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A Depicker

Publications and source records attributed to A Depicker.

At least 19 recordsLinked to original sources
PubMed1999

Gene silencing results in instability of antibody production in transgenic plants.

The stability of antibody and Fab expression was assessed in five different homozygous transgenic Arabidopsis lines. Each of these lines showed silencing of the transgenes that encode the antibody polypeptides, leading to instability of antibody production. However, each line had a different and specific instability profile. The characteristic variation in the level of antibody accumulation in each line as a function of developmental stage indicated that the T-DNA integration pattern played a role in triggering silencing, and also that the history and the integration position of simple transgene loci can influence the susceptibility to epigenetic silencing. In different lines with low antibody accumulation levels, methylation was found either in the promoter alone, in both the promoter and the transcribed region, in the transcribed region only, or in the transcribed region and downstream sequences. In conclusion, our data suggest that epigenetic effects result in different transgene expression profiles in each of the five Arabidopsis lines analyzed.

Antibody Formation↗
PubMed1999

High level accumulation of single-chain variable fragments in the cytosol of transgenic Petunia hybrida.

The accumulation of five murine single-chain variable fragments, binding to dihydroflavonol 4-reductase, was analyzed in transgenic Petunia hybrida plants. The five scFv-encoding sequences were cloned in an optimized plant transformation vector for expression in the cytosol under control of the 35S promoter. In a transient expression assay we found that the scFv expression levels were reproducible and correlated with those in stably transformed petunia. Our results show that accumulation in the cytosol strongly depends on the intrinsic properties of the scFv fragment. Three of the five scFv fragments accumulated to unexpectedly high levels in the cytosol of the primary transformants, but no phenotypic effect could be detected. Experimental results indicate that one of the scFv fragments accumulated in the cytosol to 1% of the total soluble protein as a functional antigen-binding protein in the absence of disulphide bonds. This observation supports the idea that certain antibody fragments do not need disulphide bonds to be stable and functional. Such scFv scaffolds provide new opportunities to design scFv fragments for immunomodulation in the cytosol.

Alcohol Oxidoreductases↗
PubMed1998

Accumulation pattern of IgG antibodies and Fab fragments in transgenic Arabidopsis thaliana plants.

For the further optimization of antibody expression in plants, it is essential to determine the final accumulation sites of plant-made antibodies. Previously, we have shown that, upon secretion, IgG antibodies and Fab fragments can be detected in the intercellular spaces of leaf mesophyll cells of transgenic Arabidopsis thaliana plants. However, immunofluorescence microscopy showed that this is probably not their final accumulation site. In leaves, IgG and Fab fragments accumulate also at the interior side of the epidermal cell layers and in xylem vessels. These accumulation sites correspond with the leaf regions where water of the transpiration stream is entering a space impermeable to the proteins or where water is evaporating. In roots, plant-made Fab fragments accumulate in intercellular spaces of cortex cells, in the cytoplasm of pericycle and, to a lesser extent, endodermis cells, and in cells of the vascular cylinder. In other words, antibody accumulation occurs at the sites where water passes on its radial pathway towards and within the vascular bundle. Taken together, our results suggest that, upon secretion of plant-made antibodies or Fab fragments, a large proportion of these proteins are transported in the apoplast of A. thaliana, possibly by the water flow in the transpiration stream.

Animals↗
PubMed1998

Agrobacterium tumefaciens transformation and cotransformation frequencies of Arabidopsis thaliana root explants and tobacco protoplasts.

In view of the recent finding that different T-DNAs tend to ligate and integrate as repeats at single chromosomal positions, the frequency of transformation and cotransformation was determined during cocultivation of Arabidopsis thaliana root explants and Nicotiana tabacum protoplasts with two Agrobacterium strains. The transformation frequency of unselected A. thaliana shoots was lower than 1% whereas that of cocultivated tobacco protoplasts was approximately 18%. The cotransformation frequencies, defined as the frequencies with which cells transformed with a first T-DNA contained a second unselected T-DNA, were approximately 40% reproducible, irrespective of the selection, the transformation frequency, and the plant system used. Extrapolation of these results suggests that at least two independently transferred T-DNAs were present in 64% of the transformed plant cells. Molecular analysis of cocultivated N. tabacum shoots regenerated on nonselective medium showed that only a few transformants had a silenced (2/46) or truncated (1/46) T-DNA. Therefore, most integrated T-DNAs expressed their selectable or screenable markers in primary transgenic plants. Remarkably, 10 to 30% of the selected A. thaliana shoots or progenies lost the T-DNA marker they were selected on. As these regenerants contained the unselected T-DNA with a high frequency (17%), these selected plants might result from the expression of unstable, transiently expressed T-DNAs. In conclusion, a significant part of the T-DNAs is lost from the transformed cells.

Agrobacterium tumefaciens↗
PubMed1997

Use of phage display for isolation and characterization of single-chain variable fragments against dihydroflavonol 4-reductase from Petunia hybrida.

To isolate specific single-chain variable (scFv) fragments against dihydroflavonol 4-reductase (DFR) from Petunia hybrida the phage display technology was used. DFR was overproduced in Escherichia coli, purified and used for immunization. From DFR-immunized mice, a phage display library was made starting from spleen mRNA using an optimized set of primers for V(H) and V(L) amplification. Several rounds of panning against recombinant DFR yielded five different scFv fragments, confirmed by subsequent DNA sequencing. They all specifically bound to recombinant DFR in ELISA and DFR in flower extracts on Western blot. These results show that phage display is a promising technology in plant molecular biology to obtain specific recombinant antibodies not only for ELISA and Western blot but also for in vivo applications in the long run.

Alcohol Oxidoreductases↗
PubMed1997

Post-transcriptional gene silencing in plants.

Overexpression of chimeric transgenes in plants can trigger post-transcriptional gene silencing that is dependent on epigenetic information and physiological conditions. The current view is that unproductive RNA serves as a crucial signal for gene silencing, although direct evidence is lacking for this theory. A signalling cascade then leads to strongly enhanced turnover of all RNAs that share a critical degree of sequence similarity. The molecular details of the mechanism are, however, insufficiently understood to explain the phenomenon completely and to comprehend its biological significance.

5-Methylcytosine↗
PubMed1997

T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs.

Nicotiana protoplasts and Arabidopsis leaf discs or roots were co-cultivated with two Agrobacterium strains each carrying a different T-DNA. Co-transformed plants were selected and the integration of the different T-DNAs was analysed at the genetic and genomic level. Genetic analysis showed that the T-DNAs derived from different bacteria were frequently integrated at the same locus, independent of the plant species or transformation method used. Southern analysis revealed that 12 out of 27 Arabidopsis transformants contained the co-transferred T-DNAs linked to each other in all possible configurations but with a preference for those with at least one right border involved in linkage. Overall, our data support the hypothesis that ligation of separate T-DNAs is a dominant mechanism in formation of the frequently observed repeats of identical T-DNAs. We propose a scheme which could explain the formation of T-DNA repeats and the preferential involvement of right borders in T-DNA linkages.

Arabidopsis↗
PubMed1996

Bacterial and plant-produced scFv proteins have similar antigen-binding properties.

A gene encoding a single-chain variable (scFv) antibody fragment was expressed as a cytoplasmic and endoplasmic reticulum-targeted protein in transgenic tobacco plants. In both cases, the scFv accumulated up to 0.01% of total soluble protein (TSP). The same scFv fragment was also produced in the periplasm of Escherichia coli. Measurement of the affinity by ELISA indicates that the affinity of the bacterially made scFv is about 80-fold lower than that of the parental Fab fragment. The results suggest that the affinity of the plant-produced scFv fragments is reduced to a similar extent, implying that all the plant-produced scFv fragments are antigen binding.

Amino Acid Sequence↗
PubMed1995

Polymorphic CAC/T repetitive sequences in the pig genome 1.

Three genomic clones were isolated from a size-selected pig DNA library by hybridization with a DNA-fingerprint probe. Analysis at the sequence level revealed that all three clones contain interrupted stretches of triplet repeats mainly composed of CAC and CAT triplets. Evaluation of the corresponding loci for polymorphism by Southern blot hybridization showed considerable length variation. For two loci the polymorphism was also demonstrated by polymerase chain reaction (PCR) amplification. The PiGMaP reference pedigree was typed for all three loci.

Animals↗
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↗
PubMed1993

Assembly of an antibody and its derived antibody fragment in Nicotiana and Arabidopsis.

The yield and assembly of an IgG1 antibody and its derived F(ab) fragment were compared in Nicotiana and Arabidopsis. The results obtained showed a lot of interclonal variability. For 45% of the primary transgenic calluses, antigen-binding entities represented less than 0.1% of the total soluble protein (TSP). Only two of the 103 analysed transformants contained more than 1% of antigen-binding protein, with 1.26% being the highest yield. Analogous amounts of complete antibody and F(ab) accumulated in primary callus tissue. Moreover, yields were in the same range for both species as far as primary callus tissue is concerned. However, the accumulation of the F(ab) fragment in leaf tissue of regenerated plants differed significantly between Nicotiana and Arabidopsis. The F(ab) fragment accumulated to only 0.044% of TSP in Nicotiana leaves but up to 1.3% in Arabidopsis leaves. Furthermore, both species showed differences in the assembly pattern of the complete antibody. Whereas Arabidopsis contained primarily fully assembled antibodies of 150 kDa, Nicotiana showed an abundance of fragments in the 50 kDa range.

Animals↗
PubMed1993

Characterization of porcine polymorphic microsatellite loci.

Twenty-seven (CA)n and two (GA)n microsatellite clones were isolated out of a size-selected genomic pig library. These were sequenced and the number of uninterrupted dinucleotides was found to range from 12 to 26. Flanking primers were chosen for 11 dinucleotide repeats and optimal conditions for polymerase chain reaction (PCR) amplifications were established. Different microsatellite loci were amplified simultaneously by combining primer sets. Related and unrelated pigs were screened for length polymorphisms of the different microsatellite loci. The polymorphic information content (PIC) of these loci ranged between 0.62 and 0.83. Segregation studies in pig reference families established Mendelian inheritance. Locus S0022 was found to be X-linked.

Alleles↗
PubMed1992

Effect of T-DNA configuration on transgene expression.

T-DNA vectors were constructed which carry a beta-glucuronidase (gusA) gene fused to the promoter of the nopaline synthase (nos) gene and the 3' end of the octopine synthase (ocs) gene. This reporter gene was cloned at different locations and orientations towards the right T-DNA border. For each construct, between 30 and 60 stably transformed calli were analysed for beta-glucuronidase activity. Depending on the T-DNA configuration, distinct populations of gusA-expressing calli were obtained. Placing the reporter gene in the middle of the T-DNA results in relatively low expression levels and a limited inter-transformant variability. Placing the gene with its promoter next to the right border led to an increase in both the mean activity and the variability level. With this construct, some of the calli expressed the gusA gene at levels four to five times higher than the mean. In all these series, at least 30% of the calli contained reporter gene activities that were less than half of the mean expression level. Separating the gusA gene from the right T-DNA border by an additional 3'-untranslated region, derived from the nos gene, resulted in an increase in the mean expression to a level almost four times higher than that of constructions carrying the reporter gene in the middle of the T-DNA. Moreover, the number of transformants with extremely low activities decreased by at least 50% and this resulted in significantly lower inter-transformant variability independently of the orientation of the reporter gene on the T-DNA.

Agrobacterium tumefaciens↗
PubMed1991

Transcriptional interference in transgenic plants.

When a promoterless marker gene is transformed into the plant genome using the Agrobacterium vector system, on average 30% of the T-DNA inserts produce gene fusions. This suggests that the T-DNA is preferentially integrated into transcribed regions. Here, we proposed that this transcriptional activity is responsible for some of the variation in expression frequently observed among independent transformants. Using hybrid gene constructions, we show that transcriptional readthrough into a downstream gene with opposite orientation substantially reduces expression of this gene both in transient expression and in transgenic plants. Furthermore, a poly(A) signal/terminator can block readthrough and restore the expression of the gene. Finally, enzymatic analysis of calli suggests that less variation in neomycin phosphotransferase II synthesis is observed when the gene is separated from plant DNA by promoter and terminator elements.

Agrobacterium tumefaciens↗
PubMed1991

The extensin signal peptide allows secretion of a heterologous protein from protoplasts.

Extensins are hydroxyproline-rich glycoproteins which are amongst the most abundant proteins present in the cell wall of higher plants. Here, we describe the structural analysis of an extensin-encoding gene from Nicotiana plumbaginifolia. The encoded protein (46 kDa) has a highly repetitive structure and contains 37% proline, 18.1% tyrosine, 13.4% lysine, 8.1% serine and 7.1% histidine. The extensin-encoding sequence contains a typical signal peptide for translocation of the protein to the endoplasmic reticulum. By using chimeric genes consisting of different 5' parts of the extensin-encoding gene and the neomycin phosphotransferase II-encoding gene (nptII) as reporter gene, we show that the N-terminal part of extensin can mediate the secretion of NPTII from electroporated N. tabacum protoplasts.

Amino Acid Sequence↗
PubMed1990

Cloning and sequence analysis of truncated T-DNA inserts from Nicotiana tabacum.

Transgenic plants produced by Agrobacterium-mediated transformation usually have one or a few stable and intact T-DNA insertions. However, in a significant number of the transformants Southern blot analysis has revealed the occurrence of aberrant T-DNA insertions missing one or both ends. During the study of this phenomenon, we obtained KmR Nicotiana tabacum clones after cocultivation with an Agrobacterium strain containing a promoterless nptII gene located internally in the T-DNA. Expression of this nptII gene requires a break in the T-DNA region upstream from the nptII-coding sequence and insertion of the truncated T-DNA in a transcriptionally active plant DNA region. The most conspicuous result from Southern analyses on four such KmR plant clones is that they contain several T-DNAs truncated at other positions besides the upstream region of the nptII sequence. Four truncated T-DNA insertions have been cloned. Two insertions contain the nptII gene fused to plant expression signals and are missing the right part of the T-DNA. Another is missing the left T-DNA part and the last T-DNA is lacking both ends. Sequence analysis of the T-DNA::plant junctions has shown that the T-DNA breakpoints are randomly distributed and do not show obvious homologies to one another or to the border consensus sequence. S1-type mapping of the most strongly expressed plant genome::nptII fusion revealed a specific transcription start point and putative TATA and CAAT boxes in the upstream plant DNA region; the steady-state nptII mRNA in these plants is about 20 times more abundant than in transgenic Pnos-nptII plants.

Base Sequence↗