The secretory pore array hypothesis of transmitter release.
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Animals↗
Biomedical subjects
B Keller
Publications and source records attributed to B Keller.
Colinearity and gene density in grass genomes.
Grasses are the single most important plant family in agriculture. In the past years, comparative genetic mapping has revealed conserved gene order (colinearity) among many grass species. Recently, the first studies at gene level have demonstrated that microcolinearity of genes is less conserved: small scale rearrangements and deletions complicate the microcolinearity between closely related species, such as sorghum and maize, but also between rice and other crop plants. In spite of these problems, rice remains the model plant for grasses as there is limited useful colinearity between Arabidopsis and grasses. However, studies in rice have to be complemented by more intensive genetic work on grass species with large genomes (maize, Triticeae). Gene-rich chromosomal regions in species with large genomes, such as wheat, have a high gene density and are ideal targets for partial genome sequencing.
Arabidopsis↗
Glycine-rich cell wall proteins act as specific antigen targets in autoimmune and food allergic disorders.
Our objective was to investigate the presence of a B and T cell immune response directed against the glycine-rich cell wall protein (GRP) in patients with different autoimmune disorders and with food allergy. GRP is an ubiquitous food protein that has high homology with cytokeratins and other self proteins [Epstein-Barr virus nuclear antigen-1 (EBNA-I), heterogeneous nuclear ribonucleoprotein, fibrillar collagen] which are common targets in autoimmune disorders. A peptide (GGYGDGGAHGGGYGG) derived from GRP was used to screen human sera in direct and competitive ELISA assay. Anti-GRP-specific IgG were analyzed for their ability to cross-react with autoantigens. The intracellular cytokine profiles of the peptide-specific T cell clones obtained from representative patients have been studied. BALB/c mice were immunized with the peptide coupled to the carrier protein keyhole limpet hemocyanin (KLH). Serum IgG antibodies directed against the GRP peptide were detected in several autoimmune disorders and in food allergic patients, and were able to cross-react with autoantigens including keratin, collagen and EBNA-I. Twenty-five T cell clones showed a specific proliferative response to the GRP peptide and were of the T(h)0 phenotype. Eight of the 10 BALB/c mice immunized with the peptide coupled to KLH developed an autoimmune response. Our data suggest that phylogenetically highly conserved epitopes in plants, viruses and humans may be responsible for an autoimmune response in susceptible individuals. They also indicate that the antigen spreading of a particular sequence among apparently divergent proteins may participate to initiate or amplify an immune response.
Amino Acid Sequence↗
Activation tagging of the LEAFY PETIOLE gene affects leaf petiole development in Arabidopsis thaliana.
In a screen for leaf developmental mutants we have isolated an activator T-DNA-tagged mutant that produces leaves without a petiole. In addition to that leafy petiole phenotype this lettuce (let) mutant shows aberrant inflorescence branching and silique shape. The LEAFY PETIOLE (LEP) gene is located close to the right border of the T-DNA insert linked with these dominant phenotypes and encodes a protein with a domain with similarity to the DNA binding domain of members of the AP2/EREBP family of transcription factors. Introduction of the activation-tagged LEP gene in wild-type plants conferred all the phenotypic aberrations mentioned above. The leafy petiole phenotype consists of a conversion of the proximal part of the leaf from petiole into leaf blade, which means that leaf development in let is disturbed along the proximodistal axis. Therefore, LEP is involved in either cell division activity in the marginal meristem or patterning along the proximodistal axis.
Arabidopsis↗
[Differentiation of yeasts in mastitis milk] .
Differentiating 174 yeasts from clinical samples to get a survey of those species found in mastitis milk and milking machines we identified five genera with 29 species. Species of the genus Candida dominated with 77% of all samples. More than 25% of yeasts from milk were identified as C. rugosa and more than 20% as C. catenulata. The six species found most often represented 74% of all yeasts in this group. Yeasts from milking machines were more heterogeneous.
Animals↗
Transmitter quantal size in Torpedo electrocytes is determined by frequency of release.
Miniature end-plate potentials (MEPPs) were focally recorded from the cytoplasmic surface of electrocytes in isolated columns of the Torpedo electric organ. Double electrode studies showed that the junctional area was restricted to 12 micron2. MEPP frequencies ranging from 1/min to 400/s were controlled with electrode advancement against the cytoplasmic surface. Stable membrane potentials and noise levels indicated constant intracellular, focal recording conditions. Focal MEPPs are only 1-3 mV and MEPP amplitudes smoothly decreased with an increase in MEPP frequency which demonstrates a process that meters quantal size at moment of release. Thus, release if not from a prepackaged store. MEPP interval analyses showed that events are weakly interactive at low frequencies and periodic at higher frequencies. The interdependency of MEPP amplitudes and intervals indicates that the mechanism of release controls both rate and quantal size. We propose that the amplitude and frequency dependencies of MEPPs at the Torpedo nerve-electrocyte junction are best described by a membrane channel (e.g., mediatophore, Israël and Dunant, Neurochem. Int. 28 (1996) 1-9) that meters transmitter from a presynaptic store.
Animals↗
High gene density is conserved at syntenic loci of small and large grass genomes.
Comparative genomic analysis at the genetic-map level has shown extensive conservation of the gene order between the different grass genomes in many chromosomal regions. However, little is known about the gene organization in grass genomes at the microlevel. Comparison of gene-coding regions between maize, rice, and sorghum showed that the distance between the genes is correlated with the genome size. We have investigated the microcolinearity at Lrk gene loci in the genomes of four grass species: wheat, barley, maize, and rice. The Lrk genes, which encode receptor-like kinases, were found to be consistently associated with another type of receptor-like kinase (Tak) on chromosome groups 1 and 3 in Triticeae and on chromosomes homoeologous to Triticeae group 3 in the other grass genomes. On Triticeae chromosome group 1, Tak and Lrk together with genes putatively encoding NBS/LRR proteins form a cluster of genes possibly involved in signal transduction. Comparison of the gene composition at orthologous Lrk loci in wheat, barley, and rice revealed a maximal gene density of one gene per 4-5 kb, very similar to the gene density in Arabidopsis thaliana. We conclude that small and large grass genomes contain regions that are highly enriched in genes with very little or no repetitive DNA. The comparison of the gene organization suggested various genome rearrangements during the evolution of the different grass species.
Amino Acid Sequence↗
The unitary evoked potential at the frog nerve-muscle junction results from synchronous gating of fusion pores at docked vesicles.
Exocytosis of a single vesicle has been proposed as the mechanism which determines quantal size by releasing a prepackaged and standard amount of acetylcholine. As first described by del Castillo and Katz (1954) the endplate potential is composed of 100 unitary events and the small variance suggests a binomial release from 100 "discrete patches of membrane". However, exocytosis of 100 vesicles selected randomly from 5000 docked vesicles would yield a variance that is 7 times greater than observed values. We propose that the presynaptic ridge with its compliment of docked vesicles functions as the "discrete patch of membrane" such that arrays of calcium activated fusion pores meter transmitter to form the unit of release. A model based on the synchronous flicker of a large number of fusion pores produces the small variance of both miniature end plate potentials and unitary end plate potentials. Release from a single locus (fusion pore) would generate the sub-MEPP. This model permits vesicle trafficking and vesicular content depletion during tetanic stimulation and explains the frequency dependency of MEPP amplitudes and changes in sub-MEPP to bell-MEPP class ratios.
Animals↗
Plant genomics and the genetics of disease resistance: 4th Gatersleben Research Conference: Plant Genomics--Structural Function and Applied Aspects, Schloss Meisdorf/Harz and IPK, Gatersleben, Germany, 17-21 June 1999.
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Animals↗
Why does postpsychotic IQ decline in childhood-onset schizophrenia?
OBJECTIVE: The authors' goal was to examine whether the postpsychotic decline in full scale IQ during adolescence for patients with childhood-onset schizophrenia is due to a dementing process or simply failure to acquire new information and skills. METHOD: Linear regression was used to determine the rate of change for scaled and raw scores on subtests of 31 patients with childhood-onset schizophrenia. The resulting slopes were examined and related to changes in the patients' brains determined by magnetic resonance imaging. RESULTS: Three postpsychotic subtest scaled scores declined significantly: picture arrangement, information, and block design. In contrast, there was no decline in the non-age-corrected (raw) scores for any subtest. A significant correlation was found between decrease in hippocampal volume and a smaller increase in raw score on the information subtest. CONCLUSIONS: The decline during adolescence in the full-scale IQ of patients with childhood-onset schizophrenia does not reflect dementia but, rather, an inability to acquire new information and abilities.
Adolescent↗
Construction and characterization of a bacterial artificial chromosome (BAC) library for the A genome of wheat.
A genomic bacterial artificial chromosome (BAC) library of the A genome of wheat has been constructed. Triticum monococcum accession DV92 was selected for this purpose because it is a cultivated diploid wheat and one of the parental lines used in the construction of a saturated genetic map. Leaves from this accession were used to isolate high-molecular-weight DNA from nuclei. This DNA was partially digested with restriction enzyme Hind III, subjected to double size selection, electroeluted and cloned into the pINDIGO451 BAC vector. The library consists of 276,480 clones with an average insert size of 115 kb. Excluding the 1.33% of empty clones and 0.14% of clones with chloroplast DNA, the coverage of this library is 5.6 genome equivalents. With this genome coverage the probability of having any DNA sequence represented in this library is higher than 99.6%. Clones were sorted in 720,384-well plates and blotted onto 15 high-density filters. High-density filters were screened with several single or low-copy clones and five positive BAC clones were selected for further analysis. Since most of the T. monococcum BAC ends included repetitive sequences, a modification was introduced into the classical end-isolation procedure to select low copy sequences for chromosome walking.
Chromosomes, Bacterial↗
Specific interaction of the tomato bZIP transcription factor VSF-1 with a non-palindromic DNA sequence that controls vascular gene expression.
The grp1.8 gene of French bean (Phaseolus vulgaris) is specifically expressed in vascular tissue and encodes a glycine-rich structural protein (GRP1.8) of the cell wall. Earlier promoter analysis had shown that a 28 bp fragment of the grp1.8 promoter (vs-1) confers vascular expression to heterologous minimal promoters and is bound by the tomato bZIP transcription factor VSF-1. Here, we analysed the interaction of VSF-1 with fragments of the vs-1 element and studied the molecular basis of specific binding both in the DNA sequence of the promoter element as well as in the protein. The minimal binding site of VSF-1 is a 9 bp, non-palindromic sequence with two non-identical half-sites and a central nucleotide which separates them. The amino acid sequence of the VSF-1 DNA-binding basic domain has a Lys at position -10 instead of a conserved Arg found in the other bZIP factors isolated so far. This lysine was found to be required for specific recognition of the non-palindromic binding site: a mutant VSF-1 with a Lys-to-Arg substitution at position -10 bound with higher affinity to a palindromic sequence than the wild-type protein. The minimal binding site of VSF-1 was sufficient and necessary to confer vascular-specific expression to a heterologous promoter in vivo. The vsf-1 promoter also showed vascular-specific expression in transgenic tobacco. The close similarity of these expression patterns suggests that VSF-1 is specifically involved in vascular expression of the grp1.8 gene in plants.
Basic-Leucine Zipper Transcription Factors↗
Molecular characterization of a new type of receptor-like kinase (wlrk) gene family in wheat.
In plants, several types of receptor-like kinases (RLK) have been isolated and characterized based on the sequence of their extracellular domains. Some of these RLKs have been demonstrated to be involved in plant development or in the reaction to environmental signals. Here, we describe a RLK gene family in wheat (wlrk, wheat leaf rust kinase) with a new type of extracellular domain. A member of this new gene family has previously been shown to cosegregate with the leaf rust resistance gene Lr10. The diversity of the wlrk gene family was studied by cloning the extracellular domain of different members of the family. Sequence comparisons demonstrated that the extracellular domain consists of three very conserved regions interrupted by three variable regions. Linkage analysis indicated that the wlrk genes are specifically located on chromosome group 1 in wheat and on the corresponding chromosomes of other members of the Triticeae family. The wlrk genes are constitutively expressed in the aerial parts of the plant whereas no expression was detected in roots. Protein immunoblots demonstrated that the WLRK protein coded by the Lrk10 gene is an intrinsic plasma membrane protein. This is consistent with the hypothesis that WLRK proteins are receptor protein kinases localized to the cell surface. In addition, we present preliminary evidence that other disease resistance loci in wheat contain genes which are related to wlrk.
Amino Acid Sequence↗
Comparative mapping of the two wheat leaf rust resistance loci Lr1 and Lr10 in rice and barley.
The wheat genome is large, hexaploid, and contains a high amount of repetitive sequences. In order to isolate agronomically important genes from wheat by map-based cloning, a simpler model of the genome must be used for identifying candidate genes. The objective of this study was to comparatively map the genomic regions of two wheat leaf rust disease resistance loci, Lr1 and Lr10, in the putative model genomes of rice and barley. Two probes cosegregating with the Lr1 gene on chromosome 5DL of wheat were studied. The rice sequences corresponding to the two probes were isolated and mapped. The two probes mapped to two different rice chromosomes, indicating that the organization of the region orthologous to Lr1 is different in rice and wheat. In contrast, synteny was conserved between wheat and barley in this chromosomal region. The Lrk10 gene cosegregated with Lr10 on chromosome 1AS in wheat. The rice gene corresponding to Lrk10 was mapped on rice chromosome 1, where it occurred in many copies. This region on rice chromosome 1 corresponds to the distal part of the group 3S chromosomes in Triticeae. The synteny is conserved between rice chromosome 1 and the Triticeae group 3S chromosomes up to the telomere of the chromosomes. On group 3S chromosomes, we found a gene that is partially homologous to Lrk10. We conclude that in the genomic regions studied, there is limited and only partially useful synteny between wheat and rice. Therefore, barley should also be considered as a model genome for isolating the Lr1 and Lr10 genes from wheat.
Base Sequence↗
Cell death in prion disease.
Prion diseases are neurodegenerative transmissible diseases. The infectious agent, termed prion, is thought to consist of an altered host-encoded protein. The pathogenesis of these diseases which typically in a very short time lead to rampant nerve cell death and astrocytic gliosis is poorly understood. Investigations using the in situ endlabeling technique and electron microscopy in a scrapie model in the mouse (79A strain) show that nerve cell death is due to apoptosis. A cell culture model using a synthetic peptide of the prion protein (PrP106-126) shows that this peptide is toxic only to normal neurons whereas nerve cells derived from PrP knock-out (PrP0/0) mice are unaffected by this neurotoxic effect. In addition, microglia play a crucial part in this process by secreting reactive oxygen species. Experiments in animals will have to show whether these cell culture findings adequately reflect the in vivo pathogenesis.
Amino Acid Sequence↗
Immobilization of the cell-adhesive peptide Arg-Gly-Asp-Cys (RGDC) on titanium surfaces by covalent chemical attachment.
Surface modification of acid-pretreated titanium with 3-aminopropyltriethoxylsilane (APTES) in dry toluene resulted in covalently bonded siloxane films with surface coverage that was relatively controllable by regulating the reaction conditions. A hetero-bifunctional cross-linker, N-succinimidyl-3-maleimidopropionate (SMP), reacted with the terminal amino groups, forming the exposed maleimide groups. Finally, a model cell-binding peptide, Arg-Gly-Asp-Cys (RGDC), was immobilized on the surface through covalent addition of the cysteine thiol groups to the maleimide groups. X-ray photoelectron spectroscopy, radiolabelling techniques, and ellipsometry were used to quantify and characterize the modified surfaces.
Journal Article↗
Molecular cloning of a new receptor-like kinase gene encoded at the Lr10 disease resistance locus of wheat.
More than 100 resistance genes against wheat rust pathogens have been described in wheat and its relatives. Although many of them have been extensively used in wheat resistance breeding, none of these resistance loci has yet been analyzed at the molecular level. By screening a set of near-isogenic lines carrying different leaf rust resistance genes with a wheat probe encoding a serine/ threonine protein kinase, we detected a polymorphic DNA fragment in the line with the Lr10 resistance gene. This fragment mapped to the Lr10 disease resistance locus and encodes a receptor-like protein kinase which we called LRK10. LRK10 contains a new type of extracellular domain not found in known plant or animal receptor kinases. Several conserved amino acids in S-domain glycoproteins and receptor-like kinases were also found in LRK10, suggesting that LRK10 and S-domain proteins belong to the same superfamily of specific recognition molecules in plants. Lrk10 was expressed at low levels in young seedlings and belongs to a gene family. Analysis of wheat lines with and without the Lr10 gene demonstrated that Lrk10 and Lr10 belong to the same genetic locus. We conclude that gene isolation based on protein kinase homology can identify new receptor domains and provide candidates for disease resistance genes in the complex wheat genome.
Alleles↗
Structural cell-wall proteins in protoxylem development: evidence for a repair process mediated by a glycine-rich protein.
Polyclonal antibodies were used to localize structural cell-wall proteins in differentiating protoxylem elements in etiolated bean and soybean hypocotyls at the light- and electron-microscopic level. A proline-rich protein was localized in the lignified secondary walls, but not in the primary walls of protoxylem elements, which remain unlignified, as shown with lignin-specific antibodies. Secretion of the proline-rich protein was observed during lignification in different cell types. A glycine-rich protein (GRP1.8) was specifically localized in the modified primary walls of mature protoxylem elements and in cell corners between xylem elements and xylem parenchyma cells. The protein was secreted by Golgi bodies both in protoxylem cells after the lignification of their secondary walls and in the surrounding xylem parenchyma cells. The modified primary walls of protoxylem elements were visualized under the light microscope as filaments or sheets staining distinctly with the protein stain Coomassie blue. Electron micrographs of these walls show that they are composed of an amorphous material of moderate electron-density and of polysaccharide microfibrils. These materials form a three-dimensional network, interconnecting the ring- or spiral-shaped secondary wall thickenings of protoxylem elements and xylem parenchyma cells. The results demonstrate that the modified primary walls of protoxylem cells are not simply breakdown products due to partial hydrolysis and passive elongation, as believed until now. Extensive repair processes produces cell walls with unique staining properties. It is concluded that these walls are unusually rich in protein and therefore have special chemical and physical properties.
Antibodies↗