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At least 847 records · Page 47Linked to original sources
PubMed2000

Virus-like particles assemble in plants and bacteria expressing the coat protein gene of Indian peanut clump virus.

cDNA copies of the coat protein (CP) gene of Indian peanut clump virus (IPCV)-H were introduced into cells of Nicotiana benthamiana or Escherichia coli by transformation with vectors based on pROKII or pET respectively. In both plant and bacterial cells, IPCV CP was expressed and assembled to form virus-like particles (VLP). In plant extracts, the smallest preponderant particle length was about 50 nm. Other abundant lengths were about 85 and about 120 nm. The commonest VLP length in bacterial extracts was about 30 nm. Many of the longer VLP appeared to comprise aggregates of shorter particles. The lengths of the supposed 'monomer' VLP corresponded approximately to those expected for encapsidated CP gene transcript RNA. Immunocapture RT-PCR, using primers designed to amplify the CP gene, confirmed that the VLP contained RNA encoding IPCV-H CP. The results show that encapsidation does not require the presence of the 5'-terminal untranslated sequence of the virus RNA and suggest that if there is an 'origin of assembly' motif or sequence, it lies within the CP gene. When transgenic plants expressing IPCV-H CP were inoculated with IPCV-L, a strain that is serologically distinct from IPCV-H, the virus particles that accumulated contained both types of CP.

Arachis↗
PubMed2001

RNAs 1 and 2 of Alfalfa mosaic virus, expressed in transgenic plants, start to replicate only after infection of the plants with RNA 3.

RNAs 1 and 2 of the tripartite genome of Alfalfa mosaic virus (AMV) encode the two viral replicase subunits. Full-length DNA copies of RNAs 1 and 2 were used to transform tobacco plants (R12 lines). None of the transgenic lines showed resistance to AMV infection. In healthy R12 plants, the transcripts of the viral cDNAs were copied by the transgenic viral replicase into minus-strand RNAs but subsequent steps in replication were blocked. When the R12 plants were inoculated with AMV RNA 3, this block was lifted and the transgenic RNAs 1 and 2 were amplified by the transgenic replicase together with RNA 3. The transgenic expression of RNAs 1 and 2 largely circumvented the role of coat protein (CP) in the inoculum that is required for infection of nontransgenic plants. The results for the first time demonstrate the role of CP in AMV plus-strand RNA synthesis at the whole plant level.

Alfalfa mosaic virus↗
PubMed1989

Regulation of beta-glucuronidase expression in transgenic tobacco plants by an A/T-rich, cis-acting sequence found upstream of a French bean beta-phaseolin gene.

A 0.8-kilobase fragment from the 5'-flanking region of a French bean beta-phaseolin gene yielded strong, temporally regulated, and embryo-specific expression of beta-glucuronidase (GUS) in transgenic tobacco plants, paralleling that found for the seed protein phaseolin [Sengupta-Gopalan, C., Reichert, N.A., Barker, R.F., Hall. T.C., and Kemp, J.D. (1985) Proc. Natl. Acad. Sci. USA 82, 3320-3324]. Gel retardation and footprinting assays using nuclear extracts from immature bean cotyledons revealed strong binding of nuclear proteins to an upstream region (-628 to -682) that contains two inverted A/T-rich motifs. Fusion of a 103-base pair fragment or a 55-base pair synthetic oligonucleotide containing these motifs to a minimal 35S promoter/GUS cassette yielded strong GUS expression in several tissues. A different pattern of GUS expression was obtained in immature embryos and germinating seedlings from the nominally constitutive, full-length, 35S promoter. Whereas GUS expression under the control of the 0.8-kilobase beta-phaseolin regulatory region is limited to immature embryos, expression from constructs containing the A/T-rich motifs is strongest in roots. These data, combined with S1 mapping, provide direct evidence that a plant upstream A/T-rich sequence that binds nuclear proteins can activate transcription in vivo. They also indicate that additional regulatory elements in the beta-phaseolin 5'-flanking region are required for embryo-specific gene expression.

Base Composition↗
PubMed1999

Gene silencing-mediated resistance in transgenic tobacco plants carrying potato virus Y coat protein gene.

Unlike other pathogens, plant viruses are hardly controlled by chemical agents. Potato virus Y (PVY) is distributed around the world, and causes a great loss economically. In an attempt to minimize the damage by viruses, the PVY coat protein (CP) gene was introduced into tobacco by Agrobacterium-mediated transformation. A significant proportion of the transgenic plants displayed resistance to PVY and showed substantially decreased CP transgene expression at both protein and steady-state mRNA levels compared to susceptible transgenic or nontransgenic plants. A resistant plant was selected and self-fertilized for several generations until T4 progenitor lines were obtained. Most of these T4 plants accumulated extremely low levels of CP protein and steady-state mRNA, and exhibited almost complete resistance to PVY. DNA gel blot analysis revealed that the transgenic plants typically had two or three copies of the transgene. These results are characteristic of pathogen-derived resistance, in which the resistance against virus is the consequence of post-transcriptional gene silencing directed by homologous transgenes. To uncover factors that may play roles in gene silencing, sequences in the 3' part of the transcribed region of the CP gene were transcribed in vitro and the RNA fragments were incubated with cell extracts from transgenic plants. A ribonuclease activity was detected that appeared to be specific for this transcript in the PVY-resistant transgenic plants.

Capsid↗
PubMedDate not supplied

[Creation of transgenic plants Nicotiana tabacum and Solanum tuberosum, resistant to the herbicide phosphinothricin].

The expression cassette for phosphinothricin acetyltransferase gene (PAT or bar) from S. hygroscopicus has been constructed on the basis of the pBI121 plasmid. Leaf disks of N. tabacum cv. SR1 and steam segments of S. tuberosum cv. Prigozhii-2 have been transformed using Agrobacterium. Selection of the transformed plants carried out by PCR analysis revealed 12 transgenic tobacco plants and 3 transgenic potato plants. bar gene expression and plant resistance to phosphinothricin treatment have been studied in regenerated plants. All the transgenic plants were found to be resistant to the herbicide. Mendelian inheritance of the inserted gene has been proved by analysis of the F1 progeny of transformed tobacco plants.

Acetyltransferases↗
PubMed1997

Ribozyme mediated targeting of cucumber mosaic virus RNA 1 and 2 in transgenic tobacco plants.

Hammerhead ribozymes have been extensively used to inhibit the expression of cellular genes or viral genes mainly in the animal study. In this study, we designed a ribozyme targeting the conserved leader sequences of cucumber mosaic virus (CMV) RNA 1 and 2. The ribozyme, with asymmetric lengths of flanking complementary regions, cleaved a model substrate RNA efficiently at 26 degrees C as well as at 37 degrees C or 50 degrees C in vitro. And the ribozyme encoding sequence was introduced into tobacco plants and expressed with the CaMV 35S promoter and 3' NOS terminator in a monomeric type (pBIR1), tandemly repeated type (pBIR3), and cotranscriptionally combined type (pRokR) with 2.2 copies of I17N satellite RNA. Virus challenging experiments in F1 plants of respective transformants with CMV-Y showed specific reductions of viral RNA 1 and 2 in comparison with RNA 3 or 4. Although young plants of a three-leaf-stage showed rather similar mild symptom attenuations in all constructions compared to CMV-Y inoculated wild type, fully grown plants showed a differential degree of resistance upon systemic infections of CMV-Y in pRokR, pBIR3 and pBIR1 transformed plants in a decreasing order.

Agrobacterium tumefaciens↗
PubMed1992

In vivo import of a normal or mutagenized heterologous transfer RNA into the mitochondria of transgenic plants: towards novel ways of influencing mitochondrial gene expression?

Evidence that nuclear-encoded RNAs are present inside mitochondria has been reported from a wide variety of organisms, and is presumed to be due to import of specific cytosolic RNAs. In plants, the first examples were the mitochondrial leucine transfer RNAs of bean. In all cases, the evidence is circumstantial, based on hybridization of the mitochondrial RNAs to nuclear and not mitochondrial DNA. Here we show that transgenic potato plants carrying a leucine tRNA gene from bean nuclear DNA contain RNA transcribed from the introduced gene both in the cytosol and inside mitochondria, providing proof that the mitochondrial leucine tRNA is derived from a nuclear gene and imported into the mitochondria. The same bean gene carrying a 4 bp insertion in the anticodon loop was also expressed in transgenic potato plants and the transcript found to be present inside mitochondria, suggesting that this natural RNA import system could eventually be used to introduce foreign RNA sequences into mitochondria.

Base Sequence↗
PubMed2001

The effects of the site-directed removal of N-glycosylation from cationic peanut peroxidase on its function.

Peanut peroxidase has been diffracted. The location of its heme and calcium moieties have been shown and their role demonstrated. However, the structure and role of its glycans is only now being elucidated. The role of three N-linked complex glycans on cationic peroxidase (cPrx) of peanut (Arachis hypogaea L cv. Valencia), as expressed by prxPNC1 in transgenic tobacco, was analyzed by site-directed replacement of each of the three glycosylation sites, N-60, N-144, and N-185 with Q, individually. The mutant prxPNC1 cDNAs with a 3' histidine-tag were expressed in transgenic tobacco. The effect on the catalytic ability, thermal stability, and unfolding properties of the mutant peroxidases, isolated from the medium of transgenic tobacco cell suspension cultures were compared with those of the wild cPrx from peanut. It was found that the ablation of the glycans at N-60 and N-144 influences the full expression of the cPrx catalytic ability. The glycan at N-185 is important for the thermostability, as is the removal of the carbohydrate chain at N-185, resulting in rapid enzymatic decrease at temperatures of 50 degrees C. All three glycans appeared to influence the folding of the protein.

Arachis↗
PubMed1989

Deletion analysis of the polyadenylation signal of a pea ribulose-1,5-bisphosphate carboxylase small-subunit gene.

The polyadenylation signal of a pea gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS) has been analyzed by deletion mutagenesis and Ti plasmid-mediated gene transfer. Sequences between 6 and 137 bases upstream from the normal polyadenylation sites in this gene (bases -6 to -137) are required for functioning of these sites. In addition, bases -111 to -235 can affect 3' end formation by altering the pattern of 3' termini seen in various transcription units. Sequences between 37 and 95 bases upstream from a cryptic polyadenylation site in this gene [A. G. Hunt, DNA 7: 329-336 (1988)] are necessary for mRNA 3' end formation at this site. At least two different parts of the 3' region of this rbcS gene can serve as a downstream element for polyadenylation at the normal poly(A) addition sites in this gene. Our studies indicate that: 1. the upstream sequences required for polyadenylation in plants are different from those defined in mammalian RNA polymerase II transcription units; 2. sequences 100 or more bases upstream and downstream from poly(A) addition sites in this gene can affect poly(A) addition site choice; and 3. there are apparently redundant downstream elements for polyadenylation in this gene.

Base Sequence↗
PubMed1995

Antisense suppression of S-RNase expression in Nicotiana using RNA polymerase II- and III-transcribed gene constructs.

In the Solanaceae, self-incompatibility is controlled by a single, multi-allelic ('S') locus. One product of this locus is a ribonuclease, the S-RNase, which is expressed predominantly in mature pistils and has recently been shown to cause allele-specific pollen rejection in transgenic plants. Hybrid Nicotiana plumbaginifolia x N. alata plants were used to test the effects of antisense suppression of the SA2-RNase from N. alata using three different gene constructs: two driven by RNA polymerase II-transcribed promoters, and the third, containing a truncated soybean tRNA (met-i) gene, transcribed by RNA polymerase III. All three constructs caused suppression of S-RNase activity in the transgenic plants. Unexpectedly, the CaMV 35S promoter was more effective for antisense suppression than the tissue specific tomato ChiP promoter. Antisense suppression of S-RNase correlated with low sense SA2 transcript levels and high antisense SA2 transcript levels. Untransformed hybrids that contained the N. alata SA2 allele were incompatible with N. alata SA2 pollen, while transgenic plants with suppressed SA2 gene expression accepted the pollen. The utility of this hybrid plant system for studying some aspects of antisense gene suppression is discussed.

Chimera↗
PubMed1994

Destabilization of rbcS sense transcripts by antisense RNA.

Steady-state rbcS mRNA levels are drastically reduced in transgenic tobacco plants that express rbcS antisense RNAs. We have found that these reductions are not due to an effect of the antisense RNA at the level of rbcS transcription; rather, the sense mRNAs are more actively degraded in the mutant than wild-type plants. We have examined the kinetics of this turnover process by inhibiting transcription with cordycepin, and have found that rbcS sense mRNA decay is accelerated about five-fold in the antisense plants. This provides direct evidence that antisense RNAs can serve to destabilize sense transcripts in plants.

Deoxyadenosines↗
PubMed1993

Modulation of glutamine synthetase gene expression in tobacco by the introduction of an alfalfa glutamine synthetase gene in sense and antisense orientation: molecular and biochemical analysis.

A glutamine synthetase (GS) cDNA isolated from an alfalfa cell culture cDNA library was found to represent a cytoplasmic GS. The full-length alfalfa GS1 coding sequence, in both sense and antisense orientation and under the transcriptional control of the cauliflower mosaic virus 35S promoter, was introduced into tobacco. Leaves of tobacco plants transformed with the sense construct contained greatly elevated levels of GS transcript and GS polypeptide which assembled into active enzyme. Leaves of the plants transformed with the antisense GS1 construct showed a significant decrease in the level of both GS1 and GS2 polypeptides and GS activity, but did not show any significant decrease in the level of endogenous GS mRNA. We have proposed that antisense inhibition using a heterologous antisense GS RNA occurs at the level of translation. Our results also suggest that the post-translational assembly of GS subunits into a holoenzyme requires an additional factor(s) and is under regulatory control.

Alcohol Oxidoreductases↗
PubMed2000

DNA stability in plant tissues: implications for the possible transfer of genes from genetically modified food.

The potential for transfer of antibiotic resistance genes from genetically modified (GM) plant material to microbes through genetic recombination in the human or animal gut is a consideration that has engendered caution in the use of GM foods. This study was aimed at defining the optimal physical and chemical conditions necessary to ensure sufficient fragmentation of DNA in plant tissues to a size where it would be unlikely to be stably transferred to bacterial gut microflora. The ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SS) genes are of similar size (approximately 1.4 kb) to transgenes present in GM plants. DNA analysis and PCR amplification of Rubisco SS genes showed that fresh maize and maize silage contained high molecular weight DNA and intact Rubisco SS genes. Relatively high temperatures and pressurised steam were necessary to degrade fully genomic DNA and Rubisco SS genes in maize and wheat grains, the source of most animal feedstuffs. Furthermore, chemical expulsion and extrusion of oilseeds resulted in residues with completely degraded genomic DNA. These results imply that stringent conditions are needed in the processing of GM plant tissues for feedstuffs to eliminate the possibility of transmission of transgenes.

DNA Fragmentation↗
PubMed1996

The mechanism of GT element-mediated cell type-specific transcriptional control.

Promoter studies have revealed that sequences related to the GT-1 binding site, known as GT elements, are conserved in plant nuclear genes of diverse functions. In this work, we addressed the issue of whether GT elements are involved in cell type-specific transcriptional regulation. We found that the inactivation of GT-1 site-mediated transcription in roots is correlated with the absence of the GT-1 binding activity in root extracts. In addition, the mutation of the related GT-1 (from the pea rbcs-3A) and the S1F (from the spinach rps1) sites resulted in an increase of their transcriptional activity in roots that contain a distinct GT element-binding factor, referred to as RGTF. Although specific to GT elements, RGTF has a different sequence requirement and a lower sequence specificity than GT-1. Interestingly, RGTF has a higher binding affinity to the mutant GT-1 and S1F sites than to the wild-type sequences. This correlation suggests that RGTF may have some role in transcriptional regulation in roots. Furthermore, root cellular protein extracts contain an inhibitory activity that prevents GT-1 from binding to DNA. This helps to explain the absence of the GT-1 binding activity in roots in which the gene of GT-1 is expressed. Together, these data suggest that the cell type-specific transcription modulation by GT elements is achieved by using two different strategies.

DNA-Binding Proteins↗
PubMed2000

A minimum length of N gene sequence in transgenic plants is required for RNA-mediated tospovirus resistance.

We showed previously that transgenic plants with the green fluorescent protein (GFP) gene fused to segments of the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) displayed post-transcriptional gene silencing of the GFP and N gene segments and resistance to TSWV. These results suggested that a chimeric transgene composed of viral gene segments might confer multiple virus resistance in transgenic plants. To test this hypothesis and to determine the minimum length of the N gene that could trans-inactivate the challenging TSWV, transgenic plants were developed that contained GFP fused with N gene segments of 24-453 bp. Progeny from these plants were challenged with: (i) a chimeric tobacco mosaic virus containing the GFP gene, (ii) a chimeric tobacco mosaic virus with GFP plus the N gene of TSWV and (iii) TSWV. A number of transgenic plants expressing the transgene with GFP fused to N gene segments from 110 to 453 bp in size were resistant to these viruses. Resistant plants exhibited post-transcriptional gene silencing. In contrast, all transgenic lines with transgenes consisting of GFP fused to N gene segments of 24 or 59 bp were susceptible to TSWV, even though the transgene was post-transcriptionally silenced. Thus, virus resistance and post-transcriptional gene silencing were uncoupled when the N gene segment was 59 bp or less. These results provide evidence that multiple virus resistance is possible through the simple strategy of linking viral gene segments to a silencer DNA such as GFP.

Blotting, Northern↗
PubMed1999

A species of small antisense RNA in posttranscriptional gene silencing in plants.

Posttranscriptional gene silencing (PTGS) is a nucleotide sequence-specific defense mechanism that can target both cellular and viral mRNAs. Here, three types of transgene-induced PTGS and one example of virus-induced PTGS were analyzed in plants. In each case, antisense RNA complementary to the targeted mRNA was detected. These RNA molecules were of a uniform length, estimated at 25 nucleotides, and their accumulation required either transgene sense transcription or RNA virus replication. Thus, the 25-nucleotide antisense RNA is likely synthesized from an RNA template and may represent the specificity determinant of PTGS.

Amino Acid Oxidoreductases↗
PubMed2000

Transgenic or plant expression vector-mediated recombination of Plum Pox Virus.

Different mutants of an infectious full-length clone (p35PPV-NAT) of Plum pox virus (PPV) were constructed: three mutants with mutations of the assembly motifs RQ and DF in the coat protein gene (CP) and two CP chimeras with exchanges in the CP core region of Zucchini yellow mosaic virus and Potato virus Y. The assembly mutants were restricted to single infected cells, whereas the PPV chimeras were able to produce systemic infections in Nicotiana benthamiana plants. After passages in different transgenic N. benthamiana plants expressing the PPV CP gene with a complete (plant line 4.30.45.) or partially deleted 3'-nontranslated region (3'-NTR) (plant line 17.27. 4.), characterization of the viral progeny of all mutants revealed restoration of wild-type virus by recombination with the transgenic CP RNA only in the presence of the complete 3'-NTR (4.30.45.). Reconstitution of wild-type virus was also observed following cobombardment of different assembly-defective p35PPV-NAT together with a movement-defective plant expression vector of Potato virus X expressing the intact PPV-NAT CP gene transiently in nontransgenic N. benthamiana plants. Finally, a chimeric recombinant virus was detected after cobombardment of defective p35PPV-NAT with a plant expression vector-derived CP gene from the sour cherry isolate of PPV (PPV-SoC). This chimeric virus has been established by a double recombination event between the CP-defective PPV mutant and the intact PPV-SoC CP gene. These results demonstrate that viral sequences can be tested for recombination events without the necessity for producing transgenic plants.

Base Sequence↗