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Biomedical subjects

J Schell

Publications and source records attributed to J Schell.

At least 19 recordsLinked to original sources
PubMed1992

Activation of a plant gene by T-DNA tagging: auxin-independent growth in vitro.

A transferred DNA (T-DNA) tagging vector with the potential to produce dominant mutations was used with cocultured Agrobacterium tumefaciens and protoplasts to tag genes involved in the action of the plant growth substance auxin. Transgenic calli were selected for their ability to grow in the absence of auxin in the culture media. From one experiment, 12 calli that displayed this phenotype were recovered, of which 11 were able to regenerate into plants. In one plant studied in detail, protoplast division in the absence of auxin genetically cosegregated with a single T-DNA insert. A messenger RNA encoded by a 6.4-kilobase sequence of plant genomic DNA rescued from the mutant is overexpressed relative to untransformed plants. The genomic DNA, as well as a cognate complementary DNA, once transfected into protoplasts promote growth and cell division in vitro in the absence of exogenously added auxin.

Agrobacterium tumefaciens↗
PubMed1992

Expression of a calmodulin methylation mutant affects the growth and development of transgenic tobacco plants.

Transgenic plants were constructed that express two foreign calmodulins (VU-1 and VU-3 calmodulins) derived from a cloned synthetic calmodulin gene. VU-1 calmodulin, similar to endogenous plant calmodulin, possesses a lysine residue at position 115 and undergoes posttranslational methylation. VU-3 calmodulin is a site-directed mutant of VU-1 calmodulin that is identical in sequence except for the substitution of an arginine at position 115 and thus is incapable of methylation. Both calmodulin genes, under the control of the cauliflower mosaic virus 35S promoter, were expressed in transgenic tobacco. Foreign calmodulin protein accumulated in plant tissues to levels equivalent to that of the endogenous calmodulin. All transformed lines of VU-1 plants were indistinguishable from untransformed controls with respect to growth and development. However, all transformed lines of VU-3 plants were characterized by decreased stem internode growth, reduced seed production, and reduced seed and pollen viability. The data suggest that these phenotypes are the result of the expression of the calmodulin mutant rather than the position of transferred DNA insertion or the overall alteration of calmodulin levels. Analyses of the activity of the purified transgenic calmodulins suggest that calmodulin-dependent NAD kinase is among the potential targets that may have altered regulation in VU-3 transgenic plants.

Amino Acid Sequence↗
PubMed1992

High rates of Ac/Ds germinal transposition in Arabidopsis suitable for gene isolation by insertional mutagenesis.

Overexpression of the Activator (Ac) transposase gene in Arabidopsis thaliana resulted in a minimal germinal transposition frequency of 27% in which independent Dissociation (Ds) transposition events were observed. Molecular analysis of 45 F1 generation Ac/Ds plants indicated that high rates of somatic excision had occurred, and independent germinal insertions were identified in F2 generation progeny plants. A tandem cauliflower mosaic virus (CaMV) promoter fused to two different Ac coding sequences significantly increased the rate of Ds transposition. The CaMV-Ac fusions activated single and multiple copies of two different Ds elements, DsDHFR and Ds35S-1, and reciprocal crosses resulted in similar transposition frequencies. The improved rate of independent germinal transposition observed makes Arabidopsis an ideal system for insertional mutagenesis.

Base Sequence↗
PubMed1992

Molecular cloning and structural analysis of genes from Zea mays (L.) coding for members of the ras-related ypt gene family.

We have isolated, cloned, and characterized two cDNAs from Zea mays (L.), denoted yptm1 and yptm2, encoding proteins related to the ypt protein family. Amino acid similarity scores with YPT1 from yeast and ypt from mouse are in the range of 70% for yptm1 and 74% for yptm2, respectively, whereas similarities with p21 ras and other ras-related proteins are less than 40%. Most amino acid residues showing identity are clustered in the GTP/GDP binding domain. In addition, two cysteine residues close to the C-terminal ends, known to be palmitoylated and necessary for membrane binding in all eukaryotic ras-related proteins that have been characterized so far, are conserved in the maize genes as well. Northern blot hybridization analysis of poly(A)+ mRNA from etiolated maize coleoptiles revealed single mRNA species of approximately the same size as the isolated cDNAs. The gene for yptm1 is expressed at very low levels in maize coleoptiles and tissue culture cells. The gene for yptm2 is expressed at higher levels and is differentially represented in RNAs isolated from various organs of maize plants, with its highest level in leaves and flowers. The structural similarity of the genes identified suggests that they could be involved in the control of secretory processes.

Amino Acid Sequence↗
PubMed1992

Complementation of the cs dis2-11 cell cycle mutant of Schizosaccharomyces pombe by a protein phosphatase from Arabidopsis thaliana.

The activities of type I protein phosphatases play a central role in eukaryotic cell cycle control. Here, we report the cloning and characterization from the flowering plant Arabidopsis thaliana of a cDNA clone named PP1-At which is highly homologous to protein phosphatase 1. The deduced amino acid sequence of PP1-At shows that the PP1-At protein is 318 amino acid residues long and has a molecular weight of 35,298 Da. The PP1-At protein has strong similarity to all other known protein phosphatase type 1 catalytic subunits. Approximately 62% of the amino acids are identical to type 1 protein phosphatases of rabbit, mouse, Saccharomyces cerevisiae and Schizosaccharomyces pombe. RNA blot analysis revealed a single mRNA species of approximately the same size as the cDNA isolated. The PP1-At-encoded mRNA of 1.3 kb is abundant in most vegetative Arabidopsis tissues, with the lowest level of expression in leaves. When transferred to the fission yeast S.pombe, the PP1-At-encoded protein can rescue a semidominant mutant, cold sensitive (cs) dis2-11, which under nonpermissive conditions is unable to complete chromosome disjunction.

Amino Acid Sequence↗
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↗