Structural analysis of the nit2/nit1/nit3 gene cluster encoding nitrilases, enzymes catalyzing the terminal activation step in indole-acetic acid biosynthesis in Arabidopsis thaliana.

A 13.8 kb DNA sequence containing the promoters and the structural genes of the Arabidopsis thaliana nit2/nit1/nit3 gene cluster has been isolated and characterized. The coding regions of nit2, nit1 and nit3 spanned 1.9, 1.8 and 2.1 kb, respectively. The architecture of the three genes is highly conserved. Each isoform consists of five exons separated by four introns. The introns are very similar with respect to size and position, but differ considerably in sequence composition. In contrast to the coding sequences the three promoters are very different in sequence, size and in their repertoire of cis elements, suggesting differential regulation of the three nitrilase isoenzymes by the developmental program of the plant and by diverse environmental factors. The nit1 promoter was subjected to analysis in planta. Translational fusions placing the nit1 full-length promoter and a series of 5'-deletion fragments in front of the uidA gene encoding beta-glucuronidase (GUS) were used for Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum. GUS expression was highest in fully expanded leaves and in the shoot apex as well as in the apices of developing lateral buds, whereas the GUS activity displayed by developing younger leaflets was restricted to the tips of the expanding leaves. Within the root tissue GUS expression was restricted to the root tips and the tips of newly forming lateral roots. Structural features of the nitrilase gene family and nitrilase gene expression patterns are discussed in context with current knowledge of auxin biosynthesis and auxin effects on different tissues.

Transgenic tobacco plants expressing the Arabidopsis thaliana nitrilase II enzyme.

Nitrilase (E.C. 3.5.5.1) cloned from Arabidopsis thaliana converts indole-3-acetonitrile to the plant growth hormone, indole-3-acetic acid in vitro. To probe the capacity of ths enzyme under physiological conditions in vivo, the cDNA PM255, encoding nitrilase II, was stably integrated into the genome of Nicotiana tabacum by direct protoplast transformation under the control of the CaMV-35S promotor. The regenerated plants appeared phenotypically normal. Nitrilase II was expressed, based on the occurrence of its mRNA and polypeptide. The enzyme was catalytically active, when extracted from leaf tissue of transgenic plants (specific activity: 25 fkat mg(-1) protein with indole-3-acetonitrile as substrate). This level of activity was lower than that found in A. thaliana, and this was deemed essential for the in vivo analysis. Leaf tissue from the transgenic plants converted 1-[13C]-indole-3-acetonitrile to 1-[13C]-indole-3-acetic acid in vivo as determined by HPLC/GC-MS analysis. Untransformed tobacco was unable to catalyze this reaction. When transgenic seeds were grown on medium in the absence of indole-3-acetonitrile, germination and seedling growth appeared normal. In the presence of micromolar levels of exogenous indole-3-acetonitrile, a strong auxin-overproducing phenotype developed resulting in increased lateral root formation (at 10 microM indole-3-acetonitrile). Collectively, these data prove the ability of nitrilase II to convert low micromolar levels of indole-3-acetonitrile to indole-3-acetic acid in vivo, even when expressed at subphysiological levels thereby conferring a high-auxin phenotype upon transgenic plants. Thus, the Al thaliana nitrilase activity, which exceeds that of the transgenic plants, would be sufficient to meet the requirements for auxin biosynthesis in vivo.

Structure of the gene encoding nitrilase 1 from Arabidopsis thaliana.

The nitrilases of Arabidopsis thaliana (At) catalyze the conversion of indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA), thus controlling the last step of auxin biosynthesis. A full-length genomic clone encoding the complete cluster of the At nitrilases 1 to 3 (NIT1-3), including the respective promoter regions, has been isolated and the NIT1 isoform has been sequenced. The coding region (nit1) spans about 2.3 kb and is composed of five exons separated by four introns. The exon-intron splice junctions agree with the consensus sequences typical for plant genes. In agreement with the known cDNA sequence, the exons encode a protein of 346 amino acids (aa) with a deduced molecular mass of 38.2 kDa. The transcription start point (tsp) of nit1 was determined by primer extension experiments. This tsp defines a 5' untranslated region of 36 bp and is located 32 bp downstream from a TATA box. The promoter region of nit1 is located within the approx. 1.5-kb intergenic part that separates the nit2 and nit1 coding sections.

Molecular characterization of two cloned nitrilases from Arabidopsis thaliana: key enzymes in biosynthesis of the plant hormone indole-3-acetic acid.

As in maize [Wright, A.D., Sampson, M. B., Neuffer, M. G., Michalczuk, L., Slovin, J. P. & Cohen, J. D. (1991) Science 254, 998-1000], the major auxin of higher plants, indole-3-acetic acid, is synthesized mainly via a nontryptophan pathway in Arabidopsis thaliana [Normanly, J., Cohen, J. D. & Fink, G. R. (1993) Proc. Natl. Acad. Sci. USA 90, 10355-10359]. In the latter species, the hormone may be accessible from the glucosinolate glucobrassicin (indole-3-methyl glucosinolate) and from L-tryptophan via indoleacetaldoxime under special circumstances. In each case, indole-3-acetonitrile is the immediate precursor, which is converted into indole-3-acetic acid through the action of nitrilase (nitrile aminohydrolase, EC 3.5.5.1). The genome of A. thaliana contains two nitrilase genes. Nitrilase I had been cloned earlier in our laboratory. The cDNA for nitrilase II (PM255) was cloned and encodes an enzyme that converts indole-3-acetonitrile to indole-3-acetic acid, the plant hormone. We show that the intracellular location as well as the expression pattern of the two A. thaliana nitrilases are distinctly different. Nitrilase I is soluble and is expressed throughout development, but at a very low level during the fruiting stage, while nitrilase II is tightly associated with the plasma membrane, is barely detectable in young rosettes, but is strongly expressed during bolting, flowering, and especially fruit development. The results indicate that more than one pathway of indole-3-acetic acid biosynthesis via indole-3-acetonitrile exists in A. thaliana and that these pathways are differentially regulated throughout plant development.

Functional expression and molecular characterization of AtUBC2-1, a novel ubiquitin-conjugating enzyme (E2) from Arabidopsis thaliana.

The first member of a novel subfamily of ubiquitin-conjugating E2-proteins was cloned from a cDNA library of Arabidopsis thaliana. Genomic blots indicate that this gene family (AtUBC2) consists of two members and is distinct from AtUBC1, the only other E2 enzyme known from this species to date (M.L. Sullivan and R.D. Vierstra, Proc. Natl. Acad. Sci. USA 86 (1989) 9861-9865). The cDNA sequence of AtUBC2-1 extends over 794 bp which would encode a protein of 161 amino acids and a calculated molecular mass of 18.25 kDa. The protein encoded by AtUBC2-1 is shown to accept 125I-ubiquitin from wheat E1 enzymes, when expressed from Escherichia coli hosts as fusion protein carrying N-terminal extensions. It is deubiquitinated in the presence of lysine and, by these criteria, is considered a functional E2 enzyme.

A glutathione S-transferase with glutathione-peroxidase activity from Arabidopsis thaliana. Molecular cloning and functional characterization.

A full-length cDNA clone for a novel glutathione S-transferase was isolated from Arabidopsis thaliana and characterized. The cDNA encodes a polypeptide of 218 amino acids with a calculated molecular mass of 24,363 Da. The sequence was most related to the theta class within the glutathione-S-transferase superfamily of enzymes. The protein encoded by the cDNA was functionally expressed and enzymically active in Escherichia coli; glutathione-S-transferase activity with the standard enzyme substrate 1-chloro-2,4-dinitrobenzene was demonstrated (apparent Km, 10 mM; apparent Km for glutathione, 0.08 mM). The enzyme is substrate specific and did not use several electrophilic reduced-glutathione acceptor molecules for conjugation. However, it efficiently catalyzed the conversion of 13-hydroperoxy-9,11,15-octadecatrienoic acid (Km, 0.67 mM) as well as 13-hydroperoxy-9,11-octadecadienoic acid (Km, 0.79 mM) to the corresponding hydroxy derivatives with concomitant formation of oxidized glutathione. The enzyme did not use H2O2 as substrate. Thus, the cloned A. thaliana enzyme functions as glutathione peroxidase and, in the plant cell, may be involved in the removal of reactive organic hydroperoxides, such as the products of lipid peroxidation. The enzyme is structurally and enzymatically, however, unrelated to the selenium-containing glutathione peroxidases. Enzymic and immunoblotting data suggest that the A. thaliana enzyme is soluble and constitutively expressed in vegetative rosettes, but is under developmental control during the transition to bolting and flowering.

Leucine aminopeptidase from Arabidopsis thaliana. Molecular evidence for a phylogenetically conserved enzyme of protein turnover in higher plants.

Leucine aminopeptidases are exopeptidases which are presumably involved in the processing and regular turnover of intracellular proteins; however, their precise function in cellular metabolism remains to be established. Towards this goal, a full-length complementary DNA encoding a plant leucine aminopeptidase was isolated from a cDNA library of Arabidopsis thaliana and sequenced. The nucleotide sequence showed 49.5% identity to the Escherichia coli xerB-encoded leucine aminopeptidase. Sequence analysis revealed that the cDNA encodes a polypeptide of 520 amino acids with a calculated molecular mass of 54,506 Da. The C-terminal part (amino acids 200-520) of the deduced amino acid sequence showed 43.8% sequence identity to the xerB-encoded leucine aminopeptidase and 42.6% sequence identity to the amino acid sequence of bovine lens leucine aminopeptidase (EC 3.4.11.1). No sequence similarity (not even over short sequence elements) was observed with any other known peptidase or proteinase sequence. The cDNA was expressed as a fusion protein from the lacZ promoter in E. coli. Enzymatic analysis proved that the cloned cDNA encoded an active leucine aminopeptidase. The properties of this enzyme, including metal requirements, inhibitor sensitivity, pH optimum and the remarkable temperature stability, are very similar to those reported for leucine aminopeptidases from other tissues. Amino acids involved in metal and substrate binding in bovine lens aminopeptidase are completely conserved in the plant enzyme as well as in the XerB protein. Our results show that leucine aminopeptidases form a superfamily of highly conserved enzymes, spanning the evolutionary period from the bacteria to animals and higher plants. This is the first aminopeptidase cloned from a plant.

Cloning and expression of an Arabidopsis nitrilase which can convert indole-3-acetonitrile to the plant hormone, indole-3-acetic acid.

From an Arabidopsis thaliana cDNA expression library, a cDNA clone was isolated, characterized and sequenced which, at the amino acid level, resembled the Klebsiella ozaenae bromoxynil nitrilase encoded by the bxn gene. The cDNA contained a long open reading frame, starting from two possible neighbouring ATG codons and capable of encoding 340 or 346 amino acids with calculated molecular masses of 37526 Da or 38176 Da, respectively. The sequence similarity between the deduced polypeptides from the Arabidopsis cDNA and bxn was clustered in three domains, one at the C-terminus, one in the center and one near the N-terminus of the two proteins, suggesting important functional elements in these parts of the proteins. The cDNA was cloned into different vectors under the control of the lacZ promotor and was functionally expressed by induction with isopropyl-beta-D-thiogalactoside. Using a combination of high-performance liquid chromatography, monoclonal-antibody based enzyme-linked immunosorbent assay and mass spectroscopy, it was shown that the isolated cDNA clone encodes an enzymatically active nitrilase which is able to convert indole-3-acetonitrile to the plant growth hormone, indole-3-acetic-acid.

[Possibilities in the home nursing care of tumor patients with dyspnea]. / Möglichkeiten in der Pflege von Tumorpatienten mit Dyspnoe zu Hause.

Comprehensive care of oncologic patients in the region of Basel-Stadt includes also oncologic care outside of the Hospital (SEOP BS) as a special service of the cantonal federation for domestic and community care of Basel-Stadt (KVHG). Services of the SEOP are prescribed by the family doctor and arranged by the community care organisation. Next to an education in general medical care the attendant has been educated additionally in oncologic nursing. In this article first the working hypothesis of the Basel model is presented followed by a list of those nursing measures that can easily be executed at home. General recommendations for dyspneic patients are derived from the booklet "Ateminstruktion", edited by the Bernese "Höhenklinik Heiligenschwendi". Finally some important addresses providing diverse inhalation facilities and similar means for treatment at home are mentioned.

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.

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.

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.

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.