Search PubMed⌕ Search

Biomedical subjects

J Schell

Publications and source records attributed to J Schell.

At least 181 records · Page 10Linked to original sources
PubMed1988

Relocating a gene for herbicide tolerance: A chloroplast gene is converted into a nuclear gene.

The chloroplast gene psbA codes for the photosynthetic quinone-binding membrane protein Q(B), which is the target of the herbicide atrazine. This gene has been converted into a nuclear gene. The psbA gene from an atrazine-resistant biotype of Amaranthus hybridus has been modified by fusing its coding region to transcription-regulation and transit-peptide-encoding sequences of a bona fide nuclear gene. The constructs were introduced into the nuclear genome of tobacco by using the Agrobacterium tumor-inducing (Ti) plasmid system, and the protein product of nuclear psbA has been identified in the photosynthetic membranes of chloroplasts. Recovery of atrazine-tolerant transgenic plants shows that the product of the transplanted gene functions in photosynthesis. These experiments show that it is possible to modify chloroplast-gene-specified functions via nuclear-genome transformation and also raise evolutionary questions.

Journal Article↗
PubMed1988

Rhizobium meliloti nodA and nodB genes are involved in generating compounds that stimulate mitosis of plant cells.

The nodB gene of Rhizobium meliloti encodes a 23.8-kDa protein that is conserved in several Rhizobium species. Monospecific polyclonal antibodies against NodB were used to localize this protein in the cytosol of R. meliloti and Escherichia coli cells containing nodABC genes. In comparison to the NodA and NodC proteins, NodB is synthesized in a disproportionately low amount. The NodA and NodB proteins are involved in generating small, heat-stable compounds that stimulate the mitosis of various plant protoplasts. Our experiments suggest that NodC is not involved in the synthesis of the factors. On the basis of their properties, we speculate that the factors are cytokinin-like substances.

Journal Article↗
PubMed1988

Synthesis of an opine-like compound, a rhizopine, in alfalfa nodules is symbiotically regulated.

We show that the promoter of the mos locus, which encodes genes required for the synthesis of a nodule-specific, opine-like compound, a rhizopine, in alfalfa nodules is regulated by the symbiotic nitrogen-fixation regulatory gene nifA. The 5'-regulatory region and amino-terminal end of the first open reading frame of the mos locus are highly homologous to the 5'-regulatory region and amino-terminal portion of the Rhizobium meliloti nifH gene. The coordinate regulation of mos and nif genes suggests that the mos locus plays a symbiotic role. We propose that the rhizopine enhances the survival of the bacterial partner in the symbiosis.

Bacterial Proteins↗
PubMed1988

rbcS genes in Solanum tuberosum: conservation of transit peptide and exon shuffling during evolution.

Five genes of the rbcS gene family of Solanum tuberosum (potato) were studied. One of these is a cDNA clone; the other four are located on two genomic clones representing two different chromosomal loci containing one (locus 1) and three genes (locus 2), respectively. The intron/exon structure of the three genes in locus 2 is highly conserved with respect to size and position. These genes contain two introns, whereas the gene from locus 1 contains three introns. Although in most cases the amino acid sequences in the transit peptide part of different rbcS genes from the same species varied considerably more than the corresponding mature amino acid sequences, one exception found in tomato and potato indicates that the transit peptide of rbcS could have a special function. A comparison of the rbcS genes of higher plants with those of prokaryotes offers suggestive evidence that introns first served as spacer material in the process of exon shuffling and then were removed stepwise during the evolution of higher plants.

Amino Acid Sequence↗
PubMed1988

Differential expression of genes in potato tubers after wounding.

Tubers of a common tetraploid species of Solanum tuberosum (Granola) were mechanically wounded by slicing. After 18 hr only small differences were detectable in the pattern of the steady-state protein extracted from wounded versus unwounded tubers. In contrast the protein pattern obtained by in vitro translation of mRNA isolated from wounded and unwounded tubers differed significantly. A cDNA library was established and screened for wound-induced cDNA clones by differential hybridization. Two clones, wun-1 and wun-2, were found that corresponded to genes that were highly expressed in wounded potato tubers but were not expressed in unwounded tubers. The expression of the gene corresponding to wun-1 is detectable 30 min after wounding; the expression of the gene corresponding to wun-2 is detectable 4 hr after wounding. The expression of both genes (hereafter referred to as wun-1 and wun-2) remains constant for up to 24 hr after wounding. Interestingly the RNA corresponding to patatin, a major storage protein of potato tubers, behaves in the opposite way; it decreases dramatically in tubers within 30 min after wounding. The low level of patatin mRNA observed in unwounded roots and stems also disappears after wounding. Run-off transcription experiments, performed with isolated nuclei, indicate that the activation of the wound-induced genes as well as the inhibition of the patatin gene are controlled at the transcriptional level.

Journal Article↗
PubMed1987

Improved method for the isolation of RNA from plant tissues.

A fast and efficient method for the isolation of RNA from plant tissues is described. Tuber tissue is homogenized in a guanidine hydrochloride-containing buffer followed by direct extraction with phenol/chloroform. The RNA is precipitated from the aqueous phase, washed with 3 M sodium acetate and 70% ethanol, and finally dissolved in water. The yield of RNA is up to 500 micrograms/g of tissue and several tests indicate intact and nondegraded RNA. This method can be adapted to a small-scale version by the use of 1.5-ml tubes, allowing rapid isolation of RNA from a larger number of samples. Finally, this method is of particular use for isolating RNA from tissues with a high polysaccharide and nuclease content such as wounded potato tubers.

Chloroform↗
PubMed1987

Organ-specific and dosage-dependent expression of a leaf/stem specific gene from potato after tagging and transfer into potato and tobacco plants.

ST-LS1, a single copy gene from potato displaying a leaf/stem specific gene expression, was tagged by an exon modification and introduced into both potato and tobacco cells using Agrobacterium vectors. After regeneration of whole plants, the expression of the tagged gene was analyzed with respect to its organ specificity and compared to the expression of the corresponding resident gene. The expression of the transferred gene in transgenic plants closely followed the expression of the resident gene. No marked influence of the plant species serving as host was observed. The level of expression of the introduced gene varied by a factor of at least 100 in independent transformants when normalized to the expression of the resident gene. Southern analysis performed on the transformed plants indicated a correlation between copy number of the introduced gene and its expression level. The activity of the tagged gene as well as of the resident gene was significantly inhibited by treatment of the transgenic plants with the herbicide norfluorazon, indicating that this gene activity is dependent on the presence of functional chloroplasts in the leaves.

DNA, Recombinant↗
PubMed1987

Expression of a tuber-specific storage protein in transgenic tobacco plants: demonstration of an esterase activity.

A chimaeric gene composed of the 5' upstream region of STLS1, a leaf/stem specifically expressed gene from Solanum tuberosum, and the RNA-coding as well as the 3' downstream region of patatin, the major storage protein of potato tubers, has been transferred into tobacco plants using the Agrobacterium system. The introduction of this gene led to a leaf/stem specific expression of a 42-kd large protein which immunocrossreacts with patatin antiserum. Only low amounts of immunoreacting protein of smaller size could be detected in transgenic tobacco leaves indicating that the patatin protein is fairly stable in this heterologous environment. The size of the protein as well as the size of the RNA detected in transgenic tobacco leaves using a patatin-specific probe indicates that the patatin RNA was accurately processed in both leaf and stem tissue of tobacco. The expression of the patatin gene led to the appearance of a new esterase activity in the transformed tobacco which co-migrated with a protein immunoreacting with patatin antiserum. These data therefore demonstrate that patatin in addition to serving as a storage protein displays an enzymatic activity.

Journal Article↗
PubMed1987

Phenotypic assay for excision of the maize controlling element Ac in tobacco.

We describe a phenotypic assay designed to detect excision of the maize controlling element Ac from a selectable marker gene, neomycin phosphotransferase II (NPT II). An NPT II gene which expresses kanamycin resistance in tobacco cells, and contains a unique restriction enzyme site in the untranslated leader region, was constructed. Ac, or a defective Ac element (Ac big up tri, open), was inserted into the leader region of this gene. The transposon insertions inactivated the NPT II gene as determined by transient NPT II expression assays. The three plasmids were inserted into the T DNA of Agrobacterium tumefaciens Ti plasmid vectors, and transferred to tobacco protoplasts. The transformed protoplasts were selected with 100 or 200 microg/ml kanamycin. Protoplasts transformed by the NPT II gene interrupted by Ac formed 25% as many calli resistant to 100 or 200 microg/ml kanamycin as protoplasts transformed by the uninterrupted NPT II gene. Protoplasts transformed by the NPT II gene interrupted by Ac big up tri, open did not form any calli resistant to 200 microg/ml of kanamycin when transformed under similar conditions. Southern blot hybridization analyses of seven kanamycin-resistant calli or plants obtained after transformation by the NPT II gene interrupted by Ac revealed that in all cases Ac had excised, restoring the structure of the NPT II gene. This assay is therefore useful to monitor the activity of a transposable element such as Ac and to define the regions of this element involved in transposition activity.

Journal Article↗
PubMed1987

Wound expression of a potato proteinase inhibitor II gene in transgenic tobacco plants.

A potato proteinase inhibitor II gene was transferred into tobacco plants using Agrobacterium/Ti-plasmid-mediated gene transfer techniques. Whereas no or little expression of the proteinase inhibitor II gene could be detected in non-wounded leaves, high levels of proteinase inhibitor II mRNA were detected in leaves of several transgenic tobacco plants after mechanical wounding as well as after treatment of detached leaves with oligosaccharides. Wounding of a leaf also led to a systemic induction in non-wounded leaves as well as non-wounded stem, and roots. RNA-protection experiments showed that the transcription of the proteinase inhibitor gene in transgenic tobacco plants was initiated at the same nucleotide as that of the original gene. These observations demonstrate that although no proteinase inhibitor II homologous gene can be detected in tobacco, this plant nevertheless has the capacity to regulate the expression of the potato gene in the same complex manner as in the case of potato.

Blotting, Southern↗
PubMed1987

Host-specific regulation of nodulation genes in Rhizobium is mediated by a plant-signal, interacting with the nodD gene product.

We have identified a nodD gene from the wide host-range Rhizobium strain MPIK3030 (termed nodD1) which is essential for nodulation on Macroptilium atropurpureum (siratro). Experiments with nodA-lacZ gene fusions demonstrate that the MPIK3030 nodD1 regulates expression of the nodABC genes. Additionally, we used nodC-lacZ fusions of Rhizobium meliloti to show that the MPIK3030 nodD1 gene induces expression of these fusions by interacting with plant factors from siratro and from the non-host Medicago sativa (alfalfa). The R. meliloti nodD genes, however, only interact with alfalfa exudate. In line with these results, no complementation of MPIK3030 nodD1 mutants could be obtained on siratro with the R. meliloti nodD genes, while the MPIK3030 nodD1 can complement nodD mutants of R. meliloti on alfalfa. Furthermore, R. meliloti transconjugants harbouring the MPIK3030 nodD1 efficiently nodulate the illegitimate host siratro. When compared with other nodD sequences, the amino acid sequence of the MPIK3030 nodD1 shows a conserved aminoterminus, whereas the carboxy-terminus of the putative gene product diverges considerably. Studies on a chimeric MPIK3030/R. meliloti nodD gene indicates that the carboxy-terminal region is responsible for the interaction with plant factor(s) and may have evolved in different rhizobia specifically to interact with plant-host factors.

Journal Article↗
PubMed1987

The ntrC gene of Agrobacterium tumefaciens C58 controls glutamine synthetase (GSII) activity, growth on nitrate and chromosomal but not Ti-encoded arginine catabolism pathways.

The ntrC locus of Agrobacterium tumefaciens C58 has been cloned using the Azorhizobium sesbaniae ORS571 ntrC gene as a DNA hybridization probe. Transposon Tn5 mutagenesis of the cloned ntrC locus was carried out and one Tn5 insertion within the region of highest DNA homology with A. sesbaniae ORS571 ntrC was used for gene replacement of the wild-type C58 ntrC gene. The A. tumefaciens ntrC::Tn5 mutant was found to be unable to grow on nitrate as sole nitrogen (N) source, to lack glutamine synthetase (GSII) activity and to be unable to use arginine (or ornithine) as sole N source, unless the Ti-encoded arginine catabolism pathway was induced with small amounts of nopaline. Thus the A. tumefaciens ntrC regulatory gene is essential for (transcriptional) activation of the GSII and nitrate reductase genes, as well as for the chromosomal but not the Ti-borne arginine catabolism pathways.

Agrobacterium tumefaciens↗
PubMed1987

Expression and assembly of functional bacterial luciferase in plants.

The luxA and luxB structural genes of Vibrio harveyi luciferase [alkanal,reduced FMN:oxygen oxidoreductase (1-hydroxylating, luminescing), EC 1.14.14.3] were introduced into a plant expression vector and transferred into tobacco and carrot cells by Agrobacterium-mediated or direct DNA transformation. Simultaneous expression of the luxA and luxB genes was monitored by protein immunoblot analysis. Luciferase-mediated light emission provided evidence for the assembly of the two protein subunits into a functional dimeric enzyme in plant protoplasts, in transformed calli, and in leaves of transformed plants. Bacterial luciferase may provide a useful marker-gene system for the quantitative assay of coordinate gene expression in transgenic plants.

Journal Article↗
PubMed1987

Genes for the catabolism and synthesis of an opine-like compound in Rhizobium meliloti are closely linked and on the Sym plasmid.

In alfalfa nodules induced by Rhizobium meliloti strain L5-30 the compound L-3-O-methyl-scyllo-inosamine (3-O-MSI) is synthesized. This compound is also catabolized specifically by this strain. Its biological properties are therefore similar to the Agrobacterium opines. To answer the question whether opine-like compounds ("Rhizopines") play a role in a plant symbiotic interaction, we isolated the genes for the catabolism of 3-O-MSI (moc genes) and for the induction of its synthesis in the nodule [mos gene(s)]. moc and mos genes were shown to be closely linked and located on the Sym plasmid of L5-30, suggesting that they have co-evolved and may be important in symbiosis. These genes have been cloned into a broad host-range vector that can be mobilized into other R. meliloti strains where they are expressed. The location of the mos genes in the bacteria extends the opine concept, initially developed for a plant pathological interaction, to a symbiotic one.

Journal Article↗
PubMed1987

Analysis of cis-active sequences involved in the leaf-specific expression of a potato gene in transgenic plants.

ST-LS1 is a light-inducible, single-copy gene from potato that is expressed only in photosynthetic tissues. Various sequences derived from the 5'-upstream region of this gene were fused to the coding region of the chloramphenicol acetyltransferase (CAT) gene and to the gene 7 termination region of the transfer DNA (T-DNA) from the Agrobacterium Ti plasmid pTiACH5 and transferred to tobacco using Ti-plasmid vectors. After regeneration of whole plants, tissues were assayed for the expression of the CAT gene. Sequences derived from the 5'-upstream region of the ST-LS1 gene comprising positions -334 to +11 were sufficient to confer a leaf/stem-specific as well as a light-inducible expression of the CAT gene. Destruction of chloroplasts by treatment with the herbicide norfluorazon and subsequent exposure to light drastically reduced the expression of the CAT gene indicating that this upstream sequence most likely interacts with a chloroplast-dependent signal. When sequences from position -98 to position +675 were fused to a truncated inactive fragment of the cauliflower mosaic virus 35S promoter in a head-to-head manner, the corresponding chimeric genes were again expressed in photosynthetic tissues only, indicating that these sequences have enhancer-like properties.

Journal Article↗
PubMed1987

Recombination of selectable marker DNA in Nicotiana tabacum.

A chimeric neomycin phosphotransferase II (NPT II) gene, which normally provides kanamycin resistance to transformed plant cells, was inactivated by in vitro deletions. Repair plasmids not containing plant-specific transcription signals but containing only the NPT II coding region (or parts of it) were used in co-transformation experiments involving direct DNA uptake into protoplasts isolated from Nicotiana tabacum. Recombination, or gene conversion mediated by homologous sequences produced active NPT II genes in about 1% of transformants, rendering these cells resistant to kanamycin. Analysis of the size of the active enzyme indicated that recombination had occurred producing an NPT II gene indistinguishable from the wild-type gene. Southern blot analysis revealed that the bulk of co-transformed donor plasmid DNA had suffered structural modifications; however, kanamycin resistance was inherited in a Mendelian fashion, indicating that at least one functional and structurally intact copy of the regenerated NPT II gene is integrated into the host genome.

DNA, Recombinant↗