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W D Reiter

Publications and source records attributed to W D Reiter.

17 recordsLinked to original sources
PubMed1999

Characterization of N-glycans from Arabidopsis. Application to a fucose-deficient mutant.

The structures of glycans N-linked to Arabidopsis proteins have been fully identified. From immuno- and affinodetections on blots, chromatography, nuclear magnetic resonance, and glycosidase sequencing data, we show that Arabidopsis proteins are N-glycosylated by high-mannose-type N-glycans from Man5GlcNAc2 to Man9GlcNAc2, and by xylose- and fucose (Fuc)-containing oligosaccharides. However, complex biantenary structures containing the terminal Lewis a epitope recently reported in the literature (A. -C. Fitchette-Lainé, V. Gomord, M. Cabanes, J.-C. Michalski, M. Saint Macary, B. Foucher, B. Cavalier, C. Hawes, P. Lerouge, and L. Faye [1997] Plant J 12: 1411-1417) were not detected. A similar study was done on the Arabidopsis mur1 mutant, which is affected in the biosynthesis of L-Fuc. In this mutant, one-third of the Fuc residues of the xyloglucan has been reported to be replaced by L-galactose (Gal) (E. Zablackis, W.S. York, M. Pauly, S. Hantus, W.D. Reiter, C.C.S. Chapple, P. Albersheim, and A. Darvill [1996] Science 272: 1808-1810). N-linked glycans from the mutant were identified and their structures were compared with those isolated from the wild-type plants. In about 95% of all N-linked glycans from the mur1 plant, L-Fuc residues were absent and were not replaced by another monosaccharide. However, in the remaining 5%, L-Fuc was found to be replaced by a hexose residue. From nuclear magnetic resonance and mass spectrometry data of the mur1 N-glycans, and by analogy with data reported on mur1 xyloglucan, this subpopulation of N-linked glycans was proposed to be L-Gal-containing N-glycans resulting from the replacement of L-Fuc by L-Gal.

Arabidopsis↗
PubMed1998

A rapid method to screen for cell-wall mutants using discriminant analysis of Fourier transform infrared spectra.

We have developed a rapid method to screen large numbers of mutant plants for a broad range of cell wall phenotypes using Fourier transform infrared (FTIR) microspectroscopy of leaves. We established and validated a model that can discriminate between the leaves of wild-type and a previously defined set of cell-wall mutants of Arabidopsis. Exploratory principal component analysis indicated that mutants deficient in different cell-wall sugars can be distinguished from each other. Discrimination of cell-wall mutants from wild-type was independent of variability in starch content or additional unrelated mutations that might be present in a heavily mutagenised population. We then developed an analysis of FTIR spectra of leaves obtained from over 1000 mutagenised flax plants, and selected 59 plants whose spectral variation from wild-type was significantly out of the range of a wild-type population, determined by Mahalanobis distance. Cell wall sugars from the leaves of selected putative mutants were assayed by gas chromatography-mass spectrometry and 42 showed significant differences in neutral sugar composition. The FTIR spectra indicated that six of the remaining 17 plants have altered ester or protein content. We conclude that linear discriminant analysis of FTIR spectra is a robust method to identify a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification.

Arabidopsis↗
PubMed1997

Developmental regulation of cell interactions in the Arabidopsis fiddlehead-1 mutant: a role for the epidermal cell wall and cuticle.

Although the plant epidermis serves primarily a protective role, during plant development some epidermal cells specialize, becoming competent to interact not only with pollen but also with other epidermal cells. In the former case, these interactions mediate recognition, germination, and pollen growth responses and, in the latter case, result in interorgan fusions which, most commonly, alter floral architecture in ways that are thought to promote reproductive success. In either case, all of the initial signaling events must take place across the cell wall and cuticle. In Arabidopsis, mutation of the FIDDLEHEAD gene alters the shoot epidermis such that all epidermal cells become competent to participate in both types of interactions. In fdh-1 mutants, epidermal cells manifest not only a contact-mediated fusion response but also interact with pollen. Since carpel epidermal derivatives manifest both of these properties, we postulated that fdh-1 epidermal cells were ectopically expressing a carpel-like program. In this report we demonstrate that manifestation of the fdh-1 phenotype does not require the product of the AGAMOUS gene, indicating that the phenotype is either independent of the carpel development program or that fdh-1 mutations activate a carpel-specific developmental program downstream of the AG gene. Furthermore, we demonstrate that plants bearing mutations in the fdh-1 gene show significant changes in cell wall and cuticular permeability. Biochemical analyses of the lipid composition of the crude cell wall fraction reveal that fdh-1 cell walls differ from wild-type and manifest significant changes in high-molecular-weight lipid peaks. These results suggest that cell wall and cuticular permeability may be important determinants in developmental signaling between interacting cells and implicate lipids as important factors in modulating the selectivity of the permeability barrier presented by the epidermal cell wall and cuticle.

Arabidopsis↗
PubMed1997

The MUR1 gene of Arabidopsis thaliana encodes an isoform of GDP-D-mannose-4,6-dehydratase, catalyzing the first step in the de novo synthesis of GDP-L-fucose.

GDP-L-fucose is the activated nucleotide sugar form of L-fucose, which is a constituent of many structural polysaccharides and glycoproteins in various organisms. The de novo synthesis of GDP-L-fucose from GDP-D-mannose encompasses three catalytic steps, a 4,6-dehydration, a 3,5-epimerization, and a 4-reduction. The mur1 mutant of Arabidopsis is deficient in L-fucose in the shoot and is rescued by growth in the presence of exogenously supplied L-fucose. Biochemical assays of the de novo pathway for the synthesis of GDP-L-fucose indicated that mur1 was blocked in the first nucleotide sugar interconversion step, a GDP-D-mannose-4,6-dehydratase. An expressed sequence tag was identified that showed significant sequence similarity to proposed bacterial GDP-D-mannose-4,6-dehydratases and was tightly linked to the mur1 locus. A full-length clone was isolated from a cDNA library, and its coding region was expressed in Escherichia coli. The recombinant protein exhibited GDP-D-mannose-4,6-dehydratase activity in vitro and was able to complement mur1 extracts in vitro to complete the pathway for the synthesis of GDP-L-fucose. All seven mur1 alleles investigated showed single point mutations in the coding region for the 4,6-dehydratase, confirming that it represents the MUR1 gene.

Amino Acid Sequence↗
PubMed1997

Mutants of Arabidopsis thaliana with altered cell wall polysaccharide composition.

To analyze the synthesis, structure and function of the plant cell wall by a genetic approach, 5200 chemically mutagenized Arabidopsis plants were screened for changes in the monosaccharide composition of hydrolyzed cell wall material by gas chromatography of alditol acetates This screening procedure identified 23 mutant lines representing 11 different loci designated mur1 to mur11. The mur lines fall into essentially three groups: (1) complete absence of a monosaccharide, (2) significant reduction in the amount of a single monosaccharide, and (3) complex alterations in the relative amounts of several monosaccharides. All mutants in the first category represent alleles of the mur1 locus, and are deficient in the de novo synthesis of fucose. Mutants with reductions in a single monosaccharide have been identified for fucose (mur2, mur3), arabinose (mur4, mur5, mur6, mur7), and rhamnose (mur8). Mutants with complex changes in monosaccharide composition are represented by the mur9, mur10 and mur11 loci. Most of the mutant lines did not show obvious morphological or physiological alterations; however, lines mur1, mur9 and mur10 co-segregated with reduced vigor or dwarfism of the plants. These results demonstrate the feasibility of identifying plants with altered cell wall compositions via a biochemical screening procedure. The availability of these mutants provides novel opportunities to study the functions of cell wall polysaccharides, gain insight into the biosynthesis of cell wall material, and clone cell wall-related genes.

Arabidopsis↗
PubMed1996

Substitution of L-fucose by L-galactose in cell walls of Arabidopsis mur1.

An Arabidopsis thaliana mutant (mur1) has less than 2 percent of the normal amounts of L-fucose in the primary cell walls of aerial portions of the plant. The survival of mur1 plants challenged the hypothesis that fucose is a required component of biologically active oligosaccharides derived from cell wall xyloglucan. However, the replacement of L-fucose (that is, 6-deoxy-L-galactose) by L-galactose does not detectably alter the biological activity of the oligosaccharides derived from xyloglucan. Thus, essential structural and conformational features of xyloglucan and xyloglucan-derived oligosaccharides are retained when L-galactose replaces L-fucose.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1992

Elements of an archaeal promoter defined by mutational analysis.

The sequence requirements for specific and efficient transcription from the 16S/23S rRNA promoter of Sulfolobus shibatae were analysed by point mutations and by cassette mutations using an in vitro transcription system. The examination of the box A-containing distal promoter element (DPE) showed the great importance of the TA sequence in the center of box A for transcription efficiency and the influence of the sequence upstream of box A on determining the distance between the DPE and the start site. In most positions of box A, replacement of the wild type bases by adenines or thymines are less detrimental than replacements by cytosines or guanines. The effectiveness of the proximal promoter element (PPE) was not merely determined by its high A + T content but appeared to be directly related to its nucleotide sequence. At the start site a pyrimidine/purine (py/pu) sequence was necessary for unambiguous initiation as shown by analysis of mutants where the wild type start base was replaced. The sequence of box A optimal for promoter function in vitro is identical to the consensus of 84 mapped archaeal promoter sequences.

Base Composition↗
PubMed1990

Identification and characterization of a defective SSV1 genome integrated into a tRNA gene in the archaebacterium Sulfolobus sp. B12.

Within the chromosome of the archaebacterium Sulfolobus sp. B12, a 7.4 kb region was identified which displayed extensive sequence similarities to the 15.5 kb genetic element SSV1 carried by the same strain both as a circular form and as a site-specifically integrated copy. DNA sequence analysis indicated that this 7.4 kb region (designated SSV1intB) represented an SSV1-like element distinguishable from the full-length integrated copy (designated SSV1intA) by extensive deletions and point mutations. The physical organization of DNA sequences of SSV1intB indicated that this element was integrated at the same attP site as previously identified for SSV1intA. A comparison of the DNA sequences at the left attachment sites of SSV1intA and SSV1intB revealed that they both represented very similar putative arginine tRNA genes followed by a 10 bp inverted repeat sequence. S1 nuclease mapping experiments indicated that these tRNA genes are transcribed.

Archaea↗
PubMed1990

In vitro transcription of two rRNA genes of the archaebacterium Sulfolobus sp. B12 indicates a factor requirement for specific initiation.

We describe a cell-free transcription system for the archaebacterium Sulfolobus sp. B12 that specifically initiates transcription at the 5S rRNA-encoding DNA and the 16S/23S rRNA-encoding DNA promoters of the same species. With this crude extract system, specific initiation was absolutely dependent on the box A motif, a highly conserved promoter element in archaebacteria located approximately 25 base pairs upstream of transcription initiation sites. In vitro transcription of the rRNA genes by purified RNA polymerase, however, resulted in semi-specific, box A-independent initiation, indicating that factor(s) in the crude extract were necessary for the highly specific box A-dependent transcription. Fractionation of the cell-free extract by sucrose-gradient centrifugation resulted in the identification of a low molecular weight fraction complementing purified RNA polymerase to an extract-like specificity.

Archaea↗
PubMed1990

Mutational analysis of an archaebacterial promoter: essential role of a TATA box for transcription efficiency and start-site selection in vitro.

By using a recently developed in vitro transcription assay, the 16S/23S rRNA-encoding DNA promoter from the archaebacterium Sulfolobus sp. B12 was dissected by deletion and linker substitution mutagenesis. The analysis of 5' and 3' deletion mutants defined a core promoter region between positions -38 and -2 containing all information for efficient and specific transcription. Further characterization of this region by linker substitution mutagenesis indicated two sequence elements important for promoter function--one located between positions -38 and -25 (distal promoter element) and the other one located between positions -11 and -2 (proximal promoter element). The distal promoter element encompassed the TATA-like "box A" element located approximately 26 nucleotides upstream of the majority of transcription start sites in archaebacteria (Archaeobacteria). All mutations within this box A motif virtually abolished promoter function. Complete inactivation of the proximal promoter element was dependent on extensive mutagenesis; this element is not conserved between archaebacterial promoters except for a high A + T content in stable RNA gene promoters from Sulfolobus. Mutants containing insertions or deletions between the distal and proximal promoter elements were only slightly affected in their transcription efficiency but displayed a shift in their major initiation site, retaining an essentially fixed distance between the distal promoter element and the transcription start site. Thus, efficient transcription and start-site selection were dependent on a conserved TATA-like sequence centered approximately 26 nucleotides upstream of the initiation site, a situation unlike that of eubacterial promoters but resembling the core structure of most eukaryotic RNA polymerase II (and some RNA polymerase III) promoters. This finding suggests a common evolutionary origin of these promoters consistent with the known similarities between archaebacterial and eukaryotic RNA polymerases.

Archaea↗
PubMed1989

Transfer RNA genes frequently serve as integration sites for prokaryotic genetic elements.

The DNA sequences were determined at the boundaries of the integrated copy of the archaebacterial genetic element SSV1. A 44 bp sequence present as a single copy on the 15.5 kb circular SSV1 DNA flanked the integrated copy as a direct DNA sequence repeat, suggesting that SSV1 integration occurred by recombination between this 44 bp SSV1 sequence and an identical sequence on the bacterial chromosome. At the left attachment site, a region encompassing the 44 bp attachment core sequence and the 31 nucleotides upstream of it displayed all characteristics expected for an arginine tRNA gene. An analysis of published attachment site sequences of other systems revealed that tRNA genes also constitute the bacterial attachment site in the case of three temperate phages and two transmissible plasmids in eubacteria, indicating a widespread occurrence of tRNA genes as integration target sites. This finding may be important for the understanding of mechanisms and evolution of site-specific recombination.

Archaea↗
PubMed1988

Comparative evaluation of gene expression in archaebacteria.

Gene organization, gene structure, especially regarding transcription and translation signals, and the structure of essential components of the gene expression machinery of archaebacteria are compared with those of eubacteria and eukaryotes. Many features of the genetic machinery of archaebacteria are shared either with eubacteria or with eukaryotes. For example, the translation signals including ribosome-binding sites are the same as in eubacteria, but the consensus sequence of archaebacterial promoters closely resembles that of the eukaryotic polymerase II promoters. Archaebacterial genes can be organized in transcription units resembling those of eubacteria. But the sequences of several protein components of the genetic machinery have strikingly more homology with those of their eukaryotic than with those of their eubacterial correspondents. The sequences of the large components of DNA-dependent RNA polymerases of archaebacteria closely resemble those of the eukaryotic RNA polymerases II and, somewhat less, III. In a dendrogram calculated from percentage homology data, the eukaryotic RNA polymerase I component A shares a branching point with the eubacterial component. The implications of these findings for the origin and the evolution of the eukaryotic ancestry are discussed.

Archaea↗
PubMed1988

Transcription termination in the archaebacterium Sulfolobus: signal structures and linkage to transcription initiation.

The precise map positions were determined for the 3'-termini of five transcripts of the Sulfolobus virus-like particle SSV1. In all cases analyzed, these 3'-termini mapped immediately downstream of a sequence TTTTTYT which was part of a pyrimidine-rich region of 16-19 nucleotides length. No correlation was evident between the position of the 3'-termini and possible secondary structures within the RNA. In two cases, the 3'-termini of SSV1 transcripts mapped in the immediate vicinity of transcriptional initiation sites suggesting that transcription termination can be linked to the re-initiation of RNA synthesis.

Archaea↗
PubMed1988

Analysis of transcription in the archaebacterium Sulfolobus indicates that archaebacterial promoters are homologous to eukaryotic pol II promoters.

The 5'-termini were precisely mapped for five constitutive and one UV-inducible transcript from the Sulfolobus virus-like particle SSV1. The comparison of the DNA sequences around these transcriptional initiation sites revealed the presence of two conserved sequence elements: a trinucleotide sequence close to the initiation site itself and an AT-rich hexanucleotide sequence centered about 26 nucleotides upstream of it. Similar DNA sequences were found upstream of the transcriptional start sites for the ribosomal RNA genes in Sulfolobus and upstream of transcriptional start sites in other archaebacteria, allowing the derivation of a general consensus sequence for archaebacterial promoters. This consensus sequence is unlike that found in eubacteria but it resembles promoters recognized by eukaryotic RNA polymerase II.

Archaea↗
PubMed1987

Putative promoter elements for the ribosomal RNA genes of the thermoacidophilic archaebacterium Sulfolobus sp. strain B12.

In Sulfolobus sp. strain B12, single-copy genes encode the three ribosomal RNAs. The genes for the 16S rRNA and for the 23S rRNA are closely linked but separated from the 5S rRNA gene. Transcription of the 16S/23S rRNA gene cluster starts 139 nucleotides upstream of the 5'-end of mature 16S rRNA. For the 5S rRNA gene the point of transcription initiation coincides with the 5'-end of mature 5S rRNA. The comparison of the upstream regions for these transcriptional start sites shows the presence of a completely conserved trinucleotide sequence around the point of transcription initiation and a completely conserved octanucleotide sequence about 22 nucleotides upstream of it. These sequences are only moderately homologous to putative promoter elements for stable RNA genes in the closely related archaebacterium Thermoproteus tenax (1), but they are very similar to corresponding sequences in the distantly related archaebacterium Methanococcus vannielii (2). The consensus sequence found for Sulfolobus and Methanococcus could therefore constitute the archetype of an archaebacterial promoter for stable RNA genes.

Archaea↗