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Biomedical subjects

J Schell

Publications and source records attributed to J Schell.

At least 109 records · Page 6Linked to original sources
PubMed1992

New plant binary vectors with selectable markers located proximal to the left T-DNA border.

Five new binary vectors have been constructed which have the following features: (1) different plant selectable markers including neomycin phosphotransferase (nptII), hygromycin phosphotransferase (hpt), dihydrofolate reductase (dhfr), phosphinothricin acetyl transferase (bar), and bleomycin resistance (ble); (2) selectable markers are located near the T-DNA left border and; (3) selectable marker and beta-glucuronidase (uidA) reporter genes are divergently organized for efficient expression, and can easily be removed or replaced as needed.

Agrobacterium tumefaciens↗
PubMed1992

Isolation and characterization of a cDNA from Cuphea lanceolata encoding a beta-ketoacyl-ACP reductase.

A cDNA encoding beta-ketoacyl-ACP reductase (EC 1.1.1.100), an integral part of the fatty acid synthase type II, was cloned from Cuphea lanceolata. This cDNA of 1276 bp codes for a polypeptide of 320 amino acids with 63 N-terminal residues presumably representing a transit peptide and 257 residues corresponding to the mature protein of 27 kDa. The encoded protein shows strong homology with the amino-terminal sequence and two tryptic peptides from avocado mesocarp beta-ketoacyl-ACP reductase, and its total amino acid composition is highly similar to those of the beta-ketoacyl-ACP reductases of avocado and spinach. Amino acid sequence homologies to polyketide synthase, beta-ketoreductases and short-chain alcohol dehydrogenases are discussed. An engineered fusion protein lacking most of the transit peptide, which was produced in Escherichia coli, was isolated and proved to possess beta-ketoacyl-ACP reductase activity. Hybridization studies revealed that in C. lanceolata beta-ketoacyl-ACP reductase is encoded by a small family of at least two genes and that members of this family are expressed in roots, leaves, flowers and seeds.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗
PubMed1992

The complete nucleotide sequence of RNA 1 of a German isolate of barley yellow mosaic virus and its comparison with a Japanese isolate.

The nucleotide sequence of RNA 1 of a German isolate of barley yellow mosaic virus has been determined and compared with a Japanese isolate of the same virus. The sequence identity is 93.6% at the nucleotide level and 96% at the amino acid level. Similar values have been found for the polyproteins of the RNA 2 of both isolates (95%). Both isolates show an RNA 1-encoded protein arrangement similar to that of potyviruses such as tobacco etch virus. In contrast, the polyproteins of the small RNAs (RNA 2) do not show such a similarity to the polyproteins of other potyviruses. However, there is a striking difference between the two isolates in the generally highly conserved active site of the RNA-dependent RNA polymerase. The German isolate exactly matches the consensus sequences for previously described potyviral RNA-dependent RNA polymerases, whereas the Japanese isolate does not.

Amino Acid Sequence↗
PubMed1992

Molecular analysis of an auxin binding protein gene located on chromosome 4 of Arabidopsis.

We have isolated a cDNA clone from Arabidopsis, At-ERabp1, for the Arabidopsis auxin binding protein located in the lumen of the endoplasmic reticulum (ER). This cDNA clone codes for a protein related to the major auxin binding protein from maize, Zm-ERabp1. A single open reading frame, 594 bases in length, predicts a protein of 198 amino acid residues and a molecular mass of 22,044 D. The primary amino acid sequence contains an N-terminal hydrophobic signal sequence of 33 amino acids. We demonstrated by in vitro studies that the At-ERabp1 protein is translocated into ER-derived microsomes. The protein was processed, and the cleavage site for the N-terminal signal peptide was determined by radiosequencing. The mature protein is composed of 165 amino acid residues, with a molecular mass of 18,641 D. The At-ERabp1 protein contains potential N-glycosylation sites (Asn46-Ile-Ser and Asn130-Ser-Thr). In vitro transport studies demonstrated cotranslational glycosylation. Retention within the lumen of the ER correlates with an additional signal located at the C terminus and represented by the amino acids Lys196-Asp-Glu-Leu, well known to be essential for active retrieval of proteins into the lumen of the ER. DNA gel blot analysis of genomic DNA revealed single hybridizing bands, suggesting that only a single At-ERabp1 gene is present in the Arabidopsis genome. Restriction fragment length polymorphism mapping indeed revealed a single locus mapping to chromosome 4.

Amino Acid Sequence↗
PubMed1991

Identification of NolR, a negative transacting factor controlling the nod regulon in Rhizobium meliloti.

In Rhizobium meliloti, expression of the nodulation genes (nod and nol genes) is under both positive and negative controls. These genes are activated by the products of the three related nodD genes, in conjunction with signal molecules from the host plants. We showed that negative regulation is mediated by a repressor protein, binding to the overlapping nodD1 and nodA as well as to the nodD2 promoters. The encoding gene, termed nolR, was identified and cloned from strain 41. By subcloning, deletion and Tn5 mutagenesis, a region of 594 base-pairs was found to be necessary and sufficient for repressor production in strains of R. meliloti lacking the repressor or in Escherichia coli. Sequence analysis revealed that nolR encodes a 13,349 Da protein, which is in agreement with the molecular weight of the NolR protein, determined after purification by affinity chromatography, utilizing long synthetic DNA multimers of the 21 base-pair conserved repressor-binding sequence. Our data suggest that the native NolR binds to the operator site in dimeric form. The NolR contains a helix-turn-helix motif, which shows homology to the DNA-binding sequences of numerous prokaryotic regulatory proteins such as the repressor XylR or the activator NodD and other members of the LysR family. Comparison of the putative DNA-binding helix-turn-helix motifs of a large number of regulatory proteins pointed to a number of novel regularities in this sequence. Hybridizations with an internal nolR fragment showed that sequences homologous to the nolR gene are present in all R. meliloti isolates tested, even in those that do not produce the repressor. In another species, such as Rhizobium leguminosarum, where NodD is autoregulated, however, such sequences were not detected.

Amino Acid Sequence↗
PubMed1991

Geminivirus-based shuttle vectors capable of replication in Escherichia coli and monocotyledonous plant cells.

Shuttle vectors have been constructed that are able to replicate in either Escherichia coli or plant cells. They contain the ColE1 origin of replication and parts of the wheat dwarf virus genome, a geminivirus infecting a variety of species of monocotyledonous plants. Such plasmids are able to replicate in E. coli and wheat cells. The plasmids can be rescued in E. coli and show no changes during their passage through plant cells. Such an E. coli/plant cell shuttle vector system could be used for the amplification of foreign genes in plant cells, for studies on DNA rearrangement or the isolation of plant transposons.

Bacteriocin Plasmids↗
PubMed1991

Purification of tobacco nuclear proteins binding to a CACGTG motif of the chalcone synthase promoter by DNA affinity chromatography.

The activity of various light-regulated and developmentally regulated plant gene promoters critically depends upon the presence of a conserved sequence with a central CACGTG motif. Using band-shift assays, we have identified nuclear factor(s) from Nicotiana tabacum, termed CG-1, that specifically recognize(s) this transcriptional element in the ultraviolet-light-regulated Antirrhinum majus chalcone synthase promoter. CG-1 activity is constitutively expressed in tobacco seedlings grown in the absence of ultraviolet light as well as in seedlings induced for chalcone synthase gene expression by ultraviolet light irradiation. CG-1 activity has also been detected in flower tissue. DNA-protein cross-linking studies identified three polypeptides with apparent molecular masses of 20, 32 and 42 kDa binding to the CACGTG motif. Proteins interacting with the CACGTG motif were purified from N. tabacum seedlings using differential sequence-specific DNA affinity chromatography employing wild-type and mutated CG-1-binding sites. Denaturing polyacrylamide gel electrophoresis revealed major polypeptides of approximately 20, 30 and 40 kDa which are highly enriched in the affinity-purified fractions binding specifically to the CACGTG motif.

Acyltransferases↗
PubMed1991

Reduced steady-state levels of rbcS mRNA in plants kept in the dark are due to differential degradation.

When plants are placed in the dark, the level of the abundant mRNA encoding the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS) declines rapidly. We present evidence demonstrating an active degradation of rbcS mRNA in the dark. Detailed analysis shows that transcripts originating from different members of the rbcS gene family are differentially affected by this degradation. This phenomenon is not common to all light-regulated plant genes since the mRNA for ST-LS1, another leaf-specific and light-induced gene, is not degraded in the dark within the same time scale.

Base Sequence↗
PubMed1991

The protein encoded by the rolB plant oncogene hydrolyses indole glucosides.

The rolB gene of Agrobacterium rhizogenes, whose expression stimulates the formation of roots by transformed plant tissues and other growth alterations in transgenic plants, codes for a beta-glucosidase able to hydrolyse indole-beta-glucosides. Indeed, we show that extracts of bacteria and/or plant tissue expressing the rolB protein hydrolyse indoxyl-beta-glucoside (plant indican). Because of the structural similarity between indoxyl-beta-glucoside and indole-3-acetyl-beta-glucoside (IAA-beta-glucoside), we propose that the physiological and developmental alterations in transgenic plants expressing the rolB gene could be the result of an increased intracellular auxin activity caused by the release of active auxins from inactive beta-glucosides. Thus two of the oncogenes carried by the T-DNA of the plant pathogen Agrobacterium rhizogenes (rolB and rolC) perturb plant growth and development by coding for beta-glucosidases with distinct specificities. Whereas the rolC beta-glucosidase releases cytokinins from their glucoside conjugates, the rolB encoded protein hydrolyses indole-beta-glucosides. The combined action of these two genes therefore is expected to modulate the intracellular concentration of two of the main growth factors active in plants.

Base Sequence↗
PubMed1991

T-DNA gene 5 of Agrobacterium modulates auxin response by autoregulated synthesis of a growth hormone antagonist in plants.

Oncogenes carried by the transferred DNA (T-DNA) of Agrobacterium Ti plasmids encode the synthesis of plant growth factors, auxin and cytokinin, and induce tumour development in plants. Other T-DNA genes regulate the tumorous growth in ways that are not yet understood. To determine the function of T-DNA gene 5, its coding region was expressed in Escherichia coli. Synthesis of the gene 5 encoded protein (26 kDa) correlated with a 28-fold increase in conversion of tryptophan to indole-3-lactate (ILA), an auxin analogue. Expression of chimeric gene 5 constructs in transgenic tobacco resulted in overproduction of ILA that enhanced shoot formation in undifferentiated tissues and increased the tolerance of germinating seedlings to the inhibitory effect of externally supplied auxin. Promoter analysis of gene 5 in plants revealed that its expression was inducible by auxin and confined to the vascular phloem cells. cis-regulatory elements required for auxin regulation and phloem specific expression of gene 5 were mapped to a 90 bp promoter region that carried DNA sequence motifs common to several auxin induced plant promoters, as well as a binding site for a nuclear factor, Ax-1. ILA was found to inhibit the auxin induction of the gene 5 promoter and to compete with indole-3-acetic acid (IAA) for in vitro binding to purified cellular auxin binding proteins. It is suggested therefore that ILA autoregulates its own synthesis and thereby modulates a number of auxin responses in plants.

Base Sequence↗
PubMed1991

The plant oncogene rolC is responsible for the release of cytokinins from glucoside conjugates.

The rolC gene of Agrobacterium rhizogenes, which drastically affects growth and development of transgenic plants, codes for a cytokinin-beta-glucosidase. Indeed, rolC protein expressed in Escherichia coli as a fusion protein hydrolyses cytokinin glucosides, thus liberating free cytokinins. Furthermore, beta-glucosidase activity present in E. coli extracts expressing the rolC protein was inhibited by affinity-purified antibodies specific for the rolC protein. Finally, rolC proteins expressed in transgenic plants were shown to be responsible for cytokinin-beta-glucosidase activity. Morphological and phytohormonal analysis, performed on transgenic plants that are somatic mosaics for the expression of the rolC gene, extend and confirm our interpretation that the developmental, physiological and morphological alterations caused by rolC expression in transgenic plants are primarily due to a modification of the cytokinin balance. These observations shed new light on the control of growth and differentiation in plants by growth factors.

Bacterial Proteins↗
PubMed1991

T-DNA integration: a mode of illegitimate recombination in plants.

Transferred DNA (T-DNA) insertions of Agrobacterium gene fusion vectors and corresponding insertional target sites were isolated from transgenic and wild type Arabidopsis thaliana plants. Nucleotide sequence comparison of wild type and T-DNA-tagged genomic loci showed that T-DNA integration resulted in target site deletions of 29-73 bp. In those cases where integrated T-DNA segments turned out to be smaller than canonical ones, the break-points of target deletions and T-DNA insertions overlapped and consisted of 5-7 identical nucleotides. Formation of precise junctions at the right T-DNA border, and DNA sequence homology between the left termini of T-DNA segments and break-points of target deletions were observed in those cases where full-length canonical T-DNA inserts were very precisely replacing plant target DNA sequences. Aberrant junctions were observed in those transformants where termini of T-DNA segments showed no homology to break-points of target sequence deletions. Homology between short segments within target sites and T-DNA, as well as conversion and duplication of DNA sequences at junctions, suggests that T-DNA integration results from illegitimate recombination. The data suggest that while the left T-DNA terminus and both target termini participate in partial pairing and DNA repair, the right T-DNA terminus plays an essential role in the recognition of the target and in the formation of a primary synapsis during integration.

Base Sequence↗