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A Depicker

Publications and source records attributed to A Depicker.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1990

Analysis of the stop codon context in plant nuclear genes.

A region of 18 nucleotides surrounding the stop codon (the stop codon context) in 748 plant nuclear genes was analyzed. Non-randomness was found both upstream and downstream from the stop codon, suggesting that these sequences may help in ensuring efficient termination of translation. The UAG amber codon is the least-used stop codon and the bias in the nucleotide distribution 5' and 3' to the stop codon was more pronounced for the amber codon than for the other stop codons. This might indicate that the codon context affects termination more at UAG than at UGA or UAA stop codons.

Base Composition↗
PubMed1990

Plant chromosome/marker gene fusion assay for study of normal and truncated T-DNA integration events.

During Agrobacterium tumefaciens infection, the T-DNA flanked by 24 bp imperfect direct repeats is transferred and stably integrated into the plant chromosome at random positions. Here we measured the frequency with which a promoterless reporter gene is activated after insertion into the Nicotiana tabacum SR1 genome. When adjacent to the right or left T-DNA border sequences, at least 35% of the transformants express the marker gene, suggesting preferential T-DNA insertion (greater than 70%) in transcriptionally active regions of the plant genome. When the promoterless neomycin phosphotransferase II (nptII) gene is located internally in the T-DNA, the activation frequency drops to 1% since gene activation requires T-DNA truncation. These truncation events in the nptII upstream region occur independently of the nature of the upstream sequence and of the T-DNA length. Deletion of the right border region prevents the detection of activated marker genes. Therefore, T-DNA truncation probably occurs after synthesis of a normal T-DNA intermediate during the transfer and/or integration process. In the absence of border regions, expression of the nptII selectable marker directed by the nopaline synthase promoter was detected in 1 out of 10(5) regenerated calli, suggesting the possibility that any DNA sequence from the Ti plasmid can be transformed into the plant genome, albeit at a low frequency.

Base Sequence↗
PubMed1990

A hypervariable pig DNA fragment.

A new hypervariable tandem repeat was isolated from the pig genome and characterized by DNA sequence. The use of this DNA fragment as a probe in order to follow allelic segregation and DNA fingerprinting in pigs, horses and rabbits is documented.

Animals↗
PubMed1989

Expression of dicistronic transcriptional units in transgenic tobacco.

We investigated whether the two cistrons of a dicistronic mRNA can be translated in plants to yield both gene products. The coding sequences of various reporter genes were combined in dicistronic units, and their expression was analyzed in stably transformed tobacco plants at the RNA and protein levels. The presence of an upstream cistron resulted in all cases in a drastically reduced expression of the downstream cistron. The translational efficiency of the gene located downstream in the dicistronic units was 500- to 1,500-fold lower than that in a monocistronic control; a 500-fold lower value was obtained with a dicistronic unit in which both cistrons were separated by 30 nucleotides, whereas a 1,500-fold lower value was obtained with a dicistronic unit in which the stop codon of the upstream cistron and the start codon of the downstream cistron overlapped. As a strategy to select indirectly for transformants with enhanced levels of expression of a gene which is by itself nonselectable, the gene of interest can be cloned upstream from a selectable marker in a dicistronic configuration. This strategy can be used provided that the amount of dicistronic mRNA is high. If, on the other hand, the expression of the dicistronic unit is too low, selection of the downstream cistron will primarily give clones with rearranged dicistronic units.

Base Sequence↗