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Biomedical subjects

J Schell

Publications and source records attributed to J Schell.

At least 145 records · Page 8Linked to original sources
PubMed1989

Engineering of monomeric bacterial luciferases by fusion of luxA and luxB genes in Vibrio harveyi.

Luciferase (Lux)-encoding sequences are very useful as reporter genes. However, a drawback when applying Vibrio harveyi Lux as a reporter enzyme in eukaryotic cells, is that it is a heterodimeric enzyme, thus requiring simultaneous synthesis of both Lux subunits to be active. To overcome this disadvantage, luxA and luxB genes encoding the A and B subunits of this light-emitting heterodimeric Lux, were fused and expressed in Escherichia coli. Comparative analysis of four fused monomeric Lux enzymes by in vivo enzyme assay, immunoblotting and partial enzyme purification, showed that the fused Lux were active both as AB or as BA monomers, albeit at different levels (up to 80% activity for AB and up to 2% for BA, as compared with the wild type binary A + B construct). One of the LuxAB fusion proteins was stably expressed in calli of Nicotiana tabacum, and displayed very high Lux activity, thus demonstrating its potential as a reporter enzyme in eukaryotic systems.

Base Sequence↗
PubMed1989

Both developmental and metabolic signals activate the promoter of a class I patatin gene.

Patatin is one of the major soluble proteins in potato tubers and is encoded by a multigene family. Based on structural considerations two classes of patatin genes are distinguished. The 5'-upstream regulatory region of a class I gene contained within a 1.5 kb sequence is essential and sufficient to direct a high level of tuber-specific gene activity which was on average 100- to 1000-fold higher in tubers as compared to leaf, stem and roots in greenhouse grown transgenic potato plants when fused to the beta-glucuronidase reporter gene. Histochemical analysis revealed this activity to be present in parenchymatic tissue but not in the peripheral phellem cells of transgenic tubers. Furthermore the promoter fragment can be activated in leaves under conditions that simulate the need for the accumulation of starch in storage organs, i.e. high levels of sucrose. The expression is restricted to both mesophyll and epidermal cells in contrast to vascular tissue or hair cells.

Journal Article↗
PubMed1989

Wheat dwarf virus, a geminivirus of graminaceous plants needs splicing for replication.

By analysing mRNAs with the polymerase chain reaction (PCR) and by studying in vitro generated mutants we have identified an intron in the genome of wheat dwarf virus (WDV), a geminivirus of cereals. Polypeptides whose expression is essential for the replication of the viral DNA have been defined. They are encoded by two distinct overlapping open reading frames (ORFs). The joining of these two ORFs by deletion of the intron as well as the introduction of a frameshift mutation within the intron do not prevent replication of the viral genome in suspension culture cells. In contrast to WDV, the geminiviruses of dicotyledonous plants possess a single continuous ORF, highly homologous to the two individual ones of WDV. We propose that mRNA splicing is a common feature of all geminiviruses of the Gramineae and might contribute to their host class specificity. The existence of a functional intron is a novel finding for the plant viruses.

Amino Acid Sequence↗
PubMed1989

Positive and negative control of nod gene expression in Rhizobium meliloti is required for optimal nodulation.

We show that expression of common nodulation genes in Rhizobium meliloti is under positive as well as negative control. A repressor protein was found to be involved in the negative control of nod gene expression. Whereas the activator NodD protein binds to the conserved cis-regulatory element (nod-box) required for coordinated regulation of nod genes, the repressor binds to the overlapping nodD1 and nodA promoters, at the RNA polymerase binding site. A model depicting the possible interaction of the plant-derived nod gene inducer (luteolin), the NodD and the repressor with the nod promoter elements is presented. Mutants lacking the repressor exhibited delayed nodulation phenotype, indicating that fine tuning of nod gene expression is required for optimal nodulation of the plant host.

Journal Article↗
PubMed1989

Correlation of the expression of the nuclear photosynthetic gene ST-LS1 with the presence of chloroplasts.

A detailed analysis of the expression of a chimeric gene, consisting of the upstream region of the nuclear photosynthetic gene ST-LS1, encoding a component of the water-oxidizing complex of photosystem II, fused to the coding sequence of beta-glucuronidase (GUS) as a reporter, is described. The expression of this chimeric gene at the cellular level was detected by histochemical methods and shows that the expression of this gene is correlated with the presence of chloroplasts. Interestingly, the GUS activity was not only detected in typical photosynthetic tissues, e.g. leaves and stems, but also in green roots containing chloroplasts. In contrast no activity was detected in neighbouring white root tissue which was devoid of chloroplasts. One can therefore separate the relative importance of the (morphological) differentiation steps responsible for the formation of tissues normally involved in photosynthesis, from the importance of the developmental stage (characterized by the presence of chloroplasts), for the expression of this nuclear photosynthetic gene. Our data strongly suggest that the developmental stage of the plastids is the primary determinant for the activity of this nuclear photosynthetic gene, although they do not yet allow the exclusion of the reverse type of control, i.e. control of the differentiation of the plastid by the expression of certain nuclear genes. A chimeric gene, consisting of the promoter of the 35S cauliflower mosaic virus (CaMV) gene and the GUS coding sequence, was used as a control throughout the experiments, confirming that the observed differential ST-LS1-GUS gene expression reflects the particular transcriptional regulation impacted on this gene by its cis-acting regulatory sequences.

Cell Nucleus↗
PubMed1989

Molecular cloning and structural analysis of a gene from Zea mays (L.) coding for a putative receptor for the plant hormone auxin.

The major auxin-binding protein from maize coleoptiles was purified to homogeneity. The protein has an apparent mol. wt of 22 kd and binds 1-naphthylacetic acid with a KD of 2.40 x 10(-7) M. Additional antigenically related proteins, present in very low amounts, could be demonstrated in maize coleoptiles using immunodetection. Extensive protein sequence analysis of the major auxin-binding protein allowed the construction of several synthetic oligonucleotide probes which were used to isolate a cDNA coding for this protein. The cDNA corresponds to a mRNA with a 3'-poly(A)+ sequence and a single, long open reading frame of 603 bases. The open reading frame, starting 34 residues from the 5' end of the cDNA, predicts a 21,990 Dalton protein of 201 amino acids. Comparison of this deduced amino acid sequence with the partial amino acid sequences of purified auxin-binding protein, revealed a perfect match, involving a total of 53 amino acid residues. The primary amino acid sequence includes a 38-amino-acid-long N-terminal hydrophobic leader sequence which could represent a signal for translocation of this protein to the endoplasmic reticulum. An additional signal is located at the C-terminal end, consisting of the amino acids KDEL known to be responsible for preventing secretion of proteins from the lumen of the endoplasmic reticulum in eucaryotic cells. The primary sequence contains a N-glycosylation site (-asp133-thr-thr-). This site was found to be glycosylated by a high-mannose-type oligosaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Regulation of plant genes specifically induced in nitrogen-fixing nodules: role of cis-acting elements and trans-acting factors in leghemoglobin gene expression.

Transgenic alfalfa plants harboring a gene fusion between the soybean leghemoglobin (lbc3) promoter region and the chloramphenicol acetyl transferase (cat) gene were used to determine the influence of rhizobial mutants on lb gene expression in nodules. The promoter region of the Sesbania rostrata glb3 (Srglb3) leghemoglobin gene was examined for the presence of conserved motifs homologous to binding site 1 and 2 of the soybean lbc3 promoter region, found to interact with a trans-acting factor present in soybean nodule nuclear extracts (Jensen EO, Marcker KA, Schell J, de Bruijn FJ, EMBO J 7:1265-1271, 1988). Subfragments of the S. rostrata glb3 (Srglb3) promoter region were examined for binding to trans-acting factors from nodule nuclear extracts. In addition to the binding sites previously identified (Metz BA, Welters P, Hoffmann HJ, Jensen EO, Schell J, de Bruijn FJ, Mol Gen Genet 214: 181-191), several other sites were found to interact with trans-acting factors. In most cases the same trans-acting factor(s) were shown to be involved. One fragment (202) was found to bind specifically to a different factor (protein) which was extremely heat-resistant (100 degrees C). The appearance of this factor was shown to be developmentally regulated since the expected protein-DNA complexes were first observed around 12 days after infection, concomitant with the production of leghemoglobin proteins. Fragments of the Srglb3 5' upstream region were fused to the beta-glucuronidase reporter gene with its own CAAT and TATA box region or those of the cauliflower mosaic virus 35S and nopaline synthase (nos) promoters.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1989

A class II patatin promoter is under developmental control in both transgenic potato and tobacco plants.

A new member of the patatin gene family belonging to the class II subfamily was isolated and characterized by DNA sequencing. In order to study the expression profile of this gene, the promoter was fused to the beta-glucuronidase gene and transferred to potato and tobacco. Histochemical analysis revealed high expression in a few defined cells in potato tubers and in a specific layer of both potato and tobacco root tips. In contrast to the developmentally and metabolically regulated class I patatin gene B33 this gene was not inducible by elevated levels of sucrose. Expression of this chimaeric gene was also found in callus and suspension cultures of potato.

Base Sequence↗
PubMed1989

Conserved function in Nicotiana tabacum of a single Drosophila hsp70 promoter heat shock element when fused to a minimal T-DNA promoter.

To demonstrate the extent of evolutionary conservation in the mechanism of induction of heat shock genes between plants and animals, the minimal sequence from the Drosophila hsp70 promoter sufficient to confer heat shock inducible transcription in tobacco was determined. Segments of the hsp70 promoter were fused to a minimal promoter of the T-DNA indole-3-acetamide hydrolase (iaaH) gene, in a chimaeric gene fusion to a neomycin phosphotransferase (NPT II) reporter gene. Sequences bearing one or more heat shock elements (HSEs) rendered the minimal promoter heat shock inducible, with a 37 bp fragment containing a single complete HSE sufficing. The induced NPT II mRNA peaked during the heat shock period, but the maximal level of NPT II activity was not observed until 4 h later in the recovery phase, showing that the translation of the NPT II mRNA was shifted from the heat shock period of the recovery phase. That similar sequences containing a single HSE of the Drosophila hsp70 promoter could function in both flies and tobacco indicates the high degree of homology between the two heat shock gene induction systems.

Amidohydrolases↗
PubMed1989

Nucleotide sequence and regulated expression of a wound-inducible potato gene (wun1).

Mechanical wounding of potato leaves, stems, roots and tubers leads to a rapid increase of wun1 mRNA. In potato leaves, the wound-induced accumulation of wun1 mRNA is inhibited by the addition of sucrose or other osmotically active agents. This inhibition is organ specific since sucrose does not prevent wun1 mRNA accumulation in wounded tubers. In contrast, expression of patatin was shown to be repressed in tubers by wounding and this repression was reversed by increasing osmotic pressure. Sequence data obtained from the analysis of a wun1 cDNA and a wun1 genomic clone show no homology to any gene known so far. Histochemical data demonstrate a striking analogy in cell specific expression of chimeric genes expressed under the control of the wun1 promoter and the cell specific production of callose in wounded tobacco leaves.

Amino Acid Sequence↗
PubMed1989

A functional analysis of T-DNA gene 6b: the fine tuning of cytokinin effects on shoot development.

The physiological function in planta of T-DNA gene 6b was studied under various experimental conditions. For this purpose the coding region of gene 6b was cloned behind the 1'-promoter of the TR-DNA to enhance expression of the gene product in transformed plant cells. Expression of the recombinant gene in leaf discs of Nicotiana tabacum altered the capacity for shoot formation of the discs, induced by exogenous (i.e. BAP in the growth medium or agrobacterial trans-zeatin produced under control of gene tzs) or endogenous cytokinins (i.e. isopentenyladenosine produced under control of T-DNA gene 4). The data obtained indicate a reduction of cytokinin activity within the plant cells by the product of T-DNA gene 6b.

Base Sequence↗
PubMed1989

Differential regulation of soybean chalcone synthase genes in plant defence, symbiosis and upon environmental stimuli.

Four independent recombinant lambda clones hybridizing to parsley chalcone synthase (CHS) cDNA were isolated from a soybean (Glycine max) genomic library. Restriction fragment length polymorphism (RFLP) analysis indicated that the CHS gene family comprises six members. The CHS genes were found to be clustered with three genes on a 10 kb segment and pairs on others. DNA sequences of the 5'-, the coding-, and the 3' untranslated regions were determined for three different genes. A consensus alignment of the 5' regions revealed extensive homology between them for up to 150 bp upstream of the TATA box. Developmental regulation of CHS was observed in uninfected and in rhizobium-infected roots. Regulation at the level of transcription by different stimuli was investigated in the root, stem and cotyledons of soybean seedlings. Our results suggest a co-operative induction of CHS genes by wounding and elicitor treatment of cotyledons. The most rapid transcript accumulation, however, was observed in roots and stems. The induction of CHS genes by light was found to be UV dependent. A possible involvement of different members of the CHS gene family in response to elicitor versus UV treatment was analysed by the use of gene specific probes, and unexpectedly revealed that only CHS 1 transcription was induced by either elicitor or UV treatment of seedlings.

Acyltransferases↗
PubMed1989

Interscalene brachial plexus blockade with lidocaine in chronic renal failure--a pharmacokinetic study.

Plasma lidocaine concentrations, latency of onset, and duration of anaesthesia, were determined after interscalene brachial plexus block in 16 patients presenting for elective upper limb surgery. Eight patients had normal renal function and eight had chronic renal failure, as determined by creatinine clearance. Significantly higher plasma lidocaine levels were recorded ten minutes after infiltration in patients with chronic renal failure (p less than 0.05). Cmax plasma levels for normal patients (5.6 +/- 1.1 micrograms.ml-1) and for patients with chronic renal failure (6.6 +/- 1.6 micrograms.ml-1) were not significantly different. The latency of onset and duration of anaesthesia were similar in both groups. One per cent lidocaine solution may be administered to patients with normal and impaired renal function to provide effective brachial plexus blockade for short surgical procedures.

Adult↗
PubMed1989

A CACGTG motif of the Antirrhinum majus chalcone synthase promoter is recognized by an evolutionarily conserved nuclear protein.

In the chalcone synthase gene of Antirrhinum majus (snapdragon), 150 base pairs of the 5' flanking region contain cis-acting signals for UV light-induced expression. A nuclear factor, designated CG-1, specifically recognizes a hexameric motif with internal dyad symmetry, CACGTG, located within this light-responsive sequence. Binding of CG-1 is influenced by C-methylation of the CpG dinucleotide in the recognition sequence. CG-1 is a factor found in a variety of dicotyledonous plant species including Nicotiana tabacum, A. majus, Petunia hybrida, Arabidopsis thaliana, and Glycine max. CACGTG motifs contained within trans-acting factor recognition sites in various other plant promoters can interact with CG-1. In addition, the binding site of the human adenovirus major late transcription factor USF can compete for CG-1 binding to the chalcone synthase promoter. This suggests an evolutionary conservation of trans-acting factor recognition sites involved in divergent mechanisms of gene control.

Acyltransferases↗
PubMed1989

High-frequency T-DNA-mediated gene tagging in plants.

An insertion element [transferred DNA (T-DNA)], transferred by soil agrobacteria into the nuclear genome of plants, was used for induction of gene fusions in Arabidopsis thaliana, Nicotiana tabacum, and Nicotiana plumbaginifolia. A promoterless aph(3')II (aminoglycoside phosphotransferase II) reporter gene was linked to the right end of the T-DNA and transformed into plants along with a plasmid replicon and a selectable hygromycin-resistance gene. Transcriptional and translational reporter gene fusions were identified by screening for APH(3')II enzyme activity in diverse tissues of transgenic plants. The frequency of gene fusions, estimated by determination of the copy number of T-DNA insertions, showed that on average 30% of T-DNA inserts induced gene fusions in Arabidopsis and Nicotiana. Gene fusions were rescued from plants by transformation of the T-DNA-linked plasmid and flanking plant DNA into Escherichia coli. By dissection of gene fusions and construction of chimeric genes, callus- and root-specific promoters were identified that showed an altered tissue specificity in the presence of a 3'-downstream-located 35S promoter. Transcript mapping of a gene fusion and expression of a non-frame transcriptional fusion of bacterial luciferase luxA and luxB genes demonstrated that dicistronic transcripts are translated in tobacco.

Base Sequence↗