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At least 919 records · Page 51Linked to original sources

PubMed1995

A repetitive DNA fragment carrying a hot spot for de novo DNA methylation enhances expression variegation in tobacco and petunia.

A 1.6 kb repetitive DNA sequence (RPS) from Petunia hybrida was identified that destabilizes expression of a GUS marker transgene. Following polyethylene glycol (PEG)-mediated tobacco and petunia protoplast transformations, GUS expression patterns analysed on callus and plant levels were clearly more variable when constructs contained the RPS sequence. The effect on transgene expression required chromosomal integration since the two different RPS constructs employed did not exhibit reduced levels of GUS activities in transient assays. DNA methylation analysis implies a hypermethylated default state of endogenous RPS copies present in the petunia genome. Analysis of the transgene DNA in different transgenic tobacco plants showed almost complete hypermethylation of a particular Hhal site of the RPS sequence. It is proposed that, due to the presence of specific signals within the RPS region or based on interaction of RPS with other endogenous homologous sequences, RPS functions as an initiation region for de novo methylation and induces expression variegation in adjacent sequences.

Base Sequence↗

PubMed1994

Organ-specific and auxin-inducible expression of two tobacco par A-related genes in transgenic plants.

We have isolated four genomic DNA clones that contain the transcription initiation site of the parA gene(s) from a tobacco genomic library by hybridization with the 5' segment of the parA cDNA previously isolated. They were classified into two types on the basis of their nucleotide sequences. Southern blot analysis indicated that two types of clones were respectively derived from the two parental species of tobacco, Nicotiana tomentosiformis and Nicotiana sylvestris. The genes corresponding to these clones were designated as parAt and parAs, respectively, and the parA cDNA clone was shown to code for mRNA from parAt on the basis of its nucleotide sequence. The 5' regions about 400 nucleotides upstream from the transcription initiation sites of the parAt and parAs genomic clones were highly homologous to one another, but regions further upstream showed no significant similarity. The coding sequence of the GUS (beta-glucuronidase) reporter gene was linked to the 5'-upstream regions of parAt and parAs, and the sites of expression of these fusion genes were examined in transgenic tobacco plants. In the absence of auxins, both fusion genes were expressed in capsules at a late stage of seed development, mature seeds, a root apex and a root-hair zone whereas no significant expression was seen in other organs. Their expression was enhanced by 2,4-dichlorophenoxyacetic acid in most of the organs of tobacco. The results show that expression of these genes is regulated by both organ-specific and auxin-inducible mechanisms.

Amino Acid Sequence↗

PubMed1996

Genetic and biochemical dissection of transgenic RNA-mediated virus resistance.

RNA-mediated virus resistance has been observed in transgenic plants at varying frequencies, suggesting that a nuclear requirement or other pre-condition must be met. This study was undertaken to characterize genetically transgenes that confer a highly resistant state to infection by tobacco etch virus (TEV). Transgenic tobacco line 2RC-6.13, expressing an untranslatable mRNA containing the TEV coat protein open reading frame, had three distinct transgene integration events that segregated as two linkage groups. A genetic series of plants that contained zero, one, two, or all three transgene inserts in both homozygous and heterozygous conditions was produced and examined. Genetic and biochemical data suggested that RNA-mediated virus resistance is a multigenic trait in line 2RC-6.13; three or more transgenes were necessary to establish the highly resistant state. One or two transgene copies resulted in an inducible form of resistance (i.e., recovery). Transcription rates and steady state RNA levels of the transgene-derived transcript present in different members of the genetic series supported a post-transcriptional RNA degradation process as the underlying mechanism for transgene transcript reduction and virus resistance. This degradation process appeared to initiate via cleavage of specific sites within the target RNA sequence, as determined by RNA get blot and primer extension analyses of transgene-derived mRNA from various transgenic plant lines.

Capsid↗

PubMed1995

Expression of the plum pox coat protein gene in transgenic Nicotiana tabacum plants.

Plant expression vector pBI 121 containing the gene encoding coat protein of Plum Pox Virus of the Skierniewice isolate (CP PPV-S) was prepared (clone pCM1). The construct was used for transformation of Nicotiana tabacum plants using an Agrobacterium tumefaciens based system. About 82% of kanamycin resistant plant lines contained a transgene (the sequence of CP PPV-S) but only 81% of them actively expressed the PPV-S coat protein gene as measured by RT-PCR.

Agrobacterium tumefaciens↗

PubMed1994

Expression of biologically active hordothionins in tobacco. Effects of pre- and pro-sequences at the amino and carboxyl termini of the hordothionin precursor on mature protein expression and sorting.

Hordothionins (HTHs) are small anti-bacterial proteins present in barley endosperm which are processed from larger precursor proteins, consisting of an amino-terminal signal peptide (SP), the mature highly basic HTH and a carboxy-terminal acidic peptide (AP). Different HTH precursor proteins were expressed in tobacco to study the effects of the pre-sequences (SP) and pro-sequences (AP) on expression, processing, sorting and biological activity and hence the feasibility of engineering bacterial disease resistance into crops which lack these proteins. Maximum HTH expression levels of approximately 0.7% (11 mumol/kg) of total soluble protein in young tobacco leaves were obtained using a semi-synthetic gene construct encoding a complete chimaeric HTH precursor protein. Tenfold lower HTH expression levels (maximum 1.3 mumol/kg) were obtained using synthetic gene constructs without the AP-coding sequence and no expression was found in plants containing synthetic HTH gene constructs without SP- and AP-coding sequences. In both cases where expression was found, the precursors were apparently correctly processed, although the HTH produced in plants containing a construct without AP sequence appeared to be slightly modified. No effect on plant phenotype was observed. Localization studies indicated that the HTH was in identical fractions of plants expressing the two different precursors, albeit at a different ratio, and was not secreted into the intercellular spaces of leaves or culture medium by protoplasts. Our results indicated that the AP is not involved in sorting and suggested that it might facilitate transport through membranes. The in vitro toxicity of HTH isolated from transgenic tobacco plants expressing the two different precursor proteins for the bacterial plant pathogen Clavibacter michiganensis subsp. michiganensis appeared similar to that of the HTH purified from barley endosperm.

Amino Acid Sequence↗

PubMed1995

Cis-acting elements and expression pattern of the spinach rps22 gene coding for a plastid-specific ribosomal protein.

In order to study the regulation of nuclear genes coding for plastid ribosomal proteins, we have analysed the promoter region of spinach rps22 using both in vitro and in vivo approaches. By footprinting analyses, we have identified eight DNA elements interacting with spinach leaf nuclear factors in the 300 bp promoter region upstream of the transcription start site. Among these elements, four are short AT-rich sequences and one is identical to the Hex motif characterized initially in wheat histone genes. In transgenic tobacco plants, the reporter gene coding for the beta-glucuronidase (GUS) directed by a 1.2 kb upstream region of rps22 was expressed in several plant organs, with high levels in leaf mesophyll, embryo cotyledons and root meristematic cells and very low levels in other cell types. Interestingly, when deleted to -295, the promoter, which contained all the foot-printed elements, was still able to confer the same expression pattern, although the activity was relatively lower than with the 1.2 kb promoter. When deleted further to -154, the promoter, from which the AT-rich elements were eliminated, loses its activity almost completely, suggesting that these AT-rich elements are important for the rps22 promoter activity. Altogether, our results show that rps22 gene expression is controlled by specific cis elements not present in other nuclear-encoded plastid ribosomal protein genes studied so far.

Base Sequence↗

PubMed2000

Both sense and antisense RNAs are targets for the sense transgene-induced posttranscriptional silencing mechanism.

Two stable transgenic tobacco lines were obtained as segregants from a primary transformant. Plants homozygous for a T-DNA inverted repeat locus (HOlo1) showed posttranscriptional gene silencing (PTGS) of the neomycin phosphotransferase II (nptII) transgenes, whereas HOlo2 plants, homozygous for a single T-DNA insert, expressed the nptII genes normally. Transient expression of nptII genes newly introduced into leaves of both the HOlo2 and nptII-silenced HOlo1 plants was downregulated only in the silenced background. Different chimeric beta-glucuronidase (gus) genes with parts of the nptII transgene inserted in sense or antisense orientation into the 3'-untranslated region, which encoded transcripts that had homology or complementarity to nptII transcripts. showed reduced transient expression specifically in nptII-silenced tissue. Therefore, we conclude that RNAs of both polarities are targets for PTGS-induced RNA degradation, which supports the notion that double-stranded RNA acts as an inducing signal for silencing.

Agrobacterium tumefaciens↗

PubMed1997

Transcription of genes for conglutin gamma and a leginsulin-like protein in narrow-leafed lupin.

The expression of genes encoding conglutin gamma and a leginsulin-like protein has been examined in narrow-leafed lupin, Lupinus angustifolius L. Conglutin gamma is a homologue of basic 7S globulin (Bg), the insulin and leginsulin binding protein from soybean. Accumulation of conglutin gamma mRNA, as assessed by northern assays and reverse-transcription PCR, was tightly regulated both spatially and temporally in lupin plants and was detected almost exclusively in developing seeds. Similar tissue and temporal specificity was demonstrated when 1.8 kb of the promoter region from the conglutin gamma gene was used to drive the expression of a beta-glucuronidase reporter gene in transgenic plants. In stably transformed tobacco the conglutin gamma promoter produced strong, temporally regulated and seed-specific expression of the reporter gene which was localised to the embryo tissues and to a layer of cells adjacent to the seed coat. A truncated 0.29 kb promoter fragment produced much reduced levels of expression and a loss of embryo specificity. Leginsulin-like mRNA was similarly detected in lupins only in developing seeds. The leginsulin-like gene detected in L. angustifolius showed 96% sequence identity to leginsulin from soybean within the 280 bp region amplified from lupin by PCR. The results demonstrate that both components of a Bg-leginsulin putative signal transduction pathway are present in the seeds of lupin.

Albumins↗

PubMed2000

The yeast polyadenylate-binding protein (PAB1) gene acts as a disease lesion mimic gene when expressed in plants.

We have expressed the gene (PAB1) encoding the yeast polyadenylate-binding protein (Pab1p) in tobacco. Plants that accumulate the Pab1p display a range of abnormalities, ranging from a characteristic chlorosis in leaves to a necrosis and large inhibition of growth. The severity of these abnormalities reflects the levels of yeast Pab1p expression in the transgenic plants. In contrast, no obvious differences could be seen in callus cultures between the transgene and vector control. Plants that display PAB-associated abnormalities were resistant to a range of plant pathogens, and had elevated levels of expression of a pathogenesis-related gene. These two properties--impairment of growth and induction of defense responses--indicate that the yeast PAB1 gene can act as a disease lesion mimic gene in plants.

Cell Division↗

PubMed1994

Posttranscriptional silencing of reporter transgenes in tobacco correlates with DNA methylation.

Endogenous plant genes or transgenes can be silenced on introduction of homologous gene sequences. Here we document a reporter gene-silencing event in Nicotiana tabacum that has a distinctive combination of features--i.e., (i) silencing occurs by a posttranscriptional process, (ii) silencing correlates with DNA methylation, and (iii) this de novo methylation is not restricted to cytosines located in the symmetrical motifs CG and CXG.

Arabidopsis↗

PubMed1995

Complementation of tobacco etch potyvirus mutants by active RNA polymerase expressed in transgenic cells.

A genetic complementation system was developed in which tobacco etch virus (TEV) polymerase (NIb)-expressing transgenic plants or protoplasts were inoculated with NIb-defective TEV mutants. A beta-glucuronidase (GUS) reporter gene integrated into the genomes of parental and four mutant viruses was used to assay RNA amplification. Two mutants (termed VNN and EDE) contained substitutions affecting the conserved "GDD" polymerase motif or a nuclear localization signal sequence, respectively; one (aD/b) contained a mutation debilitating the NIb N-terminal cleavage site, whereas the other (delta b) lacked the entire NIb sequence. Each mutant was unable to amplify in nontransformed tobacco protoplasts. In contrast, the VNN, EDE, and delta b mutants were complemented to various degrees in NIb-expressing cells, whereas the aD/b mutant was not complemented. The VNN mutant was complemented most efficiently, reaching an average of 11-12% the level of parental TEV-GUS, although in some experiments the level was near 100%. This mutant also replicated in, and spread through, whole transgenic plants to the same level as parental virus. The EDE mutant was complemented relatively poorly, reaching 1% or less of the level of parental TEV-GUS. Despite the close proximity of the EDE substitution to the N-terminal cleavage site, proteolytic processing of NIb was unaffected in an in vitro assay. The delta b mutant was complemented to an intermediate degree in protoplasts, reaching 3.5% the level of parental virus, and replicated and moved systemically in transgenic plants. These data indicate that free NIb supplied entirely in trans can provide all NIb functions essential for RNA amplification. The relative inefficient complementation of the EDE mutant suggests that the resulting mutant protein was transinhibitory.

Biological Assay↗

PubMed2001

HC-Pro suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal.

Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation mechanism that is widespread in eukaryotic organisms. It is often associated with methylation of the transcribed region of the silenced gene and with accumulation of small RNAs (21 to 25 nucleotides) homologous to the silenced gene. In plants, PTGS can be triggered locally and then spread throughout the organism via a mobile signal that can cross a graft junction. Previously, we showed that the helper component-proteinase (HC-Pro) of plant potyviruses suppresses PTGS. Here, we report that plants in which PTGS has been suppressed by HC-Pro fail to accumulate the small RNAs associated with silencing. However, the transgene locus of these plants remains methylated. Grafting experiments indicate that HC-Pro prevents the plant from responding to the mobile silencing signal but does not eliminate its ability to produce or send the signal. These results demonstrate that HC-Pro functions downstream of transgene methylation and the mobile signal at a step preceding accumulation of the small RNAs.

Algorithms↗

PubMed2006

Evaluation of allergenicity of genetically modified soybean protein extract in a murine model of oral allergen-specific sensitization.

BACKGROUND: With the development of genetically modified crop plants there has been a growing interest in the approaches available to assess the potential allergenicity of novel gene products. For additional assessment of the potential allergenicity of expressed proteins, informative data can be generated using animal models. Soybean is one of the major source of protein in human and animal nutrition, and has also been well characterized as a major allergenic source. Advances in biotechnology have resulted in an increasing number of genetically engineered foods, and among these soybean is one of the most widespread. OBJECTIVE: To develop and characterize a murine model of IgE-mediated soybean sensitization induced by intragastric immunization, in the presence of Cholera Toxin, with wild-type soybean extract (wt-SE) or with genetically modified soybean extract (gm-SE). METHODS: Balb/c mice born in our animal facilities, from females fed on soy-free food, were fed with the same soy-free food and used in all the experiments. Mice were sensitized by gavages with soybean extracts, and allergen-specific IgE and IgG responses were studied by direct ELISA and ELISA inhibition. Antigen-specific cell proliferation and cytokine production were evaluated in spleen cell cultures. Results Sensitization with both soybean extracts induced high levels of antigen-specific IgE and IgG1 and low levels of specific IgG2a. Both wt-SE and gm-SE were able to inhibit the binding of specific IgE from mice immunized with gm-SE to the same antigen used for the ELISA coating. A comparable proliferative response was obtained with the homologous as well as with the heterologous extracts. CONCLUSION: In sensitized mice, we observed a predominantly T-helper type 2 (Th2)-type immune response, with increased soybean-specific IgE and IgG1 antibodies and a concomitant increase of IL-4 and IL-5 production. RESULTS: obtained by specific IgE ELISA inhibition and by antigen-specific T cell proliferation demonstrated that wt-SE and gm-SE shared B and T epitopes. The present murine model of soybean sensitization established by the oral route should provide valuable information about risk assessment for food allergy from new proteins of genetically modified foods.

Administration, Oral↗

PubMed1997

Protein processing and auxin response in transgenic tobacco harboring a putative cDNA of zeatin O-xylosyltransferase from Phaseolus vulgaris.

Zeatin is rapidly metabolized to O-xylosylzeatin in Phaseolus vulgaris seeds. The zeatin O-xylosyltransferase mediating this conversion, a 50 kDa protein, occurs mainly in the endosperm, both in the cytoplasm and the nuclei. A monoclonal antibody specific to the enzyme was used to isolate cDNAs from an expression library derived from P. vulgaris seeds. Two highly homologous, full-length cDNAs were isolated. The ORFs encode proteins of 69 and 67 kDa, respectively, with 90% homology at the amino acid level. cDNA-encoded protein obtained from in vitro transcription/translation was processed to protein of 50 kDa by bean endosperm extract. Transgenic tobacco plants harboring the larger ORF under the control of the CaMV35S promoter were more sensitive to the auxin NAA than control plants. The symptoms included leaf chlorosis, restriction of root elongation, and eventual cessation of growth. The antigenic preprotein was processed, and labeled zeatin was converted to O-xylosylzeatin in transgenic plants grown on NAA-containing medium. Analyses of independently transformed families indicated that the presence of the transgene coincided with the increased auxin sensitivity and protein processing correlated with the manifestation of auxin-induced damage. These results suggest that posttranslational processing regulates enzyme activity, and offer the possibility that cytokinin-auxin balance may be affected by stimulation of cytokinin metabolic enzyme activity by auxin.

Amino Acid Sequence↗

PubMed1992

Wind-induced plant motion immediately increases cytosolic calcium.

Wind is one of the most unusual and more dramatic of the environmental signals to modify plant development. Wind-stimulated crops are also known to experience considerable reductions in growth and subsequent yield. There is at present no experimental data to suggest how wind signals are perceived and transduced by plant cells. We have genetically transformed Nicotiana plumbaginifolia to express aequorin and thus produced luminous plants that directly report cytosolic calcium by emitting blue light. With these plants we have found wind stimulation to cause immediate increases in cytosolic calcium and our evidence, based on the use of specific inhibitors, suggests that this calcium is mobilized from organelle sources. Our data further suggest that wind-induced movement of tissues, by mechanically stimulating and stressing constituent plant cells, is responsible for the immediate elevation of cytosolic calcium; increases occur only when the plant tissue is actually in motion. Repeated wind stimulation renders the cells refractory to further calcium signaling but responsiveness is rapidly recovered when stimulation is subsequently diminished. Our data suggest that mechanoperception in plant cells may possibly be transduced through intracellular calcium. Since mechanoperception and transduction are considered crucial to plant morphogenesis, our observations suggest that calcium could be central in the control and generation of plant form.

Aequorin↗

PubMed1994

The soybean SAUR open reading frame contains a cis element responsible for cycloheximide-induced mRNA accumulation.

Little is known about how mRNA stability is regulated in higher plants. The SAURs (Small Auxin-Up RNAs) are a family of highly unstable mRNAs in soybean that rapidly increase in abundance after excised organs are treated with the plant hormone auxin. The SAURs are also induced by protein synthesis inhibitors, including cycloheximide, in the absence of auxin treatment and are superinduced when organs are treated with cycloheximide plus auxin. While the induction of SAURs is transcriptionally regulated by auxin, the induction by cycloheximide is posttranscriptional. Cycloheximide as well as other protein synthesis inhibitors appear to induce SAUR accumulation by increasing the stabilities of these mRNAs. To determine whether the 5'-untranslated region, the 3'-untranslated region, or the open reading frame of these unstable mRNAs is responsible for the cycloheximide inducibility, we have used chimeric genes in transgenic tobacco plants to test each of these mRNA regions. Our results show that the SAUR open reading frame within a chimeric mRNA confers cycloheximide inducibility in transgenic tobacco plants whereas chimeric mRNAs containing the SAUR 5'-untranslated region or 3'-untranslated region as isolated elements or in combination are not induced by cycloheximide. These results suggest that the SAUR open reading frame contains sequence elements that are involved in the stability of these mRNAs.

Blotting, Northern↗

PubMed1993

Inhibition of tobacco NADH-hydroxypyruvate reductase by expression of a heterologous antisense RNA derived from a cucumber cDNA: implications for the mechanism of action of antisense RNAs.

Tobacco plants were genetically transformed to generate antisense RNA from a gene construct comprised of a full-length cucumber NADH-dependent hydroxypyruvate reductase (HPR) cDNA placed in reverse orientation between the cauliflower mosaic virus 35S promoter and a nopaline synthase termination/polyadenylation signal sequence. In vivo accumulation of antisense HPR RNA within eight independent transgenic tobacco plants resulted in reductions of up to 50% in both native HPR activity and protein accumulation relative to untransformed tobacco plants (mean transgenote HPR activity = 67% wild type, mean transgenote HPR protein = 63% wild type). However, in contrast to previous reports describing antisense RNA effects in plants, production of the heterologous HPR antisense RNA did not systematically reduce levels of native tobacco HPR mRNA (mean transgenote HPR mRNA level = 135% wild type). Simple regression comparison of the steady-state levels of tobacco HPR mRNA to those of HPR antisense RNA showed a weak positive correlation (r value of 0.548, n = 9; n is wild type control plus eight independent transformants; significant at 85% confidence level), supporting the conclusion that native mRNA levels were not reduced within antisense plants. Although all transgenic antisense plants examined displayed an apparent reduction in both tobacco HPR protein and enzyme activity, there is no clear correlation between HPR activity and the amount of either sense (r = 0.267, n = 9) or antisense RNA (r = 0.175, n = 9). This compares to a weak positive correlation between HPR mRNA levels and the amount of HPR activity observed in wild-type SR1 tobacco plants (r = 0.603, n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate Dehydrogenases↗

PubMed2000

Identification of a signal peptide for oryzacystatin-I.

A previously unidentified extension of an open reading frame from the genomic DNA of Japonica rice (Oryza sativa L.) encoding oryzacystatin-I (OC-I; access. M29259, protein ID AAA33912.1) has been identified as a 5' gene segment coding for the OC-I signal peptide. The signal peptide appears to direct a pre-protein (SPOC-I; Accession No. AF164378) to the endoplasmic reticulum, where it is processed into the mature form of OC-I. The start codon of SPOC-I begins 114 bp upstream from that previously published for OC-I. A putative proteolytic site. which may yield a mature OC-I approximately 12 residues larger than previously described, has been identified within SPOC-I between Ala-26 and Glu-27. The signal peptide sequence was amplified by polymerase chain reaction using genomic DNA from O. sativa seedlings and ligated to the 5' end of the truncated OC-I gene at the endogenous SalI site. Partially purified protein extracts from Escherichia coli expressing SPOC-I reacted with polyclonal antibodies raised against OC-I and revealed a protein of the expected molecular weight (15,355 Da). In-vitro translation of SPOC-I in the presence of microsomal membranes yielded a processed product approximately 2.7 kDa smaller than the pre-protein. Nicotiana tabacum L. cv. Xanthi plants independently transformed with the SPOC-I gene processed SPOC-I and accumulated the mature form of OC-I (approximately 12.6 kDa), which co-migrated with natural, mature OC-I extracted from rice seed when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Amino Acid Sequence↗