The NADH-binding subunit of respiratory chain complex I is nuclear-encoded in plants and identified only in mitochondria

In higher plants, genes for subunits of respiratory chain complex I (NADH:ubiquinone oxidoreductase) have so far been identified solely in organellar genomes. At least nine subunits are encoded by the mitochondrial DNA and 11 homologues by the plastid DNA. One of the 'key' components of complex I is the subunit binding the substrate NADH. The corresponding gene for the mitochondrial subunit has now been cloned and identified in the nuclear genome from potato ( Solanum tuberosum ). The mature protein consists of 457 amino acids and is preceded by a mitochondrial targeting sequence of 30 amino acids. The protein is evolutionarily related to the NADH-binding subunits of complex I from other eukaryotes and is well conserved in the structural domains predicted for binding the substrate NADH, the FMN and one iron-sulphur cluster. Expression examined in different potato tissues by Northern blot analysis shows the highest steady-state mRNA levels in flowers.
Precursor proteins translated in vitro from the cDNA are imported into isolated potato mitochondria in a ΔΨ-dependent manner. The processed translation product has an apparent molecular mass of 55 kDa, identical to the mature protein present in the purified plant mitochondrial complex I. However, the in-vitro translated protein is not imported into isolated chloroplasts. To further investigate whether the complex I-like enzyme in chloroplasts contains an analogous subunit for binding of NAD(P)H, different plastid protein fractions were tested with a polyclonal antiserum directed against the bovine 51 kDa NADH-binding subunit. In none of the different thylakoid or stroma protein fractions analysed were specific crossreactive polypeptides detected. These results are discussed particularly with respect to the structure of a potential complex I in chloroplasts and the nature of its acceptor site.     

Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis

The Staphylococcus epidermidis genes icaABC are involved in the synthesis of the polysaccharide intercellular adhesin (PIA), which is located mainly on the cell surface, as shown by immunofluorescence studies with PIA-specific antiserum. PIA was shown to be a linear β-1,6-linked glucosaminoglycan composed of at least 130 2-deoxy-2-amino-D-glucopyrano-syl residues of which 80–85% are N-acetylated, the rest being non-N-acetylated and positively charged. A transposon insertion in the icaABC gene cluster (ica, intercellular adhesion) led to the loss of several traits, such as the ability to form a biofilm on a polystyrene surface, cell aggregation, and PIA production. The mutant could be complemented by transformation with the IcaABC-carrying plasmid pCN27. Transfer of pCN27 into the heterologous host Staphylococcus carnosus led to the formation of large cell aggregates, the formation of a biofilm on a glass surface, and PIA expression. The nucleotide sequence of icaABC suggests that the three genes are organized in an operon and that they are co-transcribed from the mapped ica A promoter. Ica A contains four potential transmembrane helices, indicative of a membrane location. The deduced Ica A sequence shows similarity to those of polysaccharide-polymerizing enzymes, the most pronounced being with a Rhizobium meliloti N-acetylglucosaminyltransferase involved in lipo-chitin biosynthesis (22.5% overall identity and 37.4% overall similarity). This similarity suggests that Ica A has N-acetylglucosaminyltransferase activity in the formation     

Light-dependent and tissue-specific expression of the H-protein of the glycine decarboxylase complex.

Glycine decarboxylase is a mitochondrial enzyme complex, which is the site of photorespiratory CO2 and NH3 release. Although the proteins that constitute the complex are located within the mitochondria, because of their intimate association with photosynthesis their expression is controlled by light. Comparisons of the kinetics of mRNA accumulation between the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and the H-protein of glycine decarboxylase during the greening of etiolated Arabidopsis thaliana suggest that their expression is controlled in parallel. A genomic clone for the H-protein (gdcH) was isolated from Arabidopsis and sequenced. The upstream region from -856 to +62 was fused to the beta-glucuronidase (GUS) reporter gene, and this construct was transformed into tobacco. This 5' upstream regulatory region appears to control GUS expression in a manner very similar to that of the endogenous H-protein gene. Constructs with deletions in the 5' upstream region were transformed into tobacco. These deletions revealed that light-dependent and tissue-specific expression was largely controlled by a 259-bp region between -376 and -117 bp. This region contains several putative GT boxes with the GGTTAA consensus core sequence. Once these strong light-dependent elements were removed, a second level of control was revealed. In constructs in which the gdcH 5' regulatory region was shortened to -117 bp or less, there was more GUS activity in the roots than in the leaves, and in dark-grown plants than in light-grown plants. This suggests that more proximal control elements may be responsible for the constitutive low levels of gene expression noted in all nonphotosynthetic tissues.     

Proteinase Inhibitor II Gene Expression Induced by Electrical Stimulation and Control of Photosynthetic Activity in Tomato Plants

Mechanical damage and heat stimulation were used to activateproteinase inhibitor II (Pin2) gene expression in tomato plantsin both treated (local induction) and non-treated tissues (systemicinduction). Both stimuli have been shown to generate electricalsignals, leading to a systemic activation of gene expression.Treatment of tomato leaves with electrical current resultedin the accumulation of Pin2 mRNA in the local and systemic leaves.Additionally, all treatments inducing Pin2 gene activity gaverise to a significant alteration of stomatal aperture. However,heat stimulation provoked a different response in the stomatalparameters than mechanical wounding or electric treatment. Bothmechanical damage and electrical stimulation activated two characteristictime constants in the gas exchange relaxation kinetics. Conversely,heat stimulation resulted in only one major time constant. Theresults clearly show that direct current application to tomatoleaves initiates Pin2 mRNA accumulation locally and systemically.In addition, they suggest the participation of a second slowelectrical/hydraulic component in the wound response mechanismof tomato plants and a possible alternative pathway regulatingheat-induced Pin2 gene expression. (Received February 13, 1995; Accepted April 14, 1995)     

The rhodium(III) trisacetonitrile complexes: a re-investigation of the synthesis of fac- and mer- [RhCl3(CH3CN)3], their characterization by 2D NMR spectroscopy and the X-ray crystal structure of mer-[RhCl3(CH3CN)3] · CH3CN

It is shown that the reaction of RhCl₃·3H₂O with acetonitrile normally produces mixtures of mer- and fac-[RhCl₃(CH₃CN)₃] (1a and 1b, respectively). The IR and ¹H NMR spectra of these isomers were re-investigated. Their two-dimensional (¹⁰³Rh,¹H) NMR spectra were also recorded. Equilibrium and exchange studies of 1a and 1b in CD₃C were performed. It was found that in 1a the exchange rate of the nitrile molecule trans to Cl is much faster than those of mutually trans nitriles. Also the nitrile molecules in 1b underwent fast exchange in CD₃CN; however, their rate was slightly faster than that of the more labile CH₃CN in 1a. The X-ray crystal structure of mer-[RhCl₃(CH₃CN)₃]·CH₃CN (1c) was determined. Crystal data: triclinic space group

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Identification of a novel response regulator required for the swarmer-to-stalked-cell transition in Caulobacter crescentus.

The onset of motility late in the Caulobacter crescentus cell cycle depends on a signal transduction pathway mediated by the histidine kinase PleC and response regulator DivK. We now show that pleD, whose function is required for the subsequent loss of motility and stalk formation by the motile swarmer cell, encodes a 454-residue protein with tandem N-terminal response regulator domains D1 and D2 and a novel C-terminal GGDEF domain. The identification of pleD301, a semidominant suppressor of the pleC Mot phenotype, as a mutation predicted to result in a D-53-->G change in the D1 domain supports a role for phosphorylation in the PleD regulator. Disruptions constructed in the pleD open reading frame demonstrated that the gene is not essential and that the pleC phenotype can also be suppressed by a recessive, loss-of-function mutation. These results suggest that PleD is part of a signal transduction pathway controlling stalked-cell differentiation early in the C. crescentus cell cycle.     

Degradation of quinoline by immobilized Comamonas acidovorans in a three-phase airlift reactor

Quinolie degradation by Comamonas acidovorans was studied in a continuously operated three-phase airlift reactor. Porous glass beads were applied as support matrix for cell imobilization by colonization. Under steady-state conditions (S approximately 0), cell attachment was poor at low dilution rates but imporved considerably with increasing dilution rate. Conversion of quinoline was investigated below and above the washout for suspended culture (D(crit) = mu(max) = 0.42 h(-1)). With immobilized cells the reactor could be operated at D > mu(max), and complete conversion of quinoline was achieved as long as the specific quinoline feed rate D*S(0)/X did not exceed the maximum specific degradation rate (r(S, max)). The biofilm thickness was about 100 mum, and its efficiency was about 54% compared to suspended organisms. If quinoline overloads were supplied to the reactor, quinoline, as overloads were supplied to the reactor, quinoline, as well as its pathway intermediates, appeared in the reactor and conversion was low. Hence, the immobilized microorganisms remained viable and active. They could survive quinoline overloads. If the quinoline feed rate was reduced agains, complete conversion was reestablished. (c) 1995 John Wiley & Sons, Inc.     

Distribution and quantification of alpha1-integrin subunit in rat organs

Summary The ₁₁-integrin is known to be a receptor for collagen and laminin mediating cell-matrix interactions. A monoclonal antibody, 33.4, which specifically inhibits the ₁-integrin-mediated in vitro cell-collagen binding of rat hepatocytes and hepatoma-derived A-cells (Löster et al., 1994), was used to purify by immunoaffinity chromatography the ₁-integrin subunit from rat liver in large quantities for inducing a polyclonal antiserum. In immunoblot analysis on membrane extracts of several rat organs this polyclonal antiserum recognized only a 190 kDa-band, suggesting that it is highly specific for the ₁-integrin subunit. A sandwich-ELISA with monoclonal antibody 33.4 and the polyclonal antiserum against the ₁-integrin subunit, respectively, enabled the quantitative expression pattern of the ₁-integrin subunit to be studied in different rat organs. With the exceptions of brain (not detectable) and muscle (low concentration), the ₁-integrin subunit was detectable in almost all organs of the digestive, respiratory and urogenital system as well as in lymphatic organs. The highest relative concentrations of ₁-integrin subunit were found in uterus, lung and spleen, whereas in seminal vesicle, stomach, parotid gland, epididymis, kidney and liver only modest concentrations were evident. The organ distribution and localization of ₁-integrin subunit were studied by immunohistochemistry with monoclonal and polyclonal antibodies. Immunoreactivity was present in the plasma membranes of all smooth muscle cells, vascular endothelial cells of many organs and fibrocyte-fibroblast sheaths in the heart and kidney. Since these cells are in close contact with collagen-containing basal membranes as well as reticular fibrils, strong evidence exists that in rat tissues the ₁-integrin subunit is expressed at sites where collagen is present and might be involved in vivo in cell—ollagen binding.Dedicated to Professor Peter Sitte on the occasion of his 65th birthday     

The isolation of Ant1, a transposable element from Aspergillus niger

A transposable element has been isolated from the industrially important fungus Aspergillus niger (strain N402). The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. It had inserted at a TA site and appeared to have duplicated the target site upon insertion. The isolated element was found to be 4798 by in length and contained 37-bp inverted, imperfect, terminal repeats (ITRs). The sequence of the central region of the element revealed an open reading frame (designated ORF1) which showed similarity, at the amino acid level, to the transposase of the Tc1/mariner class of DNA transposons. Another sequence within the central region of the element showed similarity to the 3 coding and downstream untranslated region of the amyA gene of A. niger. Sequence homology and structural features indicate that this element, which has been named Ant1 (A. niger transposon 1), is related to the Tc1/mariner group of DNA transposons. Ant1 is apparently present as a single copy in strain N402 of A. niger.     

Genomic analysis of the mouse protamine 1, protamine 2, and transition protein 2 gene cluster reveals hypermethylation in expressing cells

To understand the role of chromatin structure in the expression of the mouse protamine 1, protamine 2, and transition protein 2 genes during spermatogenesis, we have examined the genomic organization of this cluster of ``haploid-specific' genes. As seen in the human genome, protamine 2, transition protein 2, and approximately 2.8 kb of a CpG island, hereafter called CpG island-dTP2, were clustered in a small region. Methylation analyses of this region have demonstrated that i) unlike most other tissue-specific genes, the protamine 1, protamine 2, and transition protein 2 genes were located in a large methylated domain in round spermatids, the cell type where they are transcribed, ii) the protamine 1 gene was only partially methylated in somatic cells and in testes from 7-day-old mice, and iii) the approximately 2 kb upstream and downstream of the CpG island-dTP2 were only partially methylated in somatic tissues. DNase I analysis revealed the presence of at least five strong DNase I hypersensitive sites over the CpG island-dTP2 in somatic tissues, but not in germ cells, and sequence analysis indicated that the CpG island-dTP2 is homologous to a CpG island located approximately 10.6 kb downstream of the human transition protein 2 gene. Although the nature of a CpG island-dTP2 and the function of a CpG island-dTP2-containing somatic tissue-specific DNase I hypersensitive sites in close proximity to the germ cell-specific gene cluster are unclear, the ``open' chromatin structure of the CpG island-dTP2 may be responsible for the partial methylation pattern of the flanking sequences including the transition protein 2 gene in somatic tissues. Received: 6 September 1996 / Accepted: 14 January 1997