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Biomedical subjects

J Schell

Publications and source records attributed to J Schell.

At least 55 records · Page 3Linked to original sources
PubMed1994

Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein.

Virulence proteins VirD1 and VirD2 are subunits of a relaxosome-like protein complex that mediates conjugational transfer of a Ti plasmid segment, the T-DNA, from Agrobacterium into higher plants. The VirD1-VirD2 complex binds to 25-bp repeats at the borders of the T-DNA and catalyzes sequence-specific nicking of the conjugative DNA strand (the T-strand) at the third base of these repeats. Nuclear localization signals present in VirD2 target the T-strand to plant cell nuclei. In addition, VirD2 probably plays a role in the high-frequency integration of the T-DNA into the plant genome by illegitimate recombination. Whereas Agrobacterium transformation of dicots is very efficient, T-DNA integration in most monocots can barely be detected. To develop an artificial T-DNA delivery system for monocots, a technique for efficient in vitro production of T-strand DNAs was established by using VirD1 and VirD2 proteins purified from overexpressing Escherichia coli strains. The topoisomerase-like VirD2 enzyme was shown to mediate precise, sequence-specific cleavage of T-DNA border sequences carried by single-stranded DNA templates, even in the absence of VirD1 protein. During this reaction, VirD2 remains covalently bound to the 5' end of artificial T-strand DNAs. In contrast, VirD2, alone or in complex with VirD1, fails to nick linear double-stranded DNA templates in vitro.

Bacterial Proteins↗
PubMed1994

Activation tagging: a means of isolating genes implicated as playing a role in plant growth and development.

Activation T-DNA tagging has been used to generate a variety of tobacco cell lines selected by their ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. The majority of the cell lines studied in detail contain single T-DNA inserts genetically co-segregating with the selected phenotype. While most of the plants regenerated from the mutant cell lines appear phenotypically normal, several display phenotypes which could be inferred to result from disturbances in the content, or the metabolism, of auxins and cytokinins, or polyamines. The tagging vector is designed to allow the isolation of tagged plant genes by plasmid rescue. Confirmation that the genomic sequence responsible for the selected phenotype has indeed been isolated is provided by PEG-mediated protoplast DNA uptake of rescued plasmids followed by selection for protoplast growth under the original selective conditions. Several plasmids have been rescued from the mutant lines which confer on transfected protoplasts the ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. This review describes the background to activation tagging and our progress in characterizing the genes that have been tagged in the mutant lines we have generated.

Cytokinins↗
PubMed1994

Sequence analysis of the 3'-terminal half of RNA 1 of wheat spindle streak mosaic virus.

cDNA complementary to the 3'-terminal half of RNA 1 of wheat spindle streak mosaic virus (WSSMV) from Southern France has been cloned and sequenced. One large open reading frame (ORF) of 4410 nucleotides and a nontranslated region (NTR) of 213 nucleotides at the 3'-end excluding the poly(A)-tail were found. Because of the amino acid sequence homology to the polyprotein of barley yellow mosaic virus (BaYMV) RNA 1, the encoded polyprotein of the sequenced region of WSSMV is supposed to comprise the C-terminal part of the putative cytoplasmic inclusion (CI) protein, the nuclear inclusion a (NIa) proteinase, the (NIb) RNA-polymerase and the capsid protein. The first 19 N-terminal amino acids of the capsid protein were determined by direct sequencing of proteins of purified WSSMV particles and confirmed this hypothesis. The deduced capsid protein has 294 amino acids and shows 74% identity with the BaYMV capsid protein sequence. This high sequence homology with BaYMV, in addition to the significant identities with barley mild mosaic virus (BaMMV, 35%) and its marginal homology to capsid protein sequences of aphid and mite-borne potyviruses (22-24%), supports the classification of WSSMV as a distinct member of the genus Baymovirus, family Potyviridae.

Amino Acid Sequence↗
PubMed1994

Promoter tagging with a promoterless ipt gene leads to cytokinin-induced phenotypic variability in transgenic tobacco plants:implications of gene dosage effects.

Tobacco plants have been transformed with a T-DNA construct harboring a promoterless cytokinin-synthesizing ipt gene close to the right T-DNA border. Eighteen out of 85 transgenic clones displayed phenotypic alternations typical for an enhanced cytokinin production. Northern blot analysis confirmed the transcriptional activation of the introduced gene by tagged plant promoters. The concentration of cytokinins, expressed as zeatinriboside equivalents, was increased up to sevenfold in transgenic tissues. These increases in cytokinin levels resulted in major developmental changes. Transgenic clones exhibited to different levels traits of a general cytokinin-syndrome, i.e. reduced root growth, reduced apical dominance, reduced leaf surface, reduced growth of the stem and retarded leaf senescence or displayed localized and developmentally specific cytokinin-induced alterations in otherwise normally developing plants. These traits were in particular a simultaneous break of dormancy in all axillary buds before or at the onset of flowering or the reorientation of the developmental pathway of secondary meristems or terminally differentiated cells. This indicates that endogenously produced cytokinins not only influence different growth parameters but have the potential to alter differentiation pattern. The results show that stably inherited developmental alterations due to a general or localized cytokinin overproduction can be obtained by the promoter-tagging approach. The investigation of gene dosage effects in homozygote plants readdresses the question of threshold levels for cytokinin effects on the developmental program of plants.

Adenosine↗
PubMed1994

In vitro uptake and processing of maize auxin-binding proteins by ER-derived microsomes.

We have analyzed auxin-binding proteins from maize encoded by the Zm-ERabp gene family. Open reading frames of cDNA clones predict proteins containing N-terminal hydrophobic signal sequences. In vitro studies show that the Zm-ERabp1 protein can be translocated into ER-derived microsomes where it is processed and glycosylated. A cDNA clone encoding the Zm-ERabp4 protein predicts an open reading frame with a signal sequence that shows striking differences in charge distribution, in comparison to the signal sequence of Zm-ERabp1. Two translation products are synthesized from the Zm-ERabp4 transcript in the in vitro system, but only one of them is translocated into maize endosperm microsomes, indicating that specific cotranslational modifications in the primary sequence remaining after processing may play a role in the cellular trafficking of the Zm-ERabp4 protein.

Amino Acid Sequence↗
PubMed1993

Genes involved in the control of growth and differentiation in plants.

The mechanisms underlying totipotency, the unique ability of isolated plant cells to regenerate into plants, offer developmental biology a unique challenge. While it has been recognised for some time that phytohormones, such as auxin and cytokinin, play a role in this process by inducing a variety of growth patterns in both isolated cells, unorganised callus and intact plants, the molecular basis of their action remains unknown. The molecular and biochemical analysis of the novel interaction between tumour-inducing soil bacteria and the wounded plant has provided a valuable insight into how plants respond to phytohormones. During tumour formation, the bacteria transfer to the genome of the host plant a variety of genes which either short circuit the normal pathways of accumulation of phytohormones or modify how the plant cell responds to them. In parallel to these studies, we have been investigating plant genes involved directly or indirectly in the mechanism of phytohormone action. Auxin-binding proteins (putative receptors) have been localised in various cellular locations and the genes encoding them are currently undergoing analysis. Recently, a novel form of T-DNA has been devised by which mutant plant cell lines can be generated which grow in culture in the absence of exogenously applied auxin. The tagged genes, which are in effect plant cellular proto-oncogenes, are likely to shed more light on how auxin serves to regulate growth and development.

Cell Differentiation↗
PubMed1993

Release of active cytokinin by a beta-glucosidase localized to the maize root meristem.

A beta-glucoside encoded by a cloned Zea mays complementary DNA (Zm-p60.1) cleaved the biologically inactive hormone conjugates cytokinin-O-glucosides and kinetin-N3-glucoside, releasing active cytokinin. Tobacco protoplasts that transiently expressed Zm-p60.1 could use the inactive cytokinin glucosides to initiate cell division. The ability of protoplasts to sustain growth in response to cytokinin glucosides persisted indefinitely after the likely disappearance of the expression vector. In the roots of maize seedlings, Zm-p60.1 was localized to the meristematic cells and may function in vivo to supply the developing maize embryo with active cytokinin.

Adenine↗
PubMed1993

Rhizobium NodB protein involved in nodulation signal synthesis is a chitooligosaccharide deacetylase.

The common nodulation genes nodABC are conserved in all rhizobia and are involved in synthesis of a lipooligosaccharide signal molecule. This bacterial signal consists of a chitooligosaccharide backbone, which carries at the nonreducing end a fatty acyl chain. The modified chitooligosaccharide molecule triggers development of nodules on the roots of the leguminous host plant. To elucidate the specific role of the NodB protein in nodulation factor synthesis, we have purified recombinant NodB and determined its biochemical role by direct assays. Our data show that the NodB protein of Rhizobium meliloti deacetylates the nonreducing N-acetylglucosamine residue of chitooligosaccharides. The monosaccharide N-acetylglucosamine is not deacetylated by NodB. In the pathway of Nod factor synthesis, deacetylation at the nonreducing end of the oligosaccharide backbone may be a necessary requirement for attachment of the fatty acyl chain.

Acetylglucosamine↗
PubMed1993

Identification of the initiation sequence for viral-strand DNA synthesis of wheat dwarf virus.

The intergenic region of the circular single-stranded DNA genome of geminiviruses contains a sequence potentially able to fold into a stem-loop structure. This sequence has been reported to be involved in viral replication by serving as the origin for rolling-circle replication. However, in wheat dwarf virus (WDV) a deletion of 128 bp, removing this sequence, surprisingly does not prevent de novo viral DNA synthesis, but instead abrogates the processing of replicative intermediates into monomeric genomes. This deletion mutant permitted us to study the initiation of viral-strand DNA synthesis independently from its termination and also to identify the sequence within which rolling-circle DNA replication of WDV begins. We have mapped the initiation site of replication to a pentanucleotide, TACCC, a sequence that occurs twice in the large intergenic region of WDV: it is found in the right half of the stem-loop sequence and again 170 bases upstream where it is part of a 15 nucleotide sequence highly homologous to the right half of the stem-loop sequence. Here we show that viral-strand DNA synthesis efficiently initiates at both sequences.

Base Sequence↗
PubMed1993

On plant growth regulators and their metabolites: a changing perspective.

From seed germination to vegetative growth and flowering virtually all aspects of plant growth and development are influenced by structurally relatively simple substances, termed phytohormones. It has been argued that the wide range of responses elicited by these substances requires a mode of action that is radically different from those of animal hormones. In contrast to animal hormones, it is often very difficult to distinguish between the site of synthesis and the site of action of phytohormones. Hence, plants may have developed their own mechanisms for synthesis, sequestration and release of active hormones. Current evidence indicates that enzymes that can synthesize and modify phytohormones and their antagonists or hydrolyze phytohormone conjugates to release active hormones which play a role in initiating important regulatory pathways. They are also likely to provide invaluable tools for studying the mechanisms underlying growth and development in plants.

Bacteria↗
PubMed1993

Transgenic tobacco plants regenerated from leaf disks can be periclinal chimeras.

Amongst rolC transgenic tobacco plants regenerated from leaf disks 6.5% are periclinal chimeras, i.e. plants with genetically different cell populations in different cell layers. The expression of the rolC gene of Agrobacterium rhizogenes causes a reduction in pigment content in leaves. The chimeric composition of the regenerated plants becomes thus apparent as light green leaf tissue in the transgenic region, tissue flanked by dark green wild-type sectors. Southern and northern blot analysis confirmed the chimeric nature of such plants. Investigation of selfed progeny of chimeric plants on selective media indicates that layer invasion in reproductive tissues can occur in tobacco early during the formation of the flower buds. The results show (1) that tobacco plants regenerated from leaf disks and grown on selective media have not necessarily the same clonal origin and (2) that they can give rise to non-transgenic offspring. The chimeric plants provide insight on the effect of rolC gene expression on microsporogenesis.

Blotting, Southern↗
PubMed1993

Two members of the ERabp gene family are expressed differentially in reproductive organs but to similar levels in the coleoptile of maize.

A Zea mays cDNA clone, ZmERabp4, coding for a new member of the auxin-binding protein family was isolated. The primary amino acid sequence contains an N-terminal hydrophobic leader sequence, a potential glycosylation site (Asn136-Thr-Thr) and a C-terminal KDEL motif known to be responsible for retention of proteins within the lumen of the ER. The expression pattern of the ZmERabp4 gene in various organs of maize differs from the expression pattern previously observed for the ZmERabp1 gene. The ZmERabp4 gene is expressed highly in male flower organs, whereas the ZmERabp1 gene shows highest expression in female flower parts. In situ hybridization and analysis by laser scanning microscopy revealed enhanced levels of expression for both genes in the coleoptile when compared with the primary leaf of etiolated maize seedlings.

Amino Acid Sequence↗