Search PubMed⌕ Search

Biomedical subjects

R G Herrmann

Publications and source records attributed to R G Herrmann.

At least 91 records · Page 5Linked to original sources
PubMed1993

Antisense RNA for components associated with the oxygen-evolving complex and the Rieske iron/sulfur protein of the tobacco thylakoid membrane suppresses accumulation of mRNA, but not of protein.

Homologous cDNA clones of the nuclear-codes 23- and 33-kDa polypeptides of the oxygen-evolving system, as well as the Rieske iron/sulfur protein, were expressed in antisense or sense orientation under the control of the 35S RNA cauliflower mosaic virus promoter in transgenic tobacco (Nicotiana tabacum, L.) in order to modulate stationary concentrations of transcripts for studying relationships between transcript and protein levels. In all instances, in individuals that contained as little as 10% or less of the transcripts of untransformed plants, the corresponding protein levels were not notably altered. This indicated that the mRNA levels for components under study are not rate-limiting for protein accumulation and that severely reduced amounts of these transcripts still allow normal plant development. Overexpression of the 23-kDa polypeptide by the corresponding cDNA integrated in sense orientation resulted in both higher mRNA and protein levels without detectable enhancement of oxygen evolution. At least some of the excess protein was found in the soluble fraction and not associated with the photosystem-II reaction center. An attempt has been made to reconcile the varied responses obtained using these approaches with different expression patterns.

Electron Transport Complex III↗
PubMed1993

Construction of a barley (Hordeum vulgare L.) YAC library and isolation of a Hor1-specific clone.

We have constructed an EcoRI-based YAC (yeast artificial chromosome) library from barley (Hordeum vulgare L. cv. Franka) using the vector pYAC4. The library consists of approximately 18,000 recombinant YACs with insert sizes ranging between 100 and 1000 kb (average of 160 kb) corresponding to 50% of the barley genome. Size fractionation after ligation resulted in an increased average insert size (av. 370 kb) but also in a substantial decrease in cloning efficiency. Less than 1% of the colonies showed homology to a plastome-specific probe; approximately 50% of the colonies displayed a signal with a dispersed, highly repetitive barley-specific probe. Using a primer combination deduced from the sequence of a member of the small Hor1 gene family coding for the C-hordein storage proteins, the library was screened by polymerase chain reaction and subsequently by the colony hybridization technique. A single YAC, designated Y66C11, with a 120 kb insert was isolated. This DNA fragment represents a coherent stretch from the terminal part of the Hor1 gene region as judged from the correspondence of the restriction patterns between Y66C11 DNA and barley DNA after hybridization with the Hor1-specific probe. Restriction with the isoschizomeric enzymes HpaII/MspI suggests a high degree of methylation of the Hor1 region in mesophyll cells but not in YAC-derived (yeast) DNA.

Base Sequence↗
PubMed1993

Subcellular location of lincomycin resistance in Nicotiana mutants.

Lincomycin-resistant Nicotiana plumbaginifolia plastid mutants were considered also to carry mitochondrial mutations on the basis of their ability to grow in the dark under selective conditions. To clarify the role of mitochondria, individual protoplasts of the green, lincomycin-resistant N. plumbaginifolia mutant LR400 were microfused with protoplasts of the N. tabacum plastid albino line 92V37, which possesses N. undulata cytoplasm. the production of lincomycin-resistant albino cybrid lines, with N. undulata plastids and recombinant mitochondria, strongly indicated a determining role for mitochondria in the lincomycin resistance. Sequence analysis of the region encompassing putative mutation sites in the 26S rRNA genes from the LR400 and several other lincomycin-resistant N. plumbaginifolia mutants revealed, however, no differences from the wild-type sequence. As an alternative source of the resistance of the fusion products, the N. tabacum fusion partner was also taken into account. Surprisingly, a natural lincomycin resistance of tobacco was detected, which was inherited as a dominant nuclear trait. This result compromises the interpretation of the fusion data suggested above. Thus, to answer the original question definitively, the mutant LR400 was crossed as a female parent with a N. plumbaginifolia line carrying streptomycin-resistant N. tabacum plastids. Calli were then induced from the seedlings. Occasional paternal plastid transmissions were selected as streptomycin-resistant calli on selective medium. These cell lines were shown by restriction enzyme analysis to contain paternal plastids and maternal mitochondria. They were tested for greening and growing ability in the presence of lincomycin. These resistance traits proved to be genetically linked and exclusively located in the plastids.

Base Sequence↗
PubMed1993

Characterization of the promoter from the single-copy gene encoding ferredoxin-NADP(+)-oxidoreductase from spinach.

We describe a genomic DNA segment from spinach that bears part of the single-copy gene for ferredoxin-NADP(+)-oxidoreductase (FNR) including a 3.4 kb promoter sequence. Dissection of this DNA segment and its analysis in GUS (beta-glucuronidase) gene fusions in transgenic tobacco demonstrated that the promoter differs in structure from all other promoters for thylakoid protein genes studied to date. Two regions with light-responsive elements were identified. One is located within the first 118 bp upstream of the transcription initiation site. A second fragment covering nucleotide positions -220 to -119 is capable of conferring light-dependent GUS gene expression on two different minimal promoters. The latter fragment binds a transacting factor in gel-shift assays. None of the fragments carries cis elements known from other genes to be involved in light-controlled expression. Comparison of the light responsiveness of GUS gene fusions controlled by the -753/+231 and -118/+231 regions indicates that they respond differentially to phytochrome-dependent signals and that their expression in tobacco is not restricted to tissue with functional chloroplasts.

Amino Acid Sequence↗
PubMed1992

The single-copy gene psbS codes for a phylogenetically intriguing 22 kDa polypeptide of photosystem II.

Recombinant phages that encode the complete precursor polypeptide for the 22 kDa polypeptide associated with photosystem II have been serologically selected from two lambda gt11 expression libraries made from polyadenylated RNA of spinach seedlings. The cDNAs hybridize to a 1.3 kb RNA species. The precursor protein is comprised of 274 amino acid residues and carries an N-terminal transit peptide of probably 69 amino acid residues. The mature protein exhibits four predicted transmembrane segments and is shown to be an integral component of photosystem II originating in a single-copy gene. The unique characteristics of this protein are: (i) it is the result of a gene-internal duplication of an ancestor with two membrane spans, (ii) a striking resemblance to LHC I/II, CP24/CP29 apoproteins, and ELIPs, although it does not bind chlorophyll and is present in cyanobacteria, and, as these proteins, (iii) it integrates into the membrane with uncleaved routing signals that display remarkable resemblance to patterns found in bipartite transit peptides.

Amino Acid Sequence↗
PubMed1992

Phosphorylation of cytochrome b6 by the LHC II kinase associated with the cytochrome complex.

The cytochrome b6 polypeptide present in cytochrome b6/f preparations from spinach thylakoids is phosphorylated concomitantly with the autophosphorylation of the 64 kDa polypeptide identified as the redox-controlled LHCII kinase. The N-terminal sequence of the 64 kDa kinase and sequence analysis of cytochrome b6 indicate the existence of putative phosphorylation sites in both proteins.

Amino Acid Sequence↗
PubMed1992

Insertion and assembly of the precursor of subunit II into the photosystem I complex may precede its processing.

The biogenesis and assembly of subunit II of photosystem I (PSI) (psaD gene product) were studied and characterized. The precursor and the mature form were produced in vitro and incubated with intact plastids or isolated thylakoids. Following import of the precursor into isolated plastids, mostly the mature form of subunit II was found in the thylakoids. However, when the processing activity was inhibited only the precursor form was present in the membranes. The precursor was processed by a stromal peptidase and processing could occur before or after insertion of the precursor into the thylakoids. Following insertion into isolated thylakoids, both the precursor and the mature form of subunit II were confined to the PSI complex. Insertion of the mature form of subunit II was much less efficient than that of the precursor. Kinetic studies showed that the precursor was inserted into the membrane. Only at a later stage, the mature form began to accumulate. These results suggest that in vivo the precursor of subunit II is inserted and embedded in the thylakoids, as part of the PSI complex. Only later, it is processed to the mature form through the action of a stromal peptidase.

Base Sequence↗
PubMed1992

Proton gradient-driven import of the 16 kDa oxygen-evolving complex protein as the full precursor protein by isolated thylakoids.

The biogenesis of the lumenal 16 kDa protein of the photosynthetic oxygen-evolving complex was analysed using an assay for the import of proteins by isolated thylakoids. The precursor protein is imported with high efficiency in the light in both the presence and absence of stromal extract. Import is almost completely blocked in the dark or if the uncoupler nigericin is present in the light. The data indicate that transport across the thylakoid membrane is driven by a proton motive force in which the proton gradient is the dominant component, and that the full precursor protein can be transported across the thylakoid membrane without prior cleavage by the stromal processing peptidase.

Biological Transport, Active↗
PubMed1992

A DNA library from an individual Beta patellaris chromosome conferring nematode resistance obtained by microdissection of meiotic metaphase chromosomes.

We have used a standard protocol established for human chromosomes to create a chromosome-specific plasmid library from a Beta patellaris chromosome conferring nematode resistance. A monosomic addition line was chosen carrying 18 sugar-beet (Beta vulgaris L.) and one wild-beet (B. patellaris) chromosome. The wild-beet chromosome can readily be identified as a univalent during metaphase I of meiosis. Highly synchronized meiotic material was used to excise the univalents from four pollen mother cells. The chromatin was lysed in a 1 nl collection drop, the DNA purified and restricted with Rsa I, ligated into a vector containing universal sequencing primers, and amplified by the polymerase chain reaction. The amplified DNA was inserted into a standard plasmid vector and cloned. Approximately 23,000 recombinant plasmids were obtained of which 15,800 could be utilized. Their insert sizes ranged from 80 to 700 bp with an average of 130 bp. 61 clones were tested in more detail by genomic Southern hybridization with sugar-beet and wild-beet DNA. Of these 32 plasmids (52%) contained single-copy inserts, 11 (18%) were specific for wild-beet DNA indicating that the DNA cloned originates in the univalent chromosome. The application of this technique for establishing high-density RFLP maps for discrete regions of plant genomes is discussed.

Animals↗
PubMed1992

Nucleotide sequence of cDNAs encoding the entire precursor polypeptide for thioredoxin m from spinach chloroplasts.

Using the expression vector lambda gt11 and immunochemical detection, six cDNA clones that encode the entire precursor polypeptides for spinach thioredoxin m were isolated and characterized. The ca. 1.0 kb cDNA sequence of the largest clone hybridizes to an RNA species of 1.1 kb. In each instance the cDNA sequences display single open reading frames encoding polypeptides of 181 amino acid residues corresponding to a molecular mass of 19.8 kDa. The sequences of the independently selected cDNAs fall into two classes that are indicative of at least two (closely related) genes for this protein. The amino acid sequences deduced from the cDNA sequences differ to some extent from the amino acid sequence published for spinach thioredoxin m. The sequences predict identical mature proteins of 112-114 amino acids corresponding to a polypeptide molecular mass of ca. 12.4-12.6 kDa, and include stroma-targeting N-terminal transit peptides of 67 residues which are removed during or after import into the organelle. Precursor protein was made in vitro from each of the different cDNA clones and imported into isolated intact chloroplasts. Independent of the cDNA clone used, two isoforms were detected in the chloroplasts after import in each instance. They comigrated with authentic thioredoxin mb and mc. These results indicate that the size variants observed for this protein in vivo result from post-translational modification and do not originate in different genes.

Amino Acid Sequence↗
PubMed1992

DNA markers closely linked to nematode resistance genes in sugar beet (Beta vulgaris L.) mapped using chromosome additions and translocations originating from wild beets of the Procumbentes section.

Genes conferring resistance to the beet cyst nematode (Heterodera schachtii Schm.) have been transferred to sugar beet (Beta vulgaris L.) from three wild species of the Procumbentes section using monosomic addition and translocation lines, because no meiotic recombination occurs between chromosomes of cultured and wild species. In the course of a project to isolate the nematode resistance genes by strategies of reverse genetics, probes were cloned from DNA of a fragmented B. procumbens chromosome carrying a resistance gene, which had been isolated by pulsed-field gel electrophoresis. One probe (pRK643) hybridized with a short dispersed repetitive DNA element, which was found only in wild beets, and thus may be used as a molecular marker for nematode resistance to progeneis of monosomic addition lines segregating resistant and susceptible individuals. Additional probes for the resistance gene region were obtained with a polymerase chain reaction (PCR)-based strategy using repetitive primers to amplify DNA located between repetitive elements. One of these probes established the existence of at least six different chromosomes from wild beet species, each conferring resistance independently of the others. A strict correlation between the length of the wild beet chromatin introduced in fragment addition and translocation lines and the repeat copy number has been used physically to map the region conferring resistance to a chromosome segment of 0.5-3 Mb.

Animals↗
PubMed1991

Transport of proteins into chloroplasts. Delineation of envelope "transit" and thylakoid "transfer" signals within the pre-sequences of three imported thylakoid lumen proteins.

The targeting of cytosolically synthesized proteins into the thylakoid lumen is mediated by an aminoterminal pre-sequence consisting of an "envelope transit" and a "thylakoid transfer" signal in tandem. We have investigated the structural characteristics of several thylakoid transfer signals by determining the intermediate sites at which the stromal processing peptidase cleaves to remove the transit sequences. Using this approach we have found that the thylakoid transfer signals of Silene pratensis plastocyanin, 23-kDa oxygen-evolving complex protein from wheat, and 33-kDa oxygen-evolving complex protein from wheat, are 25, 39, and 48 residues in length, respectively. All of the transfer signals contain hydrophobic core sequences and a "-3,-1" motif reminiscent of those found in signal sequences, but the amino-terminal regions of the transfer signals of the 23- and 33-kDa proteins are both longer and more highly charged. The net charge of each amino-terminal region of the transfer sequences is +1, including the amino-terminal amino group. In each case, the stromal processing peptidase cleaves immediately after a positively charged residue, but otherwise the cleavage sites exhibit no common elements of either primary or secondary structure.

Amino Acid Sequence↗
PubMed1991

Probing the role of lysine 116 and lysine 244 in the spinach ferredoxin-NADP+ reductase by site-directed mutagenesis.

Two mutants of the spinach ferredoxin-NADP+ reductase (FNR) were constructed, expressed by using a heterologous expression system previously described (Aliverti, A., Jansen, T., Zanetti, G., Ronchi, S., Herrmann, R. G., and Curti, B. (1990) Eur. J. Biochem. 191, 551-555), and purified to homogeneity. The mutant enzymes FNR-Lys116Gln and FNR-Lys244Gln were similar to the wild-type enzyme in the physicochemical properties measured; however, both enzymes showed a lower activity. Steady-state kinetic analyses and NADP+ binding measurements of the mutant proteins have revealed that the Lys116Gln enzyme had a catalytic efficiency (kcat/KmNADPH) 500-fold lower than that of the wild-type enzyme, and its interaction with NADP+ was greatly impaired. The Lys244Gln enzyme instead had almost the same kcat in the ferricyanide reductase reaction as the wild-type enzyme, while higher values of KmNADPH and Kd for NADP+ were observed. Thus, protein engineering has enabled us to define the role of Lys-116 and Lys-244 in ferredoxin-NADP+ reductase; whereas Lys-244 contributes to the stabilization of the Michaelis complex, the Lys-116 side chain plays an important role also in the further steps of the catalytic cycle, because both Km and kcat are highly affected by the mutation.

Binding Sites↗
PubMed1991

Different blue-light requirement for the accumulation of transcripts from nuclear genes for thylakoid proteins in Nicotiana tabacum and Lycopersicon esculentum.

We have isolated recombinant lambda gt11 phages which carry cDNA clones for the major light-harvesting chlorophyll a/b-binding proteins of photosystem I (LHCPI) and II (LHCPII), subunit II of photosystem I, a chlorophyll a/b-binding protein of photosystem II (CP24), the Rieske iron-sulphur protein of the cytochrome b6/f complex, and the 33, 23 and 16 kDa proteins of the water-oxidizing complex of photosystem II from Nicotiana tabacum. The nucleotide sequences of cDNA clones encoding the precursors for LHCPI and the FeS protein are presented. If tobacco or tomato seedlings, or seedlings of a phytochrome-deficient aurea mutant of tomato which lacks more than 95% of the phytochrome of the isogenic wild type, are kept in blue light, the transcript level of each of these genes is higher than in seedlings grown in red light suggesting the involvement of a blue-UVA-light photoreceptor. In the case of LHCPI, a 1 min blue-light pulse applied to red-light-grown seedlings is sufficient to increase the transcript levels to those present in blue-light-grown seedlings, whereas almost no increase is observed for transcripts encoding the FeS and 33 kDa proteins. If dark-grown tomato seedlings receive a single far-red-light pulse, significant stimulation is detected for LHCPI transcripts, whereas transcripts encoding the FeS and 33 kDa proteins are not stimulated. It is concluded that the lower light requirement for the increase in the LHCPI transcript level is not specific for one of the light-dependent signal transduction chains.

Amino Acid Sequence↗
PubMed1990

Expression in Escherichia coli of ferredoxin:NADP+ reductase from spinach. Bacterial synthesis of the holoflavoprotein and of an active enzyme form lacking the first 28 amino acid residues of the sequence.

A cDNA clone for the preprotein of spinach ferredoxin:NADP+ reductase has been modified to allow the expression in Escherichia coli of the mature flavoprotein form the lacks the transit peptide. An expression vector, pFNR1, was constructed by subcloning the fragment into the plasmid pDS12/RBSII, SphI. In the crude extracts of transformed cells after induction, two active holoproteins of 35 kDa and 32 kDa, respectively, were found. The 32-kDa protein, purified by immunoaffinity chromatography, was found to lack the first 28 residues of the spinach protein sequence and to have a methionine as the N-terminal residue instead of Val29. A new expression plasmid, pFNR2, was obtained by in vitro mutagenesis of the codon GTG for Val29 to the synonymous GTT; in this case, only the 35-kDa protein was expressed by transformed cells. Both the 35-kDa and 32-kDa enzymes were purified and characterized. All the properties analyzed of the cloned 35-kDa enzyme were very similar to those of the spinach flavoprotein. The 32-kDa form showed the same catalytic efficiency of the spinach enzyme as a diaphorase but its interaction with oxidized ferredoxin was partially impaired.

Amino Acid Sequence↗
PubMed1990

Analysis of the promotors of the single-copy genes for plastocyanin and subunit delta of the chloroplast ATP synthase from spinach.

The promotors of the single-copy genes for subunit delta of the chloroplast ATP synthase (atpD) and plastocyanin (PC) from spinach have been sequenced, dissected and analysed in transgenic F0 and F1 tobacco plants using the bacterial GUS gene as a reporter for promotor activity. The transcription of these genes is photo-controlled. The results have been compared with those obtained for the spinach rbcS-1 gene, one of the light-regulated genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, and for the cauliflower mosaic virus (CaMV) 35S RNA promotor. We find that the 5' upstream regions of about 1200 nucleotides contain all the sequences required for light regulation, organ-, tissue- and development-specific expression, and that they are structurally diverse. Their cis-acting elements are functionally defined. The proximal regions of the spinach promotors contain potential TATA, CAAT and T-cyt boxes at appropriate positions, but only sequence elements with low similarity to published light-responsive elements. Positive light-stimulated regions, regions with constitutive, light-independent enhancing effects and with 'silencer'-like activity in complete darkness are found in proximal and far upstream promotor segments. Highest activity of these promotors is correlated with the presence of chloroplasts but is not confined to photosynthetic tissue. Surprisingly, expression is observed in the phloem regions of transgenic leaves, leaf and floral stems, in the vascular area of anthers and in pollen. No histochemical staining has been detected in roots. The distal region of atpD located between -1137 and -590 contains elements for expression in the outer phloem, the region from -590 to -185 for activity in the inner phloem of floral stems. Similar tissue-specific patterns are observed with a fusion between the caufliflower mosaic virus 35S RNA promotor and the GUS gene.

Base Sequence↗
PubMed1989

Transport of proteins into chloroplasts. Import and maturation of precursors to the 33-, 23-, and 16-kDa proteins of the photosynthetic oxygen-evolving complex.

The 33-, 23-, and 16-kDa proteins of the photosynthetic oxygen-evolving complex are synthesized as precursors in the cytoplasm and transported into the thylakoid lumen of higher plant chloroplasts. In this report we have analyzed the import and maturation of these precursors, using reconstituted protein import assays and partially purified preparations of the processing peptidases involved. Precursors of the 33- and 23-kDa proteins from Spinacia and Triticum aestivum are processed by a stromal peptidase to intermediate forms; polypeptides of similar size are observed during the transport of these precursors and possibly that of the 16-kDa protein, into isolated chloroplasts. Complete maturation of the 33- and 23-kDa proteins is carried out by a thylakoidal peptidase shown previously to be involved in plastocyanin biogenesis. The data support an import mechanism involving successive cleavages by the stromal and thylakoidal processing peptidases.

Chlorophyll↗