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T Börner

Publications and source records attributed to T Börner.

At least 37 records · Page 2Linked to original sources
PubMed1999

Inter-organellar crosstalk in higher plants: impaired chloroplast development affects mitochondrial gene and transcript levels.

Co-ordination of gene expression between the three genomes present in plastids, mitochondria and nucleus is of crucial importance for plant cells. Previous studies revealed that in white leaves of the albostrians (Hordeum vulgare cv. Haisa) mutant, photosynthesis-related plastid and nuclear genes are expressed only at an extremely low level. The plastids of this mutant lack ribosomes, photosynthetic activity and have only rudimentary membrane systems. Here we report on the expression of mitochondrial genes in albostrians barley. Steady-state RNA levels of the mitochondrial genes encoding cytochrome oxidase or ATPase subunits, coxII, coxIII, atpA, atp6, atp9 and cob, were observed to be consistently elevated in the white leaves but not in roots. Investigation of mitochondrial DNA revealed an about three-fold enhanced mitochondrial gene copy number in white compared to green leaf cells, but no differential amplification of mitochondrial genes. Analysis of plants in which the white albostrians plastids were combined with a new nuclear background showed that the enhanced transcript levels were a consequence of the impaired plastids and not of the nuclear albostrians allele. Furthermore, plants bleached by the carotenoid biosynthesis inhibitor norflurazon also showed an enhanced mitochondrial transcript level. These findings allow the conclusion that lack of chloroplast activity in an otherwise fully differentiated leaf leads to an increase in mitochondrial gene copy number and an elevated level of mitochondrial transcripts. Our results indicate an influence of plastids on the genetic apparatus of mitochondria in leaves but not in roots.

Chloroplasts↗
PubMed1999

Role of microcystins in poisoning and food ingestion inhibition of Daphnia galeata caused by the cyanobacterium Microcystis aeruginosa.

The effects of microcystins on Daphnia galeata, a typical filter-feeding grazer in eutrophic lakes, were investigated. To do this, the microcystin-producing wild-type strain Microcystis aeruginosa PCC7806 was compared with a mcy- PCC7806 mutant, which could not synthesize any variant of microcystin due to mutation of a microcystin synthetase gene. The wild-type strain was found to be poisonous to D. galeata, whereas the mcy- mutant did not have any lethal effect on the animals. Both variants of PCC7806 were able to reduce the Daphnia ingestion rate. Our results suggest that microcystins are the most likely cause of the daphnid poisoning observed when wild-type strain PCC7806 is fed to the animals, but these toxins are not responsible for inhibition of the ingestion process.

Animals↗
PubMed1999

Nonribosomal peptide synthesis and toxigenicity of cyanobacteria.

Nonribosomal peptide synthesis is achieved in prokaryotes and lower eukaryotes by the thiotemplate function of large, modular enzyme complexes known collectively as peptide synthetases. These and other multifunctional enzyme complexes, such as polyketide synthases, are of interest due to their use in unnatural-product or combinatorial biosynthesis (R. McDaniel, S. Ebert-Khosla, D. A. Hopwood, and C. Khosla, Science 262:1546-1557, 1993; T. Stachelhaus, A. Schneider, and M. A. Marahiel, Science 269:69-72, 1995). Most nonribosomal peptides from microorganisms are classified as secondary metabolites; that is, they rarely have a role in primary metabolism, growth, or reproduction but have evolved to somehow benefit the producing organisms. Cyanobacteria produce a myriad array of secondary metabolites, including alkaloids, polyketides, and nonribosomal peptides, some of which are potent toxins. This paper addresses the molecular genetic basis of nonribosomal peptide synthesis in diverse species of cyanobacteria. Amplification of peptide synthetase genes was achieved by use of degenerate primers directed to conserved functional motifs of these modular enzyme complexes. Specific detection of the gene cluster encoding the biosynthetic pathway of the cyanobacterial toxin microcystin was shown for both cultured and uncultured samples. Blot hybridizations, DNA amplifications, sequencing, and evolutionary analysis revealed a broad distribution of peptide synthetase gene orthologues in cyanobacteria. The results demonstrate a molecular approach to assessing preexpression microbial functional diversity in uncultured cyanobacteria. The nonribosomal peptide biosynthetic pathways detected may lead to the discovery and engineering of novel antibiotics, immunosuppressants, or antiviral agents.

Bacterial Toxins↗
PubMed1999

Molecular reidentification of human pathogenic Trichoderma isolates as Trichoderma longibrachiatum and Trichoderma citrinoviride.

Several species of the well-known saprophytic genus Trichoderma have been identified as the cause of infections in immunosuppressed humans. Because the differentiation and identification of Trichoderma species based on morphological characters only, is very difficult, two molecular approaches were applied for species identification. Six human pathogenic Trichoderma isolates were investigated by PCR-fingerprinting and analysis of ribosomal DNA internal transcribed spacer (ITS) sequences and compared with the corresponding data sets established for described species of the genus. Five of these strains were identified as T. longibrachiatum, whereas one single strain turned out to be T. citrinoviride. Both species are very closely related and belong to Trichoderma section Longibrachiatum. These data indicate that the occurrence of pathogenic Trichoderma strains may be restricted to species of section Longibrachiatum.

DNA Fingerprinting↗
PubMed1998

In vitro replication of mitochondrial plasmid mp1 from the higher plant Chenopodium album (L.): a remnant of bacterial rolling circle and conjugative plasmids?

According to the endosymbiotic theory, mitochondrial genomes evolved from the chromosome of an alpha-proteobacterium-like ancestor and developed during evolution an extraordinary variation in size, structure and replication. We studied in vitro DNA replication of the mitochondrial circular plasmid mp1 (1309 bp) from the higher plant Chenopodium album (L.) as a model system that replicates in a manner reminiscent of bacterial rolling circle plasmids. Several mp1 subclones were tested for their ability to support DNA replication using a newly developed in vitro system. Neutral/neutral two-dimensional gel electrophoresis of the in vitro products revealed typical simple Y patterns of intermediates consistent with a rolling circle type of replication. Replication activity was very high for a BamHI-restricted total plasmid DNA clone, a 464 bp BamHI/KpnI fragment and a 363 bp BamHI/SmaI fragment. Further subcloning of a 148 bp BamHI/EcoRI fragment resulted in the strongest in vitro DNA replication activity, while a 1161 bp-template outside of this region resulted in a substantial loss of activity. Electron microscopic studies of in vitro DNA replication products from the highly active clones also revealed sigma-shaped molecules. These results support our in vivo data for the presence of a predominant replication origin between positions 628 and 776 on the plasmid map. This sequence shares homology with double-stranded rolling circle origin (dso) or transfer origin (oriT) nicking motifs from bacterial plasmids. mp1 is the first described rolling circle plasmid in eukaryotes.

Base Sequence↗
PubMed1998

Cloning and expression of a new cDNA from monocotyledonous plants coding for a diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase from barley (Hordeum vulgare).

From a cDNA library generated from mRNA of white leaf tissues of the ribosome-deficient mutant 'albostrians' of barley (Hordeum vulgare cv. Haisa) a cDNA was isolated carrying 54.2% identity to a recently published cDNA which codes for the diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) hydrolase of Lupinus angustifolius (Maksel et al. (1998) Biochem. J. 329, 313-319), and 69% identity to four partial peptide sequences of Ap4A hydrolase of tomato. Overexpression in Escherichia coli revealed a protein of about 19 kDa, which exhibited Ap4A hydrolase activity and cross-reactivity with an antibody raised against a purified tomato Ap4A hydrolase (Feussner et al. (1996) Z. Naturforsch. 51c, 477-486). Expression studies showed an mRNA accumulation in all organs of a barley seedling. Possible functions of Ap4A hydrolase in plants will be discussed.

Acid Anhydride Hydrolases↗
PubMed1998

Characterisation of transcript initiation sites in ribosome-deficient barley plastids.

Transcription of plastid genes in higher plants is driven by two RNA polymerases. One is encoded in the chloroplast, the other is encoded in the nucleus. RNA synthesis in ribosome-deficient plastids is performed exclusively by the nuclear-encoded enzyme. In vitro capping was used to identify the transcriptional start sites of the genes clpP and rpl23 in ribosome-free plastids of the barley mutant albostrians. No transcript initiation was found at sequences similar to eubacterial promoters. Instead, transcription started near the motif 5'-YRTA-3', which is also conserved in mitochondrial promoters of higher plants. Our data suggest that the nuclear encoded RNA polymerase is active in mature chloroplasts and is the sole polymerase involved in transcription of rpl123.

Base Sequence↗
PubMed1997

Characterization of recombinant phytochrome from the cyanobacterium Synechocystis.

The complete sequence of the Synechocystis chromosome has revealed a phytochrome-like sequence that yielded an authentic phytochrome when overexpressed in Escherichia coli. In this paper we describe this recombinant Synechocystis phytochrome in more detail. Islands of strong similarity to plant phytochromes were found throughout the cyanobacterial sequence whereas C-terminal homologies identify it as a likely sensory histidine kinase, a family to which plant phytochromes are related. An approximately 300 residue portion that is important for plant phytochrome function is missing from the Synechocystis sequence, immediately in front of the putative kinase region. The recombinant apoprotein is soluble and can easily be purified to homogeneity by affinity chromatography. Phycocyanobilin and similar tetrapyrroles are covalently attached within seconds, an autocatalytic process followed by slow conformational changes culminating in red-absorbing phytochrome formation. Spectral absorbance characteristics are remarkably similar to those of plant phytochromes, although the conformation of the chromophore is likely to be more helical in the Synechocystis phytochrome. According to size-exclusion chromatography the native recombinant apoproteins and holoproteins elute predominantly as 115- and 170-kDa species, respectively. Both tend to form dimers in vitro and aggregate under low salt conditions. Nevertheless, the purity and solubility of the recombinant gene product make it a most attractive model for molecular studies of phytochrome, including x-ray crystallography.

Apoproteins↗
PubMed1997

Mitochondrial and chloroplast phage-type RNA polymerases in Arabidopsis.

In addition to the RNA polymerases (RNAPs) transcribing the nuclear genes, eukaryotic cells also require RNAPs to transcribe the genes of the mitochondrial genome and, in plants, of the chloroplast genome. The plant Arabidopsis thaliana was found to contain two nuclear genes similar to genes encoding the mitochondrial RNAP from yeast and RNAPs of bacteriophages T7, T3, and SP6. The putative transit peptides of the two polymerases were capable of targeting fusion proteins to mitochondria and chloroplasts, respectively, in vitro. The results indicate that the mitochondrial RNAP in plants is a bacteriophage-type enzyme. A gene duplication event may have generated the second RNAP, which along with the plastid-encoded eubacteria-like RNAP could transcribe the chloroplast genome.

Amino Acid Sequence↗
PubMed1997

Splicing and intron-internal RNA editing of trnK-matK transcripts in barley plastids: support for MatK as an essential splice factor.

Group II introns frequently require assistance by specific factors, maturases, for folding and effective splicing in vivo. The only putative maturase of higher plant chloroplasts is encoded by matK, located in the intron of trnK. We show that in barley matK transcripts are modified at a first codon base by C-to-U RNA editing. The resulting H --> Y substitution restores a sequence motif that is present in maturases of yeast and plant mitochondria and of Lactococcus ltrA and that is positioned within the X domain. Processing of trnK-matK transcripts was further investigated in plastids lacking functional ribosomes due to a mutation. Absence of the intron-encoded matK gene product in these plastids is correlated with the accumulation of precursor transcripts for tRNALys(UUU)-matK, processed to different degrees, and by the lack of mature and spliced tRNA molecules. These results suggest an essential role of MatK for splicing of its own transcript in vivo. Processing of the 5' end of trnK exon 1 was found to proceed efficiently also in the mutant plastids although the two tRNA exons were separated by the 2481 nt intron. Consequently, presence of the intron does not interfere with the formation of mature 5' termini.

Amino Acid Sequence↗
PubMed1997

Cloning and characterization of a cDNA encoding a bacteriophage-type RNA polymerase from the higher plant Chenopodium album.

We have cloned a full-length cDNA from the higher plant Chenopodium album coding for a single subunit bacteriophage-type RNA polymerase. The cDNA isolated from an actively growing cell suspension culture recognized a 3.8 kb transcript on Northern blots. The open reading frame comprises 987 amino acids with a predicted molecular mass of 112 kDa. A comparison of the protein sequence with those of the two known fungal mitochondrial RNA polymerases, from Saccharomyces cerevisiae and Neurospora crassa , reveals extensive homology between the three enzymes. with complete conservation of all catalytically essential amino acids. The putative mitochondrial RNA polymerase from C.album , as well as homologous sequences from rice and barley, which have been partially cloned, lack two catalytically non-essential regions of up to 176 amino acids near the C-terminus present in the two fungal mitochondrial RNA polymerases. The extreme N-terminus of the cloned C.album RNA polymerase displays features of a potential mitochondrial transit sequence. In phylogenetic trees constructed to compare the evolutionary relationships between the different single subunit RNA polymerases the C.album sequence forms a subgroup together with the S.cerevisiae and the N.crassa mitochondrial RNA polymerases, well separating from both bacteriophage enzymes and plasmid-encoded RNA polymerases found in mitochondria of many fungi and some higher plants.

Amino Acid Sequence↗
PubMed1997

Precise branch point mapping and quantification of splicing intermediates.

Lariat intermediates of a group II intron were investigated via RT-PCR. Several reverse transcriptases appeared capable of reading through a branched nucleotide. A new method has been established that yields precise information about the location of the branch point within an intron. As an extension of our approach, antisense transcripts of the previously cloned PCR products were successfully used in RNase Protection Assays, providing a tool for quantification of splicing intermediates. Application of the method presented to other self-splicing introns as well as introns in nuclear pre-mRNAs is envisaged.

Base Sequence↗
PubMed1997

Disruption of a Synechocystis sp. PCC 6803 gene with partial similarity to phytochrome genes alters growth under changing light qualities.

A gene that may encode a novel light sensing histidine protein kinase, designated plpA (phytochrome-like protein), was isolated from the cyanobacterium Synechocystis sp. PCC 6803. The 200 COOH-terminal amino acids of the gene product show homology with conserved domains of several bacterial histidine kinases and the ethylene response gene etr1 of Arabidopsis, whereas its central region is similar to the chromophore attachment site of plant phytochromes. Interruption or partial deletion of plpA yielded mutants unable to grow under blue light.

Amino Acid Sequence↗
PubMed1997

Unique features of the mitochondrial rolling circle-plasmid mp1 from the higher plant Chenopodium album (L.).

We analyzed the structure and replication of the mitochondrial (mt) circular DNA plasmid mp1 (1309 bp) from the higher plant Chenopodium album(L.). Two dimensional gel electrophoresis (2DE) revealed the existence of oligomers of up to a decamer in addition to the prevailing monomeric form. The migration behavior of cut replication intermediates during 2DE was consistent with a rolling circle (RC) type of replication. We detected entirely single-stranded (ss) plasmid copies hybridizing only with one of the two DNA strands. This result indicates the occurence of an asymmetric RC replication mechanism. mp1 has, with respect to its replication, some unique features compared with bacterial RC plasmids. We identified and localized a strand-specific nicking site (origin of RC replication) on the plasmid by primer extension studies. Nicks in the plasmid were found to occur at any one of six nucleotides (TAAG/GG) around position 735 of the leading strand. This sequence shows no homology to origin motifs from known bacterial RC replicons. mp1 is the first described RC plasmid in a higher plant.

Base Sequence↗
PubMed1997

High content, size and distribution of single-stranded DNA in the mitochondria of Chenopodium album (L.).

Mitochondrial (mt) DNA of higher plants is unique in its large size and complexity. We report here a hitherto unknown feature, the presence of large quantities of single-stranded (ss) DNA. About 2.0-8.5% of the chromosomal mtDNA from a suspension culture (depending on the growth stage) and 6.5% of the chromosomal mtDNA from whole plants of Chenopodium album were found to be in ss form by dot-blot hybridization after neutral transfer. Similar amounts of ss mtDNA were observed by binding of the single-strand binding (SSB) protein of Escherichia coli under the electron microscope. Significantly less ssDNA was found in plastids of C. album and in E. coli cells. We observed ss regions between 100 and 22,800 bases distributed in the mt genome spaced from 0.5-100 kb apart. After pulsed-field gel electrophoresis (PFGE), the well-bound fraction of mtDNA (found to consist of circular, sigma-shaped and rosette-like molecules), contained the major part of ssDNA as opposed to the migrating linear molecules. Digestion of mtDNA by ss-specific nucleases followed by PFGE mobilized all well-bound DNA and correspondingly increased the quantity of migrating linear DNA molecules. The implications of ssDNA for the structural organization on plant mt genomes are discussed.

DNA, Mitochondrial↗
PubMed1997

Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis aeruginosa PCC 7806.

Several bloom-forming cyanobacterial genera produce potent inhibitors of eukaryotic protein phosphatases called microcystins. Microcystins are hepatotoxic cyclic heptapeptides and are presumed to be synthesized non-ribosomally by peptide synthetases. We identified putative peptide synthetase genes in the microcystin-producing strain Microcystis aeruginosa PCC 7806. Non-hepatotoxic strains of M. aeruginosa lack these genes. Strain PCC 7806 was transformed to chloramphenicol resistance. The antibiotic resistance cassette insertionally inactivated a peptide synthetase gene of strain PCC 7806 as revealed by Southern hybridization and DNA amplification. This is the first report of genetic transformation and mutation, by homologous recombination, of a bloom-forming cyanobacterium. Chemical and enzymatic analyses, including high-performance liquid chromatography (HPLC), mass spectrometry, amino acid activation, and protein phosphatase inhibition, revealed the inability of derived mutant cells to produce any variant of microcystin while maintaining their ability to synthesize other small peptides. The disrupted gene therefore encodes a peptide synthetase (microcystin synthetase) that is specifically involved in the biosynthesis of microcystins. Our results confirm that microcystins are synthesized non-ribosomally and that a basic difference between toxic and non-toxic strains of M. aeruginosa is the presence of one or more genes coding for microcystin synthetases.

Bacterial Proteins↗
PubMed1996

Coexistence of phycoerythrin and a chlorophyll a/b antenna in a marine prokaryote.

Prochlorococcus marinus CCMP 1375, a ubiquitous and ecologically important marine prochlorophyte, was bound to possess functional genes coding for the alpha and beta subunits of a phycobiliprotein. The latter is similar to phycoerythrins (PE) from marine Synechococcus cyanobacteria and bind a phycourobilin-like pigment as the major chromophore. However, differences in the sequences of the alpha and beta chains compared with known PE subunits and the presence of a single bilin attachment site on the alpha subunit designate it as a novel PE type, which we propose naming PE-III. P. marinus is the sole prokaryotic organisms known so far that contains chlorophylls a and b as well as phycobilins. These data strongly suggest that the common ancestor of prochlorophytes and the Synechococcus cyanobacteria contained phycobilins. Flow cytometric data from the tropical Pacific Ocean provide evidence that deep populations of Prochlorococcus possess low amounts of a PE-like pigment, which could serve either in light harvesting or nitrogen storage or both.

Amino Acid Sequence↗