Biomedical subjects

R G Herrmann

Publications and source records attributed to R G Herrmann.

At least 109 records · Page 6Linked to original sources

PubMed1989

Primary structure of spinach-chloroplast thioredoxin f. Protein sequencing and analysis of complete cDNA clones for spinach-chloroplast thioredoxin f.

The primary structure of thioredoxin f from spinach chloroplasts was determined by standard amino acid sequencing and furthermore by sequencing the corresponding nuclear genome region. The protein, with a calculated molecular mass of 12,564 Da and a molar absorption coefficient at 280 nm of 17,700 M-1 cm-1, consists of 113 residues and exhibits 24% residue identities with spinach chloroplast thioredoxin mb or Escherichia coli thioredoxin. A monospecific antibody elicited against thioredoxin f has been used to select recombinant phage from spinach cDNA libraries in lambda gt11. The inserts of positive clones were sequenced. They code for a polypeptide of 190 amino acids, composed of the thioredoxin f sequence (113 residues) and an upstream element (77 residues) which most probably constitutes the N-terminal transit peptide that directs the polypeptide into chloroplasts. In vitro transcription and translation of this construct generates a polypeptide of approximately 21 kDa, which is imported by isolated spinach chloroplasts and processed to the mature 12.5-kDa protein.

Amino Acid Sequence↗

PubMed1989

Domain structure of mitochondrial and chloroplast targeting peptides.

Representative samples of mitochondrial and chloroplast targeting peptides have been analyzed in terms of amino acid composition, positional amino acid preferences and amphiphilic character. No highly conserved 'homology blocks' are found in either class of topogenic sequence. Mitochondrial-matrix-targeting peptides are composed of two domains with different amphiphilic properties. Arginine is frequently found either at position -10 or -2 relative to the cleavage site, suggesting that some targeting peptides may be cleaved twice in succession by two different matrix proteases. In stroma-targeting chloroplast transit peptides three distinct regions are evident: an uncharged amino-terminal domain, a central domain lacking acidic residues and a carboxy-terminal domain with the potential to form an amphiphilic beta-strand. Targeting peptides that route proteins to the mitochondrial intermembrane space or the lumen of chloroplast thylakoids have a mosaic design with an amino-terminal matrix- or stroma-targeting part attached to a carboxy-terminal extension that shares many characteristics with secretory signal peptides.

Amino Acid Sequence↗

PubMed1989

Nucleotide sequences of cDNA clones encoding the entire precursor polypeptide for subunit VI and of the plastome-encoded gene for subunit VII of the photosystem I reaction center from spinach.

Recombinant phage which encode the entire precursor polypeptide for subunit VI of the photosystem I reaction center have been selected from a lambda gt11 cDNA expression library made from polyadenylated RNA of spinach seedlings. The sequence predicts a precursor polypeptide of 144 amino acids (Mr = 15.3 kDa), a mature protein of 95 residues (Mr = 10.4 kDa) that lacks methionine, histidine and cysteine, and a transit peptide of 49 residues (Mr = 4.9 kDa). The corresponding gene(s) is (are) designated psaH. The gene for subunit VII, psaC, has been located in the small single-copy region of the spinach plastid chromosome using a synthetic oligonucleotide and a heterologous hybridization probe. It is part of a polycistronic transcription unit that is constitutively expressed and processed. Putative processing products include a monocistronic RNA for psaC. The polypeptide chain of 18 (deduced) amino acids is highly conserved and strikingly resembles bacterial-type ferredoxins. It harbours cysteine residues that appear to be involved in the ligation of the two 4Fe4S centres A and B in photosystem I. None of the two subunits appears to be membrane-spanning, and subunit VI, as subunit VII, is located at the reducing (stromal) side of the reaction center. All available information on the major subunits of photosystem I from spinach has been combined into a (revised) topographic model. Evidence that the innermost - plastome-encoded - core of photosystem I represents an old bacterial heritage in present day chloroplasts is discussed.

Amino Acid Sequence↗

PubMed1988

Nucleotide sequence of cDNA clones encoding the entire precursor polypeptides for subunits IV and V of the photosystem I reaction center from spinach.

Using lambda gt11 expression cloning and immunoscreening, cDNA-containing recombinant phages for subunits IV and V of the photosystem I reaction center were isolated, sequenced and used to probe Northern blots of polyadenylated RNA prepared from spinach seedlings. The mRNA sizes for both components are approximately 1000 and 850 nucleotides, respectively. The 968 nucleotide cDNA sequence and derived amino acid sequence for subunit IV predict a single open reading frame of 231 amino acid residues (25.4 kDa). Comparison with a 13-residue N-terminal amino acid sequence determined for subunit IV suggests a mature protein of 17.3 kDa (154 residues) and a transit sequence of 77 amino acids (8.1 kDa). The corresponding data for subunit V are 677 bp (cDNA), 167 residues for the precursor protein (18.2 kDa), 98 residues for the mature polypeptide (10.8 kDa) and 69 residues for the transit peptide (7.4 kDa). Secondary structure predictions indicate that both proteins possess greatly different transit sequences and that none is membrane-spanning.

Amino Acid Sequence↗

PubMed1988

Nucleotide sequence of cDNA clones encoding the complete precursor for the "10-kDa" polypeptide of photosystem II from spinach.

By screening lambda gt11 cDNA expression libraries prepared from poly(A+) RNA of illuminated (4 and 16 h) spinach seedlings (Spinacia oleracea var. Monatol), we have recovered phage containing inserts corresponding to the complete "10-kDa" precursor polypeptide associated with the water oxidizing photosystem II. The amino acid sequence deduced from the nucleotide sequence indicates a polypeptide of 140 amino acid residues (15.0 kDa). The mature protein is predicted to consist of 99 amino acid residues (10.8 kDa). The identity of the recombinant DNAs was established serologically and by determining an N-terminal sequence of 25 amino acid residues from the purified authentic protein. This sequence was found in the cDNA-derived amino acid sequence. In addition, a monospecific antiserum detected the appropriate polypeptides when copy-RNA was translated into protein which was imported into isolated unbroken chloroplasts. The transit sequence (41 amino acid residues; 4.2 kDa) is markedly dissimilar to those of the three extrinsic, luminal photosystem II proteins. Secondary structure prediction suggests a transmembrane arrangement of the 10-kDa polypeptide in the thylakoid membrane, with the N terminus in the thylakoid lumen and a 17-amino acid C-terminal sequence at the outside partitions. This is consistent with biochemical and functional data for the protein. A major transcript of 1.0 kilobases has been observed in polyadenylated RNA.

Amino Acid Sequence↗

PubMed1988

Correlation of the in vivo anticoagulant, antithrombotic, and antimetastatic efficacy of warfarin in the rat.

Fibrin formation has been hypothesized to be an element of the metastatic process in cancer, and pharmacological interference with such fibrin formation has been proposed as a means of antimetastatic therapy. We have tested this hypothesis through an in vivo study of warfarin in two independent rat disease models--a model of chemical-injury-induced arterial thrombosis, and a model of spontaneous metastasis. We found 0.50 mg/kg-day warfarin to be uniformly lethal after two weeks treatment. The chronic dose of 0.25 mg/kg-day was non-toxic and produced effective anticoagulation and marked antithrombotic and antimetastatic activity. The 0.125 mg/kg-day dose produced a reduction in factor IIc (50%) and factor VIIc (70%), and resulted in statistically significant antithrombotic and antimetastatic activity. The 0.0625 mg/kg-day dose failed to reduce the vitamin K-dependent clotting factors, and failed to produce any antithrombotic or antimetastatic effects. The substantial correlation (very similar dose-response effects) among the anticoagulant, antithrombotic and antimetastatic efficacies of warfarin in the rat suggests that anticoagulation provides the pharmacological mechanism underlying both the antithrombotic and the antimetastatic effects. The poor therapeutic index we observed in the rat may be the attribute which limits the efficacy of warfarin in the treatment of human cancer.

Animals↗

PubMed1988

Complex RNA maturation in chloroplasts. The psbB operon from spinach.

The psbB operon of the spinach plastid chromosome encodes the genes for the 51-kDa chlorophyll a apoprotein (psbB), the 10-kDa phosphoprotein (psbH), both associated with photosystem II, as well as cytochrome b6 (petB) and subunit IV (petD) of the cytochrome b/f complex in the order given. These genes are not expressed coordinately. The RNA pattern of this DNA region is complex and resolves into eighteen major RNA species. Using northern and S1 protection analysis we demonstrate (a) that all RNA species derive from one DNA strand and hybridize in an overlapping fashion; and (b) that they arise by processing rather than by multiple transcription initiation/termination. (c) The operon is bordered by a single prokaryote-like promotor in front of psbB, and by a putative factor-independent terminator with characteristic sequence elements following petD. The terminator appears to function bidirectionally. (d) At least four distinct modification activities operate on the putative primary transcript of 5650 nucleotides and on the processing intermediates, including a novel endonucleolytic activity cleaving within a characteristic hexanucleotide motif, 3'-exonucleolytic activity at discrete RNA ends, 5' shortage of mRNA (psbB), and excision of class II intervening sequences (petB and petD). (e) Kinetically, maturation of the primary transcript is largely a stochastic process. (f) Processing results ultimately in the formation of monocistronic mRNAs for each of the two photosystem II polypeptides and a bicistronic mRNA encoding both subunits of the cytochrome b/f complex. We postulate that these RNA species represent the translationally active components in the non-coordinate dark/light expression of these genes. (g) Light is without any noticeable effect on posttranscriptional modification. Under our conditions it appears to operate at a translational rather than a transcriptional or posttranscriptional level indicating that the biogenesis of thylakoid membranes is regulated at various levels.

Amino Acid Sequence↗

PubMed1988

Nucleotide sequences of cDNAs encoding the entire precursor polypeptides for subunits II and III of the photosystem I reaction center from spinach.

Several cDNA clones encoding the complete subunit II and III precursor polypeptides of the photosystem I reaction center were isolated from two spinach lambda gt1 1 expression libraries by immunoscreening and homologous hybridization. The identity of the recombinant cDNAs was verified by an N-terminal amino acid sequence of 14 and 20 residues for the respective mature spinach proteins. The ca. 880 nucleotide long sequence and derived amino acid sequence for subunit II predict a precursor of 23.2 kDa (212 residues) and a positively charged, mature protein of 17.9 kDa (162 residues). The corresponding data for subunit III are ca. 710 nucleotides (cDNA), 13.4 kDa (125 residues, precursor polypeptide) and, again, a positively charged, mature protein of 9.7 kDa (91 residues). Secondary structure predictions indicate that both subunits are extramembraneous components of photosystem I. Subunit II is probably located on the matrix-side, subunit III in the lumen of stroma lamellae which is consistent both with biochemical findings and the proposed roles of these proteins in the electron transition from and to photosystem I, respectively. Major transcripts of 1.1 kb (subunit II) and 0.8 kb (subunit III) have been observed by RNA-DNA hybridization.

Amino Acid Sequence↗

PubMed1988

Amino acid identities in the three redox center-carrying polypeptides of cytochrome bc1/b6f complexes.

The comparison of primary structures is extended to 22 cytochromes b or b6, 12 cytochromes c1 or f, and 8 Rieske FeS proteins. Conclusions are drawn as to their phylogenetic relationship as well as on conserved, functionally important amino acids and secondary structures. The results are in favor of two independent quinone binding sites at opposite surfaces of the membrane, topping one of the two hemes of cytochrome b each.

Amino Acid Sequence↗

PubMed1988

Analysis of cDNA clones encoding the entire precursor-polypeptide for ferredoxin:NADP+ oxidoreductase from spinach.

In this paper, we report the structural characterization of several spinach ferredoxin-NADP+ oxidoreductase (FNR) cDNAs ranging in size from 0.9 to 1.5 kilobases. A comparison of the deduced amino acid sequence with the known amino acid sequence determined for the spinach protein establishes that 1.4-1.5 kpb inserts span the full length of the mature protein (314 amino acid residues; Mr = 35,382). These also include an N-terminal 55 amino acid transit peptide as well as maximally 171 and 214 nucleotide 5' and 3' untranslated sequences, respectively. Evidence has been obtained that various forms of FNR arise from at least two similar genes. The FNR precursor (369 amino acid residues) has a calculated molecular mass of 41.2 kDa. Comparison of the transit peptide with transit peptides from two other stromal proteins shows little similarity at the level of primary sequence but some common features in secondary structure predictions.

Amino Acid Sequence↗

PubMed1986

Spinach plastid genes coding for initiation factor IF-1, ribosomal protein S11 and RNA polymerase alpha-subunit.

The nucleotide sequence of 2.5 kbp from the cloned SalI fragments 8 and 11 of spinach plastid DNA has been determined. This region was found to encode three open reading frames for hydrophilic polypeptides of 77, 138, and 335 amino acids. Using the computer search algorithm of Lipman and Pearson (Science 227, 1435, 1985), these genes were identified as coding for homologues of E. coli initiation factor IF-1 (inFA), 30S ribosomal protein S11 (rps11), and the alpha-subunit of DNA-dependent RNA polymerase (rpoA). The spinach plastid gene organization is inFA - 381 bp spacer - rps11 - 72 bp spacer - rpoA. The genes are transcribed in vivo and appear to encode functional proteins. These findings imply that plastid chromosomes code for components of the organelle transcription apparatus.

Amino Acid Sequence↗

PubMed1986

Cytochrome b-559 genes from Oenothera hookeri and Nicotiana tabacum show a remarkably high degree of conservation as compared to spinach. The enigma of cytochrome b-559: highly conserved genes and proteins but no known function.

Recent data suggest that cytochrome b-559, an intrinsic membrane protein of the oxygen-evolving photosystem II in chloroplasts, is a heme cross-linked heteromeric polypeptide unit (Herrmann et al. 1984, FEBS Lett 176:239-244). The genes for this cytochrome, designated psbE and psbF, have been located on the chloroplast chromosome of Oenothera hookeri and Nicotiana tabacum by hybridization with fragments of the corresponding spinach genes, and characterized. In both cases, the nucleotide sequence discloses 2 uninterrupted reading frames of 83 and 39 codons separated by a few nucleotides, as in spinach. The amber translation stop codon of psbE overlaps the putative ribosome-binding site for psbF in all cases. The predicted molecular weights of the proteins are 9.2 and 4.3 kd, respectively. In each of the three plant species, and the direction of transcription is opposite to that of the gene for cytochrome f which is located distal to the b-559 genes. Comparison of the deduced amino acid sequences with those from the corresponding spinach genes shows 97% homology. The ubiquitous presence and remarkably high degree of structural and functional conservation of this gene cluster supports the hypothesis of a heteromeric assembly and an important functional role for cytochrome b-559.

Amino Acid Sequence↗

PubMed1986

Location and nucleotide sequence of the pre-apocytochrome f gene on the Oenothera hookeri plastid chromosome (Euoenothera plastome I)

The gene for pre-apocytochrome f has been mapped by blot hybridization on a 2.4 kbp HindIII fragment of the circular plastid chromosome of Oenothera hookeri employing probes from the corresponding spinach gene. The gene is located distal to the gene for the ATP synthase subunit alpha, at the border of the 45 kbp inversion that distinguishes spinach and Oenothera plastid chromosomes. Both genes are transcribed in the same direction. Nucleotide sequence analysis reveals a single open reading frame encoding 318 amino acids of which 285 comprise the mature polypeptide and another 33 residues represent probably a N-terminal signal sequence. The putative pre-sequence is 2 residues shorter than those known from the spinach, wheat and pea protein. The deduced amino acid sequences of f cytochromes from the four plant species show over 80% conservation, maintaining the structural characteristics of the protein.

Amino Acid Sequence↗

PubMed1986

The gene for the Mr 10,000 phosphoprotein associated with photosystem II is part of the psbB operon of the spinach plastid chromosome.

We present the nucleotide and derived amino acid sequence of the gene for the 10 kd phosphoprotein associated with photosystem II. This gene was identified by comparing the recently published first nine amino acid residues for the 10 kd phosphoprotein of spinach (Farchaus and Dilley 1986) with available sequence data from the spinach plastid chromosome. The gene, designated psbH, is part of an operon that encodes the 51 kd chlorophyll a apoprotein of the photosystem II reaction center (psbB), the phosphoprotein (73 codons), cytochrome b6 (petB) and subunit IV (petD) of the cytochrome b/f complex in the order given. Northern blot analysis revealed a complex in vivo RNA pattern for this DNA segment resulting from an extensive modification of a 5.6 kb long putative primary transcript which includes a monocistronic RNA species for the phosphoprotein of approximately 420 bases. The deduced amino acid sequence for the phosphoprotein indicates a polypeptide corresponding to a molecular weight of 7.8 kb. Secondary structure predictions place all potential phosphorylation sites on the stromal side of the membrane.

Amino Acid Sequence↗

PubMed1986

Plastocyanin is encoded by an uninterrupted nuclear gene in spinach.

Plastocyanin is a member of photosynthetic electron transport chains that transfers electrons from cytochrome f to the oxidized P700 chlorophyll a pigment of the photosystem I reaction center. We have isolated and characterized cDNA- and genomic clones from spinach (Spinacia oleracea) encoding the complete plastocyanin-precursor polypeptide. The amino acid sequence derived from the nucleotide sequence shows that the precursor consists of 168 amino acid residues including a transit sequence of 69 residues. The precursor polypeptide has a predicted Mr of 16,917, the mature protein of 10,413. The available data indicate that plastocyanin derives probably from a single-copy gene. The coding region contains no intron. The size of the mRNA as determined by S1 nuclease protection experiments is approximately 660 nucleotides, although analysis of different cDNA clones suggests that longer RNA species do exist, approaching the size of the mRNA (850 bases) estimated by Northern blot techniques.

Amino Acid Sequence↗

PubMed1985

Plastome mutation affecting the chloroplast ATP synthase involves a post-transcriptional defect.

In a plastid genome (plastome) mutation of Oenothera hookeri, at least two of the plastome-coded polypeptides (the beta and epsilon subunits) of the chloroplast ATP synthase are directly affected. As in other plastid chromosomes, the genes for the beta and epsilon subunits are located next to each other on the Oenothera ptDNA molecule and are cotranscribed. Immunoanalysis and peptide mapping of in vivo products suggests that a fusion of the two genes may have occurred in the plastome mutant. In contrast to the in vivo data, in vitro translation of the RNA using a heterologous system results in polypeptides which cannot be distinguished from those of wild-type. In addition, neither the mRNA sizes nor plastid DNA restriction fragment patterns differ from wild-type. To reconcile the paradox of these results, it is suggested that either a defect in a translational signal or some other post-transcriptional event is responsible for the mutant phenotype.

Chloroplasts↗

PubMed1984

Nucleotide sequence of the gene for the P680 chlorophyll alpha apoprotein of the photosystem II reaction center from spinach.

The DNA sequence of 2210 nucleotides including the gene for the "51 kd" chlorophyll alpha-conjugated thylakoid membrane protein associated with the photosystem II reaction center of spinach has been determined. This protein is functionally identical with the P680 chlorophyll alpha apoprotein that catalyses the primary light-induced photochemical processes of photosystem II (Camm, E.L. and Green, B.R. (1983) Biochim. Biophys. Acta 724 291-293). The only large open reading frame in the sequence consists of 508 triplets encoding a protein of molecular mass of 56,246 kd. The deduced amino acid sequence shows clustering of hydrophobic residues into seven core regions which probably traverse the membrane, and a large hydrophilic domain of about 200 amino acids interspersed between span VI and VII. Potential transcription promotor and terminator signals flanking the structural gene show prokaryotic-like features. Seven discrete RNA species ranging in size from 2.0 to over 5.0 kilobases display complementarity to apoprotein coding sequences implying that the region can be polycistronically transcribed. The primary transcript includes information for at least two further genes coding for subunits of the cytochrome b/f complex.

Amino Acid Sequence↗

PubMed1984

Sequence homology and structural similarity between cytochrome b of mitochondrial complex III and the chloroplast b6-f complex: position of the cytochrome b hemes in the membrane.

The amino acid sequences of cytochrome b of complex III from five different mitochondrial sources (human, bovine, mouse, yeast, and Aspergillus nidulans) and the chloroplast cytochrome b6 from spinach show a high degree of homology. Calculation of the distribution of hydrophobic residues with a "hydropathy" function that is conserved in this family of proteins implies that the membrane-folding pattern of the 42-kilodalton (kDa) mitochondrial cytochromes involves 8-9 membrane-spanning domains. The smaller 23-kDa chloroplast cytochrome appears to fold in five spanning domains that are similar to the first five of the mitochondria. Four highly conserved histidines are considered to be the likely ligands for the two hemes. The positions of the histidines along the spanning segments and in a cross section of the membrane-spanning alpha helices implies that two ligand pairs, His-82-His-197/198 and His-96-His-183, bridge the spanning peptides II and V, and the two hemes reside on opposite sides of the hydrophobic membrane core. In addition, the 17-kDa protein of the chloroplast b6-f complex appears to contain one or more of the functions of the COOH-terminal end of the mitochondrial cytochrome b polypeptide.

Amino Acid Sequence↗