbs(+). t ...19938352655
plants transformed with a cistron of a potato virus y protease (nia) are resistant to virus infection.an oligonucleotide carrying signals for translation initiation in plants was engineered upstream to a cdna clone containing nucleotides 5812-7260 of the potato virus y (pvy) genome. this fragment contains all but the first 100 5' terminal bases of the cistron encoding one of the pvy proteases (nia) as well as the first 251 bases of the next cistron (nib). nicotiana tabacum cv. sr1 plants were transformed with this fragment. the presence of the nia sequences in transformed plants was determined b ...19938356047
expression of the "helper component" protein of potato virus y (pvy) in e. coli: possible involvement of a third protease.transmission of potyviruses by aphids depends on the presence of a virus encoded helper-component protein (hc) that also exhibits protease activity. hc was expressed in e. coli from two types of clones: a full-length cdna clone of pvy and two 5' end clones containing the first three cistrons (3.6-3.7 kbp). the clones derived from the 5' end of pvy expressed hc of the size of the mature component. other proteins reacting with antibodies to hc were also observed, and their sizes corresponded with ...19938367942
molecular cloning, sequencing, and expression in escherichia coli of the potato virus y cytoplasmic inclusion gene.complete nucleotide sequences of cytoplasmic inclusion (ci) genes of two strains of potato virus y (pvy) were determined from six polymerase chain reaction (pcr)-amplified cdna clones. the size of the ci genes of both ordinary (pvy-o) and necrotic strains (pvy-t13) was 1902 nucleotides, with a sequence homology of 83.4%. comparison of the predicted amino acid sequences showed more than 90% homology. when these were compared with those of other potyviruses, the homology ranged from 53 to 61%. cdn ...19938418790
cloning and sequencing of potato virus y (hungarian isolate) genomic rna.a sequence of 9703 nucleotides (nt) is reported for the genomic rna of potato virus y (hungarian isolate, pvy-h), which causes necrotic rings around the buds on the tubers and mottling of leaves. the sequence contains one large open reading frame of 3061 amino acids (aa), a noncoding region of 189 nt at the 5' end and a 330-nt 3' nontranslated region. the nt sequence and the predicted aa sequence of the polyprotein of pvy-h were analysed pairwise with the only available complete sequence of pvy ...19938428653
complex formation determines the activity of ribozymes directed against potato virus yn genomic rna sequences.a ribozyme was synthesized against a conserved region in the rna-dependent rna-polymerase encoding cistron of the important plant pathogen potato virus y (pvy). this ribozyme was shown to cleave pvy-specific rna-transcripts efficiently in vitro, with up to 95% of the substrate rna being cleaved within 2 h incubation at 37 degrees c. a second ribozyme, designed with much shorter viral complementary arms in an attempt to optimize the efficiency of the cleavage reaction, surprisingly failed to clea ...19938460529
expression and assembly of the potato virus y (pvy) coat protein (cp) in escherichia coli cells.a clone harboring the full-length cdna of potato virus y in a lambda-dash vector under the control of a t7 promoter was introduced into escherichia coli carrying the t7-rna-polymerase gene on a plasmid. the viral coat protein was expressed and the product was of the same size as the corresponding mature protein in infected plants. immunoelectronmicroscopy of transfected cell extracts revealed virus-like particles, indicating that the proteins involved in its processing and the viral coat protein ...19938493811
a simplified procedure for the subtractive cdna cloning of photoassimilate-responding genes: isolation of cdnas encoding a new class of pathogenesis-related proteins.transgenic tobacco plants (ppa-1) constitutively expressing escherichia coli pyrophosphatase behind the 35s camv promoter accumulate high levels of soluble sugars in their leaves [27]. these plants were considered a tool to study adaptation of leaves to photoassimilate accumulation at the molecular level. by differential hybridization of a subtractive library enriched for transcripts present in the transgenic plants 12 different cdnas were isolated. by sequence analysis four cdnas could be ident ...19958555446
cell-free cloning and biolistic inoculation of an infectious cdna of potato virus y.potato virus y (pvy) full-length cdna has been found to be refractory to cloning in escherichia coli cells. a full-length 9.7 kb pvy cdna was obtained by reverse transcription polymerase chain reaction (rt-pcr) from the rna of pvy (tuber necrotic strain, pvyntn). double-stranded dna fragments were used as primers (ds megaprimers), to include signals for transcription in vivo (a cauliflower mosaic virus 35s rna promoter and a nopaline synthase terminator) in the final pcr product. biolistic bomba ...19968601790
loss of potyvirus transmissibility and helper-component activity correlate with non-retention of virions in aphid stylets.the hypothesis that loss of aphid transmissibility of potyvirus mutants is due to non-retention of virions in the mouthparts was tested by feeding aphids through membranes on purified virions of aphid transmissible (at or hat) and non-aphid-transmissible (nat) tobacco vein mottling virus (tvmv) or tobacco etch virus (tev), in the presence of functional [potato virus y (pvy) hc or tvmv hc] or non-functional (pvc hc) helper component (hc). tvmv virions were detected, by electron microscopic examin ...19968609482
nucleic acid-binding properties of a bacterially expressed potato virus y helper component-proteinase.the potyvirus helper component-proteinase (hc-pro) is a multifunctional protein previously reported to have affinity for polyribonucleotides. to investigate further the ability of hc-pro to bind nucleic acids, the potato virus y (pvy) lye84 isolate hc-pro gene was amplified, cloned in an escherichia coli expression vector and sequenced. hc-pro was expressed as a fusion with the maltose-binding protein and purified by affinity chromatography. electrophoretic mobility-shift assays demonstrated tha ...19968609483
switches in the mode of transmission select for or against a poorly aphid-transmissible strain of potato virus y with reduced helper component and virus accumulation.a poorly aphid-transmissible potato virus y (pvy-pat) variant emerged after several cycles of mechanical transmission of an initially aphid-transmissible (at) isolate. sequence analysis of the n-terminal region of the helper component-proteinase (hc-pro) gene revealed a lys to glu change at a position previously found to abolish the hc-pro aphid transmission activity in several potyviruses. two cycles of aphid transmission allowed the virus population to evolve towards an at form (pvy-atnew) whe ...19968757973
frequent occurrence of recombinant potyvirus isolates.we have performed a systematic search for recombination in the region encoding coat protein and the 3' non-translated region in natural isolates of potyviruses, the largest group of plant rna viruses. the presence of recombination, and the localization of the cross-over points, were confirmed statistically, by three different methods. recombination was detected or suspected in 18 out of 109 potyvirus isolates tested, belonging to four out of eight virus species, and was most prevalent in potato ...19968760448
the complete nucleotide sequence of yam mosaic virus (ivory coast isolate) genomic rna.the complete nucleotidic sequence of the yam mosaic virus (ymv) rna was determined following the cloning of partial segments of the genome by reverse transcription and polymerase chain reactions (rt-pcr) using degenerate and/or specific oligonucleotide primers. ymv genomic rna is 9,608 nucleotides in length and contains one open reading frame (orf) encoding a polyprotein of 3,103 amino acids (aa) with a calculated mr of 350,915. the 5' leader sequence of ymv rna preceding the orf is 134 nucleoti ...19968774686
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