Publications
| Title | Abstract | Year Filter | PMID(sorted ascending) Filter |
|---|
| suppression of long-distance movement of tobacco etch virus in a nonsusceptible host. | to investigate host functions involved in the tobacco etch potyvirus (tev) infection process, a tobacco line (v20) with a strain-specific defect in supporting systemic infection was analyzed. using a modified tev encoding a reporter protein, beta-glucuronidase (gus), genome amplification, cell-to-cell movement, and long-distance movement were measured in v20 and a susceptible line, havana425. comparable levels of tev-gus genome amplification were measured in inoculated protoplasts from both toba ... | 1996 | 8642685 |
| rna-mediated resistance with nonstructural genes from the tobacco etch virus genome. | sense rna-mediated virus resistance has been described for transgenic plants expressing potyviral capsid protein sequences. this study was undertaken to determine if expression of other viral sequences could induce this type of virus resistance. plants showing highly resistant or 'recovery' phenotypes were generated by expressing the tobacco etch virus (tev) 6 kda/21 kda reading frames. expression of translatable or untranslatable versions of this tev sequence produced resistant lines. highly re ... | 1995 | 8664491 |
| roles of the sequence encoding tobacco etch virus capsid protein in genome amplification: requirements for the translation process and a cis-active element. | the roles of the capsid protein (cp) and the cp coding sequence of tobacco etch potyvirus (tev) in genome amplification were analyzed. a series of frameshift-stop codon mutations that interrupted translation of the cp coding sequence at various positions were introduced into the tev genome. a series of 3' deletion mutants that lacked the cp coding sequence beyond each of the frameshift-stop codon mutations were also produced. in addition, a series of 5' cp deletion mutants were generated. amplif ... | 1996 | 8676460 |
| a mammalian 2-5a system functions as an antiviral pathway in transgenic plants. | resistance to virus infections in higher vertebrates is mediated in part through catalysis of rna decay by the, interferon-regulated 2-5a system. a functional 2-5a system requires two enzymes, a 2-5a synthetase that produces 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5a) in response to double-stranded rna, and the 2-5a-dependent rnase l. we have coexpressed these human enzymes in transgenic tobacco plants by using a single plasmid containing the cdnas for both human rnase l and a low mol ... | 1996 | 8692895 |
| analysis of the vpg-proteinase (nia) encoded by tobacco etch potyvirus: effects of mutations on subcellular transport, proteolytic processing, and genome amplification. | a mutational analysis was conducted to investigate the functions of the tobacco etch potyvirus vpg-proteinase (nia) protein in vivo. the nia n-terminal domain contains the vpg attachment site, whereas the c-terminal domain contains a picornavirus 3c-like proteinase. cleavage at an internal site separating the two domains occurs in a subset of nia molecules. the majority of nia molecules in tev-infected cells accumulate within the nucleus. by using a reporter fusion strategy, the nia nuclear loca ... | 1996 | 8794348 |
| functions of the tobacco etch virus rna polymerase (nib): subcellular transport and protein-protein interaction with vpg/proteinase (nia). | the nib protein of tobacco etch potyvirus (tev) possesses several functions, including rna-dependent rna polymerase and nuclear translocation activities. using a reporter protein fusion strategy, nib was shown to contain two independent nuclear localization signals (nls i and nls ii). nls i was mapped to a sequence within amino acid residues 1 to 17, and nls ii was identified between residues 292 and 316. clustered point mutations resulting in substitutions of basic residues within the nlss were ... | 1997 | 8995687 |
| potyvirus transmission is not increased by pre-acquisition fasting of aphids reared on artificial diet. | aphids (myzus persicae), fasted after removal from healthy rearing plants, transmitted tobacco etch potyvirus (tev) more efficiently than unfasted aphids whether virus acquisition was from infected leaves or through membranes. there was no difference in uptake of 125i-labelled tev by fasted or unfasted aphids as measured by liquid scintillation counting. when aphids acquired 125i-labelled tev, label was retained in the stylets (as determined by autoradiographic light microscopy) by 51 % of 272 f ... | 1996 | 9000109 |
| genome amplification and long-distance movement functions associated with the central domain of tobacco etch potyvirus helper component-proteinase. | the tobacco etch potyvirus (tev) helper component-proteinase (hc-pro, 460 amino acid residues) is a multifunctional protein involved in aphid-mediated transmission, genome amplification, polyprotein processing, and long-distance movement. to investigate the interrelationships between three of these functions, 25 alanine-scanning mutations affecting clusters of charged residues were introduced into the hc-pro coding sequence. the resulting mutants were analyzed with respect to hc-pro proteolytic ... | 1997 | 9123832 |
| mutations in the region encoding the central domain of helper component-proteinase (hc-pro) eliminate potato virus x/potyviral synergism. | coinfection of tobacco plants with potato virus x (pvx) and any of several members of the potyvirus group causes a synergistic disease characterized by a dramatic increase in symptom severity correlated with a 3- to 10-fold increase in the accumulation of pvx in the first systemically infected leaves. we have recently shown that pvx/potyviral synergistic disease is mediated by expression of potyviral 5'-proximal sequences encoding p1, helper component-proteinase (hc-pro), and a fraction of p3 (t ... | 1997 | 9143300 |
| two separate regions in the genome of the tobacco etch virus contain determinants of the wilting response of tabasco pepper. | infection of tabasco pepper by the tobacco etch virus (tev) typically causes wilting associated with root necrosis. however, a strain of tev, designated tev nonwilting (tev nw), is able to infect tabasco pepper plants but does not cause wilting. in order to locate the genetic determinants responsible for the wilting response, a full-length cdna clone of tev nw from which infectious transcripts can be derived was made. a number of chimeric constructs were prepared by substituting cdna fragments b ... | 1997 | 9150596 |
| immunocytology shows the presence of tobacco etch virus p3 protein in nuclear inclusions. | intracellular localization studies of various potyvirus proteins have been made in hope of finding clues to their function(s). immunocytological studies localized many of the tobacco etch virus (tev)-encoded proteins in infected cells. we used antiserum against the nonstructural p3 protein of tev to determine the subcellular location of the p3 protein in ultrathin sections of virus-infected cells. immunogold labeling with the antiserum showed labels associated with nucleoli, nuclei, or nis, abso ... | 1997 | 9169234 |
| transgenic accumulation of two plant virus coat proteins on a single self-processing polypeptide. | an expression cassette based on the highly specific tobacco etch potyvirus (tev) nuclear inclusion (nia) proteinase has been developed to produce multiple proteins through the translation of a single self-processing polypeptide. gene constructs encoding tev nia, the tobacco mosaic tobamovirus (tmv) coat protein (cp) and the soybean mosaic potyvirus (smv) cp were used to develop transgenic tobacco plants. proper processing of the multifunctional polypeptide was demonstrated, leading to accumulati ... | 1997 | 9225054 |
| formation of plant rna virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein. | the mechanisms that direct positive-stranded rna virus replication complexes to plant and animal cellular membranes are poorly understood. we describe a specific interaction between a replication protein of an rna plant virus and membranes in vitro and in live cells. the tobacco etch virus (tev) 6 kda protein associated with membranes as an integral protein via a central 19 amino acid hydrophobic domain. in the presence or absence of other viral proteins, fluorescent fusion proteins containing t ... | 1997 | 9233814 |
| suppression of potyvirus infection by coexpressed closterovirus protein. | a tobacco etch virus (tev)-based expression vector has been used for insertion of several orfs derived from the unrelated beet yellows virus (byv). hybrid tev variants expressing the byv capsid protein, 20-kda protein, or hsp70 homolog systemically infected nicotiana tabacum and stably retained byv sequences. in contrast, insertion of the orf encoding byv leader proteinase (l-pro) resulted in severely impaired systemic transport and accumulation of recombinant tev. progeny of this virus underwen ... | 1997 | 9268155 |
| vpg of tobacco etch potyvirus is a host genotype-specific determinant for long-distance movement. | the v20 cultivar of nicotiana tabacum was shown previously to exhibit a strain-specific restriction of long-distance movement of tobacco etch potyvirus (tev). in v20, both tev-hat and tev-oxnard strains are capable of genome amplification and cell-to-cell movement, but only tev-oxnard is capable of systemic infection by vasculature-dependent long-distance movement. to investigate the basis for host-specific movement of tev, chimeric virus genomes were assembled from tev-hat and tev-oxnard. virus ... | 1997 | 9343220 |
| rna binding activity of nia proteinase of tobacco etch potyvirus. | the c-terminal domain of nia protein (niapro) from tobacco etch potyvirus (tev) is a sequence-specific proteinase required for processing of the viral polyprotein. this proteinase also interacts with nib, the tev rna-dependent rna polymerase. niapro and two niapro-containing polyproteins (nia and 6/nia) were analyzed from extracts of recombinant escherichia coli. using rna-protein blot and uv-crosslinking assays, niapro and the niapro-containing polyproteins were shown to possess rna-binding act ... | 1997 | 9356344 |
| tntin and tntap: mini-transposons for site-specific proteolysis in vivo. | tobacco etch virus (tev) protease recognizes a 7-aa consensus sequence, glu-xaa-xaa-tyr-xaa-gln-ser, where xaa can be almost any amino acyl residue. cleavage occurs between the conserved gln and ser residues. because of its distinct specificity, tev protease can be expressed in the cytoplasm without interfering with viability. polypeptides that are not natural substrates of tev protease are proteolyzed if they carry the appropriate cleavage site. thus, this protease can be used to study target p ... | 1997 | 9371808 |
| charge changes near the n terminus of the coat protein of two potyviruses affect virus movement. | mutants of tobacco vein mottling virus (tvmv) with substitutions of lys or arg for asp in the dag motif at position 5 in the coat protein (cp) failed to infect tobacco plants systemically, but replicated and produced virions in protoplasts. occasional systemic infections occurred when nicotiana benthamiana or transgenic tobacco plants expressing wild-type tvmv cp were inoculated with these mutants, but viral progeny contained reversions to negatively or non-charged amino acids at position 5 or s ... | 1998 | 9460938 |
| a hypersensitive response-like mechanism is involved in resistance of potato plants bearing the ry(sto) gene to the potyviruses potato virus y and tobacco etch virus. | potato plants carrying the ry(sto) gene from solanum stoloniferum are extremely resistant to a number of potyviruses, but it is not known at what stage of infection the resistance is expressed. the resistance may be due to ry(sto) or to a closely linked gene. in this investigation, we used potato virus y (pvy) and a tobacco etch virus construct that encodes beta-glucuronidase (tev-gus) to monitor virus infections of potato plants. systemic spread of either virus in resistant potato plants was no ... | 1998 | 9460939 |
| simultaneous accumulation of multiple viral coat proteins from a tev-nia based expression vector. | we previously described an expression cassette that relies on the tobacco etch virus (tev) nuclear inclusion a (nia) protease and leads to the coordinated accumulation of multiple proteins through self processing of a polyprotein [21]. however, low levels of proteins accumulated when the full-length protease was encoded within the polyprotein [22]. studies were conducted to evaluate whether the disruption of nia nuclear localization would affect the levels of proteins produced via the cassette. ... | 1998 | 9484436 |
| secondary structures in the capsid protein coding sequence and 3' nontranslated region involved in amplification of the tobacco etch virus genome. | the 3'-terminal 350 nucleotides of the tobacco etch potyvirus (tev) genome span the end of the capsid protein (cp)-coding sequence and the 3' nontranslated region (ntr). the cp-coding sequence within this region contains a 105-nucleotide cis-active element required for genome replication (s. mahajan, v. v. dolja, and j. c. carrington, j. virol. 70:4370-4379, 1996). to investigate the sequence and secondary structure requirements within the cp cis-active region and the 3' ntr, a systematic linker ... | 1998 | 9557696 |
| identification and characterization of a locus (rtm1) that restricts long-distance movement of tobacco etch virus in arabidopsis thaliana. | screens of arabidopsis thaliana for susceptibility to tobacco etch virus (tev) revealed that each of 10 ecotypes were able to support genome replication and cell-to-cell movement in inoculated leaves. however, only four ecotypes, including c24 and la-er, supported complete infections in which tev was able to replicate and move from cell to cell and long distances through the vasculature. the rates of cell-to-cell movement of a reporter-tagged tev strain (tev-gus) in inoculated leaves of c24 and ... | 1998 | 9628015 |
| role of the helper component in vector-specific transmission of potyviruses. | four aphid species were tested for their ability to transmit tobacco etch (tev) and turnip mosaic (tumv) potyviruses. myzus persicae and aphis gossypii transmitted both viruses efficiently from infected plants, whereas lipaphis erysimi transmitted only tumv and myzus ascalonicus was a poor or non-transmitter of either virus. similar electrically monitored probing patterns were produced by m. persicae, l. erysimi and m. ascalonicus, ruling out behavioural differences as the cause of differential ... | 1998 | 9634096 |
| functional characterization and localization of protein phosphatase type 2c from paramecium. | we cloned a protein phosphatase 2c gene from paramecium (ptpp2c), which codes for one of the smallest pp2c isoforms (klumpp, s., hanke, c., donella-deana, a., beyer, a., kellner, r., pinna, l. a., and schultz, j. e. (1994) j. biol. chem. 269, 32774-32780). after mutation of 9 ciliate q codons (taa) to caa ptpp2c was expressed as an active protein in escherichia coli. the catalytic core region contains 284 amino acids as defined by c- and n-terminal deletions. the c terminus from amino acid 200-3 ... | 1998 | 9668103 |
| genetic evidence for an essential role for potyvirus ci protein in cell-to-cell movement. | the potyvirus cylindrical inclusion (ci) protein, an rna helicase required for genome replication, was analyzed genetically using alanine-scanning mutagenesis. thirty-one mutations were introduced into the ci protein coding region of modified tobacco etch virus (tev) genomes expressing either beta-glucuronidase or green fluorescent protein reporters. twelve of the mutants were replication-defective in protoplast inoculation assays. among the 19 replication-competent mutants, several possessed ce ... | 1998 | 9670556 |
| non-toxic concentrations of cadmium inhibit systemic movement of turnip vein clearing virus by a salicylic acid-independent mechanism. | systemic movement of plant viruses is a central event in viral infection. to better understand this process, the heavy metal cadmium was used to inhibit systemic spread of turnip vein clearing virus (tvcv), a tobamovirus, in tobacco plants. study of the mechanism by which cadmium exerts this inhibitory effect may provide insights into the essential steps of the tvcv systemic movement pathway. our results demonstrated that cadmium treatment did not affect tvcv transport from the inoculated non-va ... | 1998 | 9807823 |
| a counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing. | posttranscriptional gene silencing (ptgs) in plants inactivates some aberrant or highly expressed rnas in a sequence-specific manner in the cytoplasm. a silencing mechanism similar to ptgs appears to function as an adaptive antiviral response. we demonstrate that the p1/hc-pro polyprotein encoded by tobacco etch virus functions as a suppressor of ptgs. a locus comprised of a highly expressed beta-glucuronidase (gus) transgene was shown to exhibit ptgs. genetic crosses and segregation analyses re ... | 1998 | 9827799 |
| ultrastructural localization of nonstructural and coat proteins of 19 potyviruses using antisera to bacterially expressed proteins of plum pox potyvirus. | antisera to the bacterially expressed nonstructural proteins (nsp) hc-pro, ci, nia, and nib and the coat protein (cp) of plum pox potyvirus (ppv) were used for analysing the composition of virus-induced cytoplasmic and nuclear inclusions by electron microscopy. the antisera reacted with nsp and cp of ppv on immunogold-labelled ultrathin sections. antiserum to cp reacted with virions of seven out of 18 other potyviruses. cp was distributed throughout the cytoplasm of infected cells. antisera to p ... | 1998 | 9856098 |
| isolation and stability of histidine-tagged proteins produced in plants via potyvirus gene vectors. | a system for the expression and purification of histidine-tagged proteins from plants has been developed using a tobacco etch potyvirus (tev)-derived gene vectors. the vectors offered a convenient polylinker and a choice of histidine tagging at the recombinant proteins' n or c termini. these vectors were utilized for expression of proteins encoded by beet yellows closterovirus (byv). approximately 4 micrograms/g of 20-kda byv protein was readily isolated from plants systemically infected by hybr ... | 1998 | 9875335 |
| mutations in the potyvirus helper component protein: effects on interactions with virions and aphid stylets. | mutations of k --> e in the highly conserved 'kitc' motif of the potyvirus helper component (hc) protein result in loss of hc function in aphid transmission, presumably because of inability to interact with virions, stylets or both. in this study we show that hc of potato virus c (pvc), a naturally occurring variant of potato virus y (pvy) that has the k --> e mutation, lacks the ability to be retained in stylets, whereas pvy hc is retained. the k --> e mutation in either pvc or a site-directed ... | 1998 | 9880030 |
| selectable viruses and altered susceptibility mutants in arabidopsis thaliana. | the genetic basis for susceptibility or nonsusceptibility of plants to viruses is understood poorly. two selectable tobacco etch virus (tev) strains were developed for identification of arabidopsis thaliana mutants with either gain-of-susceptibility or loss-of-susceptibility phenotypes. these strains conferred a conditional-survival phenotype to arabidopsis based on systemic expression of herbicide resistance or proherbicide sensitivity genes, thereby facilitating mass selections and screens for ... | 1999 | 9892709 |
| histidine-tagging and purification of tobacco etch potyvirus helper component protein. | the coding sequence for a series of six histidines (his-tag) was inserted near the 5' terminus of the helper component (hc) coding region of tobacco etch potyvirus (tev). full length genomic clones containing the his-tag coding sequence were infectious and produced symptoms in tobacco (nicotiana tabacuma) similar to those induced by wild-type tev. the modified virus was genetically stable and the his-tag sequence was maintained through at least four cycles of aphid transmission. a protocol for p ... | 1999 | 10029320 |
| imaging fluorescence resonance energy transfer between two green fluorescent proteins in living yeast. | we show that fluorescence resonance energy transfer between two mutants of the green fluorescent protein (gfp) can be monitored by imaging microscopy in living yeast. this work is based on the constitutive expression of a gfp-containing fusion protein and the inducible expression of the tobacco etch virus (tev) protease. in the fusion protein, the p4.3 gfp mutant is linked to the ys65t gfp mutant by a spacer bearing the tev protease-specific cleavage site. | 1999 | 10218581 |
| a novel motif mediates the targeting of the arabidopsis cop1 protein to subnuclear foci. | the constitutive photomorphogenesis 1 (cop1) protein of arabidopsis thaliana accumulates in discrete subnuclear foci. to better understand the role of subnuclear architecture in cop1-mediated gene expression, we investigated the structural motifs of cop1 that mediate its localization to subnuclear foci using mutational analysis with green fluorescent protein as a reporter. in a transient expression assay, a subnuclear localization signal consisting of 58 residues between amino acids 120 and 177 ... | 1999 | 10480941 |
| regulation of closterovirus gene expression examined by insertion of a self-processing reporter and by northern hybridization. | a reporter open reading frame (orf) coding for a fusion of bacterial beta-glucuronidase (gus) with a proteinase domain (pro) derived from tobacco etch potyvirus was utilized for tagging individual genes of beet yellows closterovirus (byv). insertion of this reporter orf between the first and second codons of the byv orfs encoding the hsp70 homolog (hsp70h), a major capsid protein (cp), and a 20-kda protein (p20) resulted in the expression of the processed gus-pro reporter from corresponding subg ... | 1999 | 10482546 |
| functional analysis of the interaction between vpg-proteinase (nia) and rna polymerase (nib) of tobacco etch potyvirus, using conditional and suppressor mutants. | the tobacco etch potyvirus (tev) rna-dependent rna polymerase (nib) has been shown to interact with the proteinase domain of the vpg-proteinase (nia). to investigate the significance of this interaction, a saccharomyces cerevisiae two-hybrid assay was used to isolate conditional nia mutant proteins with temperature-sensitive (ts) defects in interacting with nib. thirty-six unique tsnia mutants with substitutions affecting the proteinase domain were recovered. most of the mutants coded for protei ... | 1999 | 10482627 |
| aids vaccination studies using feline immunodeficiency virus as a model: immunisation with inactivated whole virus suppresses viraemia levels following intravaginal challenge with infected cells but not following intravenous challenge with cell-free virus. | the feline immunodeficiency virus (fiv) provides an excellent model system for aids vaccination studies. in the present experiments we investigated the immunogenicity and the protective activity of two inactivated vaccines prepared from a primary virus isolate. one vaccine was composed of whole virus inactivated with paraformaldehyde and then purified (wiv) and the other of viral proteins extracted with tween-ether (tev). both vaccines elicited robust antiviral responses, but neither conferred a ... | 1999 | 10501242 |
| targeted rnases: a feasibility study for use in hiv gene therapy. | a targeted rnase would be ideal for gene therapy of several acquired and inherited disorders. such an rnase may be engineered to contain a ribonucleolytic domain and a specific target rna binding domain. to demonstrate the feasibility of this approach, an rnase targeted against human immunodeficiency virus (hiv) rna--tev-rnase t1--was designed and tested for its use in hiv-1 gene therapy. a human cd4+ t lymphoid (mt4) cell line and human peripheral blood lymphocytes (pbls) were transduced with r ... | 1999 | 10505117 |
| identification and characterization of the functional elements within the tobacco etch virus 5' leader required for cap-independent translation. | translation in plants is highly cap dependent, and the only plant mrnas known to naturally lack a cap structure (m(7)gpppn) are viral in origin. the genomic rna of tobacco etch virus (tev), a potyvirus that belongs to the picornavirus superfamily, is a polyadenylated mrna that is naturally uncapped and yet is a highly competitive mrna during translation. the 143-nucleotide 5' leader is responsible for conferring cap-independent translation even on reporter mrnas. we have carried out a deletion a ... | 1999 | 10516014 |
| context of the coat protein dag motif affects potyvirus transmissibility by aphids. | previous work with tobacco vein mottling virus (tvmv) has established that a highly conserved three amino acid motif, asp-ala-gly (dag), located near the n terminus of the coat protein (cp), is important for aphid transmission. however, several other potyviruses which have motifs other than dag are aphid-transmissible. creation of these motifs in tvmv through site-directed mutagenesis failed to render tvmv aphid-transmissible from infected plants, and the creation of a putative complementary mot ... | 1999 | 10567662 |
| the use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants. | as the processing mechanism of all known potyviruses involves the activity of cysteine proteinases, we asked whether constitutive expression of a rice cysteine proteinase inhibitor gene could induce resistance against two important potyviruses, tobacco etch virus (tev) and potato virus y (pvy), in transgenic tobacco plants. tobacco lines expressing the foreign gene at varying levels were examined for resistance against tev and pvy infection. there was a clear, direct correlation between the leve ... | 1999 | 10585723 |
| a modular set of prokaryotic and eukaryotic expression vectors. | a modular series of versatile expression vectors is described for improved affinity purification of recombinant fusion proteins. special features of these vectors include (i) serial affinity tags (hexahistidine-gst) to yield extremely pure protein even with very low expression rates, (ii) highly efficient proteolytic cleavage of affinity tags under a variety of conditions by hexahistidine-tagged tobacco etch virus (tev) protease, (iii) pcr cloning design that results in a product of proteolytic ... | 2000 | 10610695 |
| cloning of the arabidopsis rtm1 gene, which controls restriction of long-distance movement of tobacco etch virus. | the locus rtm1 is necessary for restriction of long-distance movement of tobacco etch virus in arabidopsis thaliana without causing a hypersensitive response or inducing systemic acquired resistance. the rtm1 gene was isolated by map-based cloning. the deduced gene product is similar to the alpha-chain of the artocarpus integrifolia lectin, jacalin, and to several proteins that contain multiple repeats of a jacalin-like sequence. these proteins comprise a family with members containing modular o ... | 2000 | 10618445 |
| expression, purification, and initial structural characterization of yadq, a bacterial homolog of mammalian clc chloride channel proteins. | yadq of escherichia coli is a homolog of the mammalian chloride channels of the clc family. the yadq gene was cloned as a fusion protein with a hexahistidine tag and tobacco etch virus protease site for the removal of the tag. the protein was expressed in the membrane of e. coli and extracted with decylmaltoside. purification was achieved by metal affinity chromatography followed by cation exchange. circular dichroism revealed a high alpha-helical content. size exclusion chromatography suggests ... | 2000 | 10648805 |
| synthesis of (-)-strand rna from the 3' untranslated region of plant viral genomes expressed in transgenic plants upon infection with related viruses. | when expressed in transgenic tobacco plants, transgene mrna that includes the 3' untranslated region (3' utr) of lettuce mosaic virus served as template for synthesis of complementary (-)-strand rna following an infection by tobacco etch virus, tobacco vein mottle virus or pepper mottle virus, but not when infected with cucumber mosaic virus. deletion of the 3' utr from the transgene abolished the synthesis of (-)-strand transcripts. similar results were obtained in transgenic tobacco plants exp ... | 2000 | 10725441 |
| arabidopsis rtm2 gene is necessary for specific restriction of tobacco etch virus and encodes an unusual small heat shock-like protein. | arabidopsis plants have a system to specifically restrict the long-distance movement of tobacco etch potyvirus (tev) without involving either hypersensitive cell death or systemic acquired resistance. at least two dominant genes, rtm1 and rtm2, are necessary for this restriction. through a series of coinfection experiments with heterologous viruses, the rtm1/rtm2-mediated restriction was shown to be highly specific for tev. the rtm2 gene was isolated by a map-based cloning strategy. isolation of ... | 2000 | 10760245 |
| production and characterization of biologically active human gm-csf secreted by genetically modified plant cells. | human granulocyte-macrophage colony-stimulating factor (gm-csf), a hemopoietic growth factor, was produced and secreted from tobacco cell suspensions. the gm-csf cdna was carried by a binary vector under the control of the camv 35s promoter and the t7 terminator. in addition, a 5'-nontranslated region from the tobacco etch virus (tev leader sequence) was fused to the n-terminal end of the gm-csf transgene. for ease of purification, a 6-his tag was added to the 3' end of the gm-csf cdna. addition ... | 2000 | 10833400 |
| a trimmed viral cap-independent translation enhancing sequence for rapid in vitro gene expression. | we prepared a short (29 nucleotides) 5' utr that enhanced cap-independent translation in a wheat germ translation system by trimming the tobacco etch virus 5' utr. the trimmed sequence, designated as te(37-65), was obtained from a conserved region among several potyviruses. the productivities of uncapped reporter mrnas carrying the te(37-65) sequence were comparable to those of capped counterparts, in that 5-20 microg of proteins were synthesized per 1 ml of translation reaction mixture. the rib ... | 2000 | 10835258 |
| molecular cloning, expression, and purification of nuclear inclusion a protease from tobacco vein mottling virus. | the gene encoding the c-terminal protease domain of the nuclear inclusion protein a (nia) of tobacco vein mottling virus (tvmv) was cloned from an isolated virus particle and expressed as a fusion protein with glutathione s-transferase in escherichia coli xl1-blue. the 27-kda protease was purified from the fusion protein by glutathione affinity chromatography and mono s chromatography. the purified protease exhibited the specific proteolytic activity towards the nonapeptide substrates, ac-glu-as ... | 2000 | 10850655 |
| controlled intracellular processing of fusion proteins by tev protease. | here we describe a method for controlled intracellular processing (cip) of fusion proteins by tobacco etch virus (tev) protease. a fusion protein containing a tev protease recognition site is expressed in escherichia coli cells that also contain a tev protease expression vector. the fusion protein vector is an iptg-inducible cole1-type plasmid, such as a t7 or tac promoter vector. in contrast, the tev protease is produced by a compatible p15a-type vector that is induced by tetracyclines. not onl ... | 2000 | 10873547 |
| novel topological features of fhac, the outer membrane transporter involved in the secretion of the bordetella pertussis filamentous hemagglutinin. | many pathogenic gram-negative bacteria secrete virulence factors across the cell envelope into the extracellular milieu. the secretion of filamentous hemagglutinin (fha) by bordetella pertussis depends on the pore-forming outer membrane protein fhac, which belongs to a growing family of protein transporters. protein alignment and secondary structure predictions indicated that fhac is likely to be a beta-barrel protein with an odd number of transmembrane beta-strands connected by large surface lo ... | 2000 | 10906141 |
| strain-specific interaction of the tobacco etch virus nia protein with the translation initiation factor eif4e in the yeast two-hybrid system. | the nia protein of potyviruses provides vpg and proteolytic functions during virus replication. it has also been shown to confer host genotype-specific movement functions in plants. specifically, nia from tobacco etch virus (tev)-oxnard, but not from most other strains, confers the ability to move long distances in nicotiana tabacum cultivar "v-20." this led to the hypothesis that all or part of nia may interact with one or more cellular factors. to identify cellular proteins that interact with ... | 2000 | 10915600 |
| conversion of compatible plant-pathogen interactions into incompatible interactions by expression of the pseudomonas syringae pv. syringae 61 hrma gene in transgenic tobacco plants. | the hrma gene from pseudomonas syringae pv. syringae has previously been shown to confer avirulence on the virulent bacterium p. syringae pv. tabaci in all examined tobacco cultivars. we expressed this gene in tobacco plants under the control of the tobacco delta0. 3 tobrb7 promoter, which is induced upon nematode infection in tobacco roots (opperman et al. 1994, science, 263, 221-223). a basal level of hrma expression in leaves of transgenic plants activated the expression of pathogenesis-relat ... | 2000 | 10929114 |
| an ry-mediated resistance response in potato requires the intact active site of the nia proteinase from potato virus y. | ry confers extreme resistance to all strains of potato virus y (pvy). to identify the elicitor of the ry-mediated resistance against pvy in potato, we expressed each of the pvy-encoded proteins in leaves of pvy-resistant (ry) and -susceptible (ry) plants. for most of the proteins tested, there was no evident response. however, when the nia proteinase was expressed in leaves of ry plants, there was a hypersensitive response (hr). proteinase active site mutants failed to induce the ry-mediated res ... | 2000 | 10972891 |
| reduced global genomic repair of ultraviolet light-induced cyclobutane pyrimidine dimers in simian virus 40-transformed human cells. | the p53 tumor-suppressor gene has been implicated in the inducible activation of excision repair of ultraviolet (uv)-induced cyclobutane pyrimidine dimers (cpds) in human cells. because the large t antigen (ltag) of the simian virus 40 (sv40) binds p53 protein and can interfere with its function, it was of interest to study dna repair in normal human fibroblasts that had been transformed by sv40 compared with that in their nontransformed parental counterparts and to determine whether such transf ... | 2000 | 11020243 |
| expression, purification, refolding, and characterization of recombinant human interleukin-13: utilization of intracellular processing. | interleukin-13 (il-13) is a pleiotropic cytokine that elicits both proinflammatory and anti-inflammatory immune responses. recent studies underscore its role in several diseases, including asthma and cancer. solution studies of il-13 and its soluble receptors may facilitate the design of antagonists/agonists which would require milligram quantities of specifically labeled protein. a synthetic gene encoding human il-13 (hil-13) was inserted into the pmal-c2 vector with a cleavage site for the tob ... | 2000 | 11049743 |
| virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway. | certain plant viruses encode suppressors of posttranscriptional gene silencing (ptgs), an adaptive antiviral defense response that limits virus replication and spread. the tobacco etch potyvirus protein, helper component-proteinase (hc-pro), suppresses ptgs of silenced transgenes. the effect of hc-pro on different steps of the silencing pathway was analyzed by using both transient agrobacterium tumefaciens-based delivery and transgenic systems. hc-pro inactivated ptgs in plants containing a pree ... | 2000 | 11078509 |
| a mammalian expression vector for expression and purification of secreted proteins for structural studies. | a mammalian expression vector with features optimized for simple expression and purification of secreted proteins has been developed. this vector was constructed to facilitate x-ray crystallographic studies of cysteine-rich glycoproteins that are difficult to express by other means. proteins expressed with this vector possess an n-terminal human growth hormone domain and an octahistidine tag separated from the desired polypeptide sequences by a tobacco etch virus protease recognition site. advan ... | 2000 | 11087690 |
| identification of the genome-linked protein in virions of potato virus a, with comparison to other members in genus potyvirus. | viruses of the genus potyvirus, the largest genus of plant-infecting viruses, have a messenger-polarity ssrna genome encapsidated by approximately 2000 units of the viral coat protein (cp), resulting in filamentous virions. only few studies have examined potyvirus virions for the presence of other structural proteins. a protein linked covalently to the 5'-end of the genome has been identified in tobacco vein mottling virus (tvmv) and tobacco etch virus (tev). in tev, it is either the viral nia p ... | 2001 | 11172914 |
| expression, purification and characterization of the structure and disulfide linkages of insulin-like growth factor binding protein-4. | insulin-like growth factor binding protein-4 (igfbp-4), like the other five igfbps, is a critical regulator of the activity of insulin-like growth factor (igf)-i and igf-ii. however igfbp-4 seems to be the only igfbp with no potential to enhance the mitogenic actions of the igfs. igfbp-1 to -3 and -5 each contain 18 conserved cysteine residues, igfbp-6 lacks two of the twelve n-terminal cysteines, while igfbp-4 has two additional cysteines in the central region. a plasmid was constructed to expr ... | 2001 | 11182766 |
| system for cleavable fc fusion proteins using tobacco etch virus (tev) protease. | we describe a novel fc fusion protein system that can be cleaved by tobacco etch virus (tev) protease. this system is desirable because it takes advantage of the high specificity of tev protease and its activity at 4 degrees c. we produced two tev-fc fusion proteins that contain the first three ig domains and all six ig domains of the cell adhesion molecule l1. both proteins were efficiently cleaved by tev protease at 4 degrees c. functional analysis of the cleavage products in neurite outgrowth ... | 2001 | 11196321 |
| a new rt-pcr method for the identification of reoviruses in seawater samples. | the frequent occurrence of reoviruses in environmental samples could be a potential source of interference with enterovirus detection, especially when enterovirus isolation on cell culture is required. in order to evaluate new virus-based criteria for enforcing recreational water quality standards, a new method based on a broad reverse transcribed polymerase chain reaction (rt-pcr) was set up to detect reoviruses. two primers were engineered to amplify a 538 base pair fragment of the sigma 2 gen ... | 2001 | 11229010 |
| the venus's-flytrap and cysteine-rich domains of the human ca2+ receptor are not linked by disulfide bonds. | the extracellular n-terminal domain of the human ca(2+) receptor (hcar) consists of a venus's-flytrap (vft) domain and a cysteine-rich (cys-rich) domain. we have shown earlier that the cys-rich domain is critical for signal transmission from the vft domain to the seven-transmembrane domain. the vft domain contains 10 cysteines: two of them (cys(129) and cys(131)) were identified as involved in intermolecular disulfide bonds necessary for homodimerization, and six others (cys(60)-cys(101), cys(35 ... | 2001 | 11238442 |
| large-scale purification of a stable form of recombinant tobacco etch virus protease. | tobacco etch virus nia proteinase (nia-pro) has become the enzyme of choice for removing tags and fusion domains from recombinant proteins in vitro. we have designed a mutant nia-pro that resists autoproteolytic inactivation and present an efficient method for producing large amounts of this enzyme that is highly pure, active, and stable over time. histidine-tagged forms of both wild-type and mutant nia-pro were overexpressed in e. coli under conditions in which greater than 95% of the protease ... | 2001 | 11252791 |
| virus-specific spatial differences in the interference with silencing of the chs-a gene in non-transgenic petunia. | potyviruses, such as potato virus y and tobacco etch virus, as well as cucumber mosaic cucumovirus, interfere with post-transcriptional gene silencing (ptgs). when redstar-type petunia hybrida cultivars, whose flowers have alternating white and pigmented sectors, were infected with these viruses, each virus induced a different pattern of restoration of floral anthocyanin pigmentation. local reversion to coloured phenotypes in the white sectors, which occurred through interference with ptgs of th ... | 2001 | 11297699 |
| functional role of the hiv-1 rev exon 1 encoded region in complex formation and trans-dominant inhibition. | to study functional aspects of the exon 1 encoded region of the human immunodeficiency virus type 1 rev protein, the viral tev protein which exhibits low rev activity but lacks the rev exon 1 encoded region was examined. neither rev-tev heteromer complex formation nor inhibition of rev by an export deficient tev mutant was observed. insertion of the rev exon 1 encoded region into the tev mutant allowed it to oligomerize with rev and act as a trans-dominant negative mutant. this showed that the e ... | 2001 | 11322956 |
| a novel method to determine the topology of peroxisomal membrane proteins in vivo using the tobacco etch virus protease. | most proteins essential for the biogenesis of peroxisomes (peroxins) that are identified to date are associated with or are integral components of the peroxisomal membrane. a prerequisite in elucidating their function is to determine their topology in the membrane. we have developed a novel tool to analyze the topology of peroxisomal membrane proteins in the yeast hansenula polymorpha in vivo using the 27-kda nia protease subunit from the tobacco etch virus (tevp). tevp specifically cleaves pept ... | 2001 | 11443138 |
| analysis of the p1 gene sequences and the 3'-terminal sequences and secondary structures of the single-stranded rna genome of potato virus v. | the immunocapture reverse transcriptase pcr method (ic-rt-pcr) was used to selectively amplify specific genome sequences of an isolate of potato virus v (pvv, genus potyvirus) from a potato plant infected by multiple viruses in the field in finland. the sequences of the 5'- and 3'-non-translated regions (ntr) and the p1-and coat protein (cp)-encoding sequences were determined because they are the most variable genomic regions in potyviruses. the sequences of the new finnish pvv isolate obtained ... | 2001 | 11450952 |
| silencing on the spot. induction and suppression of rna silencing in the agrobacterium-mediated transient expression system. | the agrobacterium-mediated transient expression assay in intact tissues has emerged as a rapid and useful method to analyze genes and gene products in plants. in many cases, high levels of active protein can be produced without the need to produce transgenic plants. in this study, a series of tools were developed to enable strong or weak induction of rna silencing and to suppress rna silencing in the absence of stable transgenes. transient delivery of a gene directing production of a double-stra ... | 2001 | 11457942 |
| production and characterization of the recombinant sphingomonas chlorophenolica pentachlorophenol 4-monooxygenase. | pentachlorophenol 4-monooxygenase (pcp4mo) from sphingomonas chlorophenolica is a flavoprotein that hydroxylates pcp in the presence of nadph and oxygen. in order to investigate the structure and function of active site, recombinant pcp4mo (repcp4mo) was produced in escherichia coli as a glutathione s-transferase (gst) fusion protein. moreover, a tobacco etch virus (tev) protease cleavage site (eklyfqg) was introduced into gst-pcp4mo and a his-tagged tev protease was employed. hence, a two-step ... | 2001 | 11708794 |
| cap-independent translation conferred by the 5' leader of tobacco etch virus is eukaryotic initiation factor 4g dependent. | the 5' leader of tobacco etch virus (tev) genomic rna directs efficient translation from the naturally uncapped viral mrna. two distinct regions within the tev 143-nucleotide leader confer cap-independent translation in vivo even when present in the intercistronic region of a discistronic mrna, indicating that the tev leader contains an internal ribosome entry site (ires). in this study, the requirements for tev ires activity were investigated. the tev ires enhanced translation of monocistronic ... | 2001 | 11711605 |
| functional specialization and evolution of leader proteinases in the family closteroviridae. | members of the closteroviridae and potyviridae families of the plant positive-strand rna viruses encode one or two papain-like leader proteinases. in addition to a c-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic n-terminal domain. we compared functions of the several leader proteinases using a gene swapping approach. the leader proteinase (l-pro) of beet yellows virus (byv; a closterovirus) was replaced with l1 or l2 proteinases of citrus tristeza virus (ctv; ... | 2001 | 11711606 |
| evidence for rna-mediated defence effects on the accumulation of potato leafroll virus. | in plants infected with potato leafroll virus (plrv), or other luteoviruses, infection is very largely confined to cells in the vascular system. even in tobacco plants transformed with plrv full-length cdna, in which all mesophyll cells should synthesize infectious plrv rna transcripts, only a minority of the mesophyll cells accumulate detectable amounts of virus. we have explored this phenomenon further by transforming a better plrv host, nicotiana benthamiana, with the same transgene, by super ... | 2001 | 11714988 |
| arabidopsis rtm1 and rtm2 genes function in phloem to restrict long-distance movement of tobacco etch virus. | restriction of long-distance movement of tobacco etch virus (tev) in arabidopsis ecotype col-0 plants requires the function of at least three genes: rtm1 (restricted tev movement 1), rtm2, and rtm3. the mechanism of tev movement restriction remains poorly understood, although it does not involve a hypersensitive response or systemic acquired resistance. a functional characterization of rtm1 and rtm2 was done. the rtm1 protein was found to be soluble with the potential to form self-interacting co ... | 2001 | 11743111 |
| host-specific involvement of the hc protein in the long-distance movement of potyviruses. | plum pox virus (ppv) is a member of the potyvirus genus that, in nature, infects trees of the prunus genus. although ppv infects systemically several species of the nicotiana genus, such as n. clevelandii and n. benthamiana, and replicates in the inoculated leaves of n. tabacum, it is unable to infect systemically the last host. the long-distance movement defect of ppv was corrected in transgenic tobacco plants expressing the 5"-terminal region of the genome of tobacco etch virus (tev), a potyvi ... | 2002 | 11799187 |
| mapping of the p1 proteinase cleavage site in the polyprotein of wheat streak mosaic virus (genus tritimovirus). | monopartite members of the family potyviridae utilize three virus-encoded proteinases to cleave the viral polyprotein into mature proteins. the amino-terminal region of the viral polyprotein is autolytically cleaved by the p1 proteinase. a domain required for p1 proteinase activity of wheat streak mosaic virus (wsmv) was mapped using a series of templates with nested 3'-truncations or 5'-deletions to program in vitro transcription-translation reactions. the wsmv p1 proteinase cleavage site was m ... | 2002 | 11807238 |
| tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. | because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (tev) is a useful reagent for cleaving genetically engineered fusion proteins. however, a serious drawback of tev protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. the rate of autoinactivation is proportional to the concentration of tev protease, implying a bimolecular reaction mechanism. yet, a ... | 2001 | 11809930 |
| processive degradation of nascent polypeptides, triggered by tandem aga codons, limits the accumulation of recombinant tobacco etch virus protease in escherichia coli bl21(de3). | due to its high degree of sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (tev protease) is a useful reagent for cleaving genetically engineered fusion proteins. however, the overproduction of tev protease in escherichia coli has been hampered in the past by low yield and poor solubility. here we demonstrate that the low yield can be attributed to the presence of arginine codons in the tev protease coding sequence that are rarely used in e. co ... | 2002 | 11812224 |
| a combination anti-hiv-1 gene therapy approach using a single transcription unit that expresses antisense, decoy, and sense rnas, and trans-dominant negative mutant gag and env proteins. | oncoretroviral vectors were engineered to allow constitutive expression of an antisense rna and the trans-activator of transcription (tat)-inducible expression of a mrna containing the trans-activation response (tar) element, the rev response element (rre), and the efficient packaging signal (psi(e) of human immunodeficiency virus-1 (hiv-1) rna. nuclear export of this mrna by the regulator of expression of virion proteins (rev) would allow its translation into wild type (wt) (motn-ti-ge-ri- ter) ... | 2002 | 11815282 |
| combined use of regulatory elements within the cdna to increase the production of a soluble mouse single-chain antibody, scfv, from tobacco cell suspension cultures. | in order to facilitate production and secretion of a soluble form of a small, single-chain antibody scfv (32 kda) in tobacco cell suspension culture, several modifications were made simultaneously to the antibody cdna that included elements that have been shown to regulate the expression of proteins in plants. the scfv cdna was initially ligated into a binary vector under the control of the camv 35s promoter and the t7 terminator for expression in tobacco suspension culture. subsequently, modifi ... | 2002 | 11922754 |
| the amplicon-plus system for high-level expression of transgenes in plants. | many biotechnological applications require high-level expression of transgenes in plants. one strategy to achieve this goal was the production of potato virus x (pvx) "amplicon" lines: transgenic lines that encode a replicating rna virus vector carrying a gene of interest. the idea was that transcription of the amplicon transgene would initiate viral rna replication and gene expression, resulting in very high levels of the gene product of interest. this approach failed, however, because every am ... | 2002 | 12042869 |
| a new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site. | to establish high-throughput methods for protein crystallography, all aspects of the production and analysis of protein crystals must be accelerated. automated, plate-based methods for cloning, expression, and evaluation of target proteins will help researchers investigate the vast numbers of proteins now available from sequenced genomes. ligation-independent cloning (lic) is well suited to robotic cloning and expression, but few lic vectors are available commercially. we have developed a new li ... | 2002 | 12071693 |
| the p1' specificity of tobacco etch virus protease. | affinity tags have become indispensable tools for protein expression and purification. yet, because they have the potential to interfere with structural and functional studies, it is usually desirable to remove them from the target protein. the stringent sequence specificity of the tobacco etch virus (tev) protease has made it a useful reagent for this purpose. however, a potential limitation of tev protease is that it is believed to require a gly or ser residue in the p1' position of its substr ... | 2002 | 12074568 |
| loss-of-susceptibility mutants of arabidopsis thaliana reveal an essential role for eif(iso)4e during potyvirus infection. | the arabidopsis thaliana-potyvirus system was developed to identify compatibility and incompatibility factors involved during infection and disease caused by positive-strand rna viruses. several arabidopsis mutants with increased susceptibility to tobacco etch potyvirus (tev) were isolated previously, revealing a virus-specific resistance system in the phloem. in this study, arabidopsis mutants with decreased susceptibility to turnip mosaic potyvirus (tumv) were isolated. three independent mutan ... | 2002 | 12123581 |
| a protease assay for two-photon crosscorrelation and fret analysis based solely on fluorescent proteins. | gfp and the red fluorescent protein, dsred, have been combined to design a protease assay that allows not only for fluorescence resonance energy transfer (fret) studies but also for dual-color crosscorrelation analysis, a single-molecule-based method that selectively probes the concomitant movement of two distinct tags. the measurement principle is based on a spectrally resolved detection of single molecules diffusing in and out of a diffraction-limited laser focus. double-labeled substrate mole ... | 2002 | 12209012 |
| characterization of post-transcriptionally suppressed transgene expression that confers resistance to tobacco etch virus infection in tobacco. | tobacco lines expressing transgenes that encode tobacco etch virus (tev) coat protein (cp) mrna with or without nonsense codons give rise to tev-resistant tissues that have reduced levels of tev cp mrna while maintaining high levels of transgene transcriptional activity. two phenotypes for virus resistance in the lines containing the transgene have been described: immune (no virus infection) and recovery (initial systemic symptoms followed by gradual recovery over several weeks). here, we show t ... | 1997 | 12237389 |
| induction of a highly specific antiviral state in transgenic plants: implications for regulation of gene expression and virus resistance. | transgenic tobacco plants expressing either a full-length form of the tobacco etch virus (tev) coat protein or a form truncated at the n terminus of the tev coat protein were initially susceptible to tev infection, and typical systemic symptoms developed. however, 3 to 5 weeks after a tev infection was established, transgenic plants "recovered" from the tev infection, and new stem and leaf tissue emerged symptom and virus free. a tev-resistant state was induced in the recovered tissue. the resis ... | 1993 | 12271055 |
| structural basis for the substrate specificity of tobacco etch virus protease. | because of its stringent sequence specificity, the 3c-type protease from tobacco etch virus (tev) is frequently used to remove affinity tags from recombinant proteins. it is unclear, however, exactly how tev protease recognizes its substrates with such high selectivity. the crystal structures of two tev protease mutants, inactive c151a and autolysis-resistant s219d, have now been solved at 2.2- and 1.8-a resolution as complexes with a substrate and product peptide, respectively. the enzyme does ... | 2002 | 12377789 |
| amino acid substitutions within the cys-rich domain of the tobacco etch potyvirus hc-pro result in loss of transmissibility by aphids. | we examined the role of several amino acid residues located at the n-terminus of the tobacco etch potyvirus (tev) helper component-proteinase (hc-pro) in virus transmissibility by aphids. site-directed mutagenesis resulted in changes affecting amino acids that appear highly conserved among a number of potyviruses. the tev hc-pro amino acid residues gly343, val345, ala346, ile348, pro355, lys358, and ile359 were arranged within a cys-rich domain in a region dispensable for tev infectivity. two hc ... | 2002 | 12491103 |
| inhibition of tobacco etch virus protease activity by detergents. | affinity tags such as polyhistidine greatly facilitate recombinant protein production. the solubility of integral membrane proteins is maintained by the formation of protein-detergent complexes (pdcs), with detergent present at concentration above its critical micelle concentration (cmc). removal of the affinity tag necessitates inclusion of an engineered protease cleavage site. a commonly utilized protease for tag removal is tobacco etch virus (tev) protease. tev is available in a recombinant f ... | 2003 | 12509992 |
| the intramitochondrial dynamin-related gtpase, mgm1p, is a component of a protein complex that mediates mitochondrial fusion. | a balance between fission and fusion events determines the morphology of mitochondria. in yeast, mitochondrial fission is regulated by the outer membrane-associated dynamin-related gtpase, dnm1p. mitochondrial fusion requires two integral outer membrane components, fzo1p and ugo1p. interestingly, mutations in a second mitochondrial-associated dynamin-related gtpase, mgm1p, produce similar phenotypes to fzo1 and ugo cells. specifically, mutations in mgm1 cause mitochondrial fragmentation and a lo ... | 2003 | 12566426 |
| recessive resistance genes against potyviruses are localized in colinear genomic regions of the tomato ( lycopersicon spp.) and pepper ( capsicum spp.) genomes. | resistance against both potato virus y (pvy) and tobacco etch virus (tev) was identified in the wild tomato relative lycopersicon hirsutum pi247087. analysis of the segregation ratio in f(2)/f(3) and bc(1) interspecific progenies indicated that a single recessive gene, or two very tightly linked recessive loci, are involved in resistance to both potyviruses. this locus was named pot-1. using amplified fragment length polymorphism markers and a set of l. hirsutum introgression lines, pot-1 was ma ... | 2002 | 12582910 |
| new donor vector for generation of histidine-tagged fusion proteins using the gateway cloning system. | an optimized donor/shuttle vector, pentr-his-ccdb, was generated that readily produces a histidine-tagged recombinant protein in multiple expression systems using gateway technology. in the current gateway system, six histidines and the tobacco etch virus protease cleavage site are encoded upstream of the attr1 recombination site such that the histidine-tagged destination/expression vector adds 15 residues to the amino-terminus of recombinant proteins. our new vector introduces the histidine tag ... | 2003 | 12726771 |
| a novel co-delivery system consisting of a tomato bushy stunt virus and a defective interfering rna for studying gene silencing. | virus induced gene silencing (vigs) and suppression are rna-specific defense and counter-defense circuits in plant-virus interactions. these phenomena have been investigated extensively with an agrobacterium-mediated transient expression system. in this study, a virus-based transient expression system was developed to study these phenomena. a tomato bushy stunt virus (tbsv) viral vector with an inactivated p19 suppressor gene, referred to as phst2-14, was chosen to express the p1 of tobacco etch ... | 2003 | 12821195 |
| structure-function analysis of the bestrophin family of anion channels. | the bestrophins are a newly described family of anion channels unrelated in primary sequence to any previously characterized channel proteins. the human genome codes for four bestrophins, each of which confers a distinctive plasma membrane conductance on transfected 293 cells. extracellular treatment with methanethiosulfonate ethyltrimethylammonium (mtset) of a series of substitution mutants that eliminate one or more cysteines from human bestrophin1 demonstrates that cysteine 69 is the single e ... | 2003 | 12907679 |
| identification of novel protein-protein interactions using a versatile mammalian tandem affinity purification expression system. | identification of protein-protein interactions is essential for elucidating the biochemical mechanism of signal transduction. purification and identification of individual proteins in mammalian cells have been difficult, however, due to the sheer complexity of protein mixtures obtained from cellular extracts. recently, a tandem affinity purification (tap) method has been developed as a tool that allows rapid purification of native protein complexes expressed at their natural level in engineered ... | 2003 | 12963786 |
| electron microscopy of host cells infected with tobacco etch virus. ii. fine structures of leaf cells before and after the appearance of external systems. | 1964 | 14197607 | |
| [tobacco etch virus proteinase: crystal structure of the active enzyme and its inactive mutant]. | tobacco etch virus protease (tev protease) is widely used as a tool for separation of recombinant target proteins from their fusion partners. the crystal structures of two mutants of tev protease, active autolysis-resistant mutant tev-s219d in complex with the proteolysis product, and inactive mutant tev-c151a in complex with a substrate, have been determined at 1.8 and 2.2 a resolution, respectively. the active sites of both mutants, including their oxyanion holes, have identical structures. th ... | 2003 | 14601399 |
| dual topology of the escherichia coli tata protein. | the escherichia coli tat system has unusual capacity of translocating folded proteins across the cytoplasmic membrane. the tata protein is the most abundant known tat component and consists of a transmembrane segment followed by an amphipathic helix and a hydrophilic c terminus. to study the operation mechanism of the tat apparatus, we analyzed the topology of tata. intriguingly, alkaline phosphatase (phoa)-positive fusions were obtained at positions gly-38, lys-40, asp-51, and thr-53, which are ... | 2004 | 14701831 |
| expression of functionally active helper component protein of tobacco etch potyvirus in the yeast pichia pastoris. | tobacco etch potyvirus (tev) is transmitted by aphids in a non-persistent manner with the assistance of a virus-encoded protein known as helper component (hc-pro). to produce a biologically active form of recombinant tev hc-pro protein, heterologous expression in the methylotrophic yeast pichia pastoris was used. a cdna encoding the tev hc-pro region, fused to a saccharomyces cerevisiae alpha-mating factor secretory peptide coding region, was inserted into the p. pastoris genome using a modified ... | 2004 | 14718639 |