The plant biotin synthase reaction. Identification and characterization of essential mitochondrial accessory protein components

In plants, the last step of the biotin biosynthetic pathway is localized in mitochondria. This chemically complex reaction is catalyzed by the biotin synthase protein, encoded by the bio2 gene in Arabidopsis thaliana. Unidentified mitochondrial proteins in addition to the bio2 gene product are obligatory for the reaction to occur. In order to identify these additional proteins, potato mitochondrial matrix was fractionated onto different successive chromatographic columns. Combination experiments using purified Bio2 protein and the resulting mitochondrial matrix subfractions together with a genomic based research allowed us to identify mitochondrial adrenodoxin, adrenodoxin reductase, and cysteine desulfurase (Nfs1) proteins as essential components for the plant biotin synthase reaction. Arabidopsis cDNAs encoding these proteins were cloned, and the corresponding proteins were expressed in Escherichia coli cells and purified. Purified recombinant adrenodoxin and adrenodoxin reductase proteins formed in vitro an efficient low potential electron transfer chain that interacted with the bio2 gene product to reconstitute a functional plant biotin synthase complex. Bio2 from Arabidopsis is the first identified protein partner for this specific plant mitochondrial redox chain.     

Isolation and analysis of a cDNA clone that encodes an alfalfa (Medicago sativa) aspartate aminotransferase

Summary Nitrate reductase (NR) assays revealed a bi-specific NAD(P)H-NR (EC 1.6.6.2.) to be the only nitrate-reducing enzyme in leaves of hydroponically grown birches. To obtain the primary structure of the NAD(P)H-NR, leaf poly(A)⁺ mRNA was used to construct a cDNA library in the lambda gt11 phage. Recombinant clones were screened with heterologous gene probes encoding NADH-NR from tobacco and squash. A 3.0 kb cDNA was isolated which hybridized to a 3.2 kb mRNA whose level was significantly higher in plants grown on nitrate than in those grown on ammonia. The nucleotide sequence of the cDNA comprises a reading frame encoding a protein of 898 amino acids which reveals 67%–77% identity with NADH-nitrate reductase sequences from higher plants. To identify conserved and variable regions of the multicentre electron-transfer protein a graphical evaluation of identities found in NR sequence alignments was carried out. Thirteen well-conserved sections exceeding a size of 10 amino acids were found in higher plant nitrate reductases. Sequence comparisons with related redox proteins indicate that about half of the conserved NR regions are involved in cofactor binding. The most striking difference in the birch NAD(P)H-NR sequence in comparison to NADH-NR sequences was found at the putative pyridine nucleotide binding site. Southern analysis indicates that the bi-specific NR is encoded by a single copy gene in birch. These sequence data appeared in the EMBL/GenBank/DDBJ nucleotide sequence data bases under the accession number X54097     

MATING SYSTEM AND ASYMMETRIC HYBRIDIZATION IN A MIXED STAND OF EUROPEAN OAKS

The sessile (Quercus petraea [Matt.] Liebl.) and pedunculate (Quercus robur L.) oaks are two closely related species having a wide sympatric distribution over Europe. Under natural conditions, they frequently form mixed forests, where hybridization is suspected to occur. In this paper, two different approaches have been applied to the study of the mating system and the interspecific gene flow in a mixed stand formed by the two species. The mating systems of both species have been studied separately by means of the mixed-mating model. The relative contribution of the parental species to the progenies have been estimated with two different methods. The first uses the admixture model. The second is an extension of the mixed-mating model and subdivides the outcrossing rate into intra- and interspecific components. The two species were almost completely outcrossing. This high level of outcrossing and interspecific gene flow could play an important role in the maintenance of the genetic diversity in these long-lived forest tree species. The contribution of the sessile oak to the pedunculate oak progenies varied from 17% to 48%. In contrast, ovules of sessile oak trees appear to be preferentially fertilized by other extreme sessile genotypes. We suggest that interspecific and directional gene flow was responsible for such patterns. Pedunculate oak is considered as a pioneer species and is progressively replaced by sessile oak. Our present findings add a further genetic component to this succession scheme, suggesting that unidirectional gene flow reinforces succession between the two species.     

Cysteine 647 in the insulin receptor is required for normal covalent interaction between alpha- and beta-subunits and signal transduction.

We have investigated the structural and functional properties of two mutant insulin receptors in which Cys647 and Cys682,683,685 have been replaced with Ser (IRS647 and IRS682,683,685, respectively). Compared with the wild-type receptor (IRWT), both mutant receptors displayed altered sensitivities to dithiothreitol with respect to insulin binding and reduction of oligomeric forms. Subunit composition of the oligomeric forms of the receptors as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-labeled receptors indicated that Cys682,683,685 are required for normal heterotetrameric structure and that Cys647 plays a major role in the normal covalent association of the alpha- and beta-subunits. Under nonreducing conditions, the affinity-labeled IRS647 migrated, almost exclusively, as a 230-kDa species which appeared to represent an alpha 2 form of the receptor. Furthermore, Chinese hamster ovary cells expressing IRS647 did not exhibit basal or insulin-stimulated autophosphorylation, suggesting that Cys647 is also required for signal transduction.     

Augmented desensitization to epidermal growth factor (EGF) immediate actions: a novel mechanism for altered EGF growth response in mutant A431 cells.

Epidermal growth factor (EGF) may either stimulate or inhibit cell growth. To elucidate the mechanism of these varied effects, we compared EGF action in parental A431 cells in which cell growth is inhibited, and clone 15, a mutant of these cells resistant to EGF growth inhibition. In both lines, EGF receptor was present in similar concentrations and underwent tyrosine phosphorylation to the same extent. Likewise, in both lines, acute exposure to EGF stimulated an increase in free cytoplasmic [Ca2+], as well as a similar increase in phosphorylation of lipocortin 1, a major substrate for the EGF receptor kinase whose phosphorylation is calcium-dependent. On the other hand, pretreatment of clone 15 cells with EGF for 72 h abolished EGF-induced phosphorylation of lipocortin 1 and led to a loss of the increase in cytoplasmic free [Ca2+], whereas no such desensitization was seen in the parental A431 cells. These data indicate a link between EGF-induced increase in cytoplasmic calcium, lipocortin phosphorylation, and cell growth and suggest that differences in mechanisms of desensitization to these immediate actions of EGF may lead to altered growth response to this hormone.     

Expression profiling in reference bacteria: dreams and reality

Profiling of gene expression in bacteria is now being used to uncover unknown genes expressed in particular genetic backgrounds or environmental conditions. Obtaining the best possible information from the expected avalanche of such experiments will require standardization of both experimental approach and statistical analysis. The first such experiments reveal challenges, pitfalls and reasonable solutions.     

Molecular cloning and sequence analysis of cDNA for human transferrin

A cDNA clone for human transferrin was identified from a human liver cDNA library by pre-screening with different ss-cDNA probes against length-fractionated liver mRNAs, positive hybridization-selection and nucleotide sequence analysis. The insert was of 1 kb, encoding human transferrin from aminoacid 403 through the COOH terminus, with a 3' non coding region of 166 nucleotides. This insert hybridized with a single major mRNA species of about 2.4 kb and several genomic DNA restriction fragments. Hybridization of the Southern blots with different parts of the transferrin insert and at different stringences suggest that the various bands observed correspond to splice sites inside one gene rather than to hybridization to several related genes. Finally, a single or a low number of transferrin gene copies seem to exist in the human genome.