Evidence for the presence of complex high-molecular mass N-linked oligosaccharides in intranuclear glycoproteins from HeLa cells.

Nonhistone proteins were extracted in 0.4 M NaCl from membrane-depleted nuclei of HeLa cells grown in the presence or the absence of [5,6-3H]fucose. Control experiments strongly suggest that most extracted proteins were indeed nuclear components. Several proteins, present in the 0.4 M NaCl nuclear extract, with M(r) ranging from 35,000 to 115,000 were identified on Western blots as fucosylated glycoproteins owing to their binding to the fucose-specific lectin, Ulex europeus agglutinin I. Results of experiments involving mild alkaline treatment and peptide N-glycosidase F digestion showed that the carbohydrate moieties of these fucosylated nuclear glycoproteins were N-linked to the polypeptide backbone. Analysis of the N-glycans revealed the presence of two populations of sialylated oligosaccharides on the basis of their relative molecular masses. The sensitivity of the high-M(r) oligosaccharides to endo-beta-galactosidase and their incorporation of [3H]glucosamine suggest that they could contain repeating N-acetyllactosamine units. [3H]Fucose incorporated into nuclei was confined to the nucleoli, as judged by autoradiography of sections cut through cells grown in the presence of [3H]fucose. Electron microscopy autoradiography showed that the fibrillar centers were never labeled, while silver grains were observed on the dense and the granular components of nucleoli. Taking into account of these data most nuclear fucosylated glycoproteins extracted in 0.4 M NaCl might be nucleolar ribonucleoproteins.     

Expression, isolation and purification of antibody fragments fused to maltose-binding protein in Escherichia coli]

We have fused the variable domains of a mouse antibody to the C-terminal end of the maltose-binding protein (malE), at the genetic level. The hybrid proteins were expressed in E. coli under control of the malEp promoter, and exported to the periplasm, at low temperature. They were purified by affinity chromatography on cross-linked amylose. When the two variable domains were fused together through a peptide link, the hybrid displayed similar affinity and specificity to the antigen as the native antibody.     

The gene for X-linked hydrocephalus maps to Xq28, distal to DXS52.

We report the study of five independent X-linked hydrocephalus (HSAS1) families with polymorphic DNA markers of the Xq28 region. A total of 58 individuals, including 7 living affected males and 22 obligate carriers, have been studied. Maximum lod score was 7.21 at theta = 2.40% for DXS52 (St14-1). A single recombination event was observed between this marker and the HSAS1 locus. Other markers studied were DXS296 (Z = 2.02 at theta = 2.5%), DXS304 (Z = 4.37 at theta = 7.8%), DXS74 (Z = 3.50 at theta = 0%), DXS15 (Z = 1.96 at theta = 5.7%), DXS134 (Z = 3.31 at theta = 0%), and F8C (Z = 5.79 at theta = 0%). These data confirm the localization of the HSAS1 gene to Xq28 and provide evidence for genetic homogeneity of this syndrome. In addition, examination of two obligate recombinant meioses along with multipoint linkage analysis supports the distal localization of the HSAS1 locus with respect to the DXS52 cluster. These observations are of potential interest for future studies aimed at HSAS1 gene characterization.     

Stereoselective biliary elimination of disopyramide and mono-N-desisopropyldisopyramide in humans.

The results of a previous pharmacokinetic study of disopyramide (DP) enantiomers in humans suggested that DP and/or mono-N-desisopropyldisopyramide (MND) may show stereoselective extrarenal elimination. Thus, the present study investigates the biliary elimination of DP and MND enantiomers in three patients who had undergone cholecystectomy for cholelithiasis. DP and MND enantiomers displayed biliary elimination. In both subjects, this elimination pathway showed the same characteristics: (1) biliary elimination of DP and MND was stereoselective, (2) the stereoselectivity was opposite to that observed for the metabolic and renal elimination pathways, i.e., the elimination of the (-)-(R)-enantiomer was higher than that of the (+)-(S)-enantiomer, and (3) biliary elimination of MND was higher than that of DP, for both enantiomers. Estimates of the relative contribution of the biliary clearance in the total clearance of DP and MND indicated that this elimination pathway was secondary, especially for DP. The biliary clearance (expressed as % of total clearance) was 1.9 to 4.0% for (-)-(R)-DP, 1.2 to 1.7% for (+)-(S)-DP, 7.8 to 22.9% for (-)-(R)-MND, and 5.2 to 10.5% for (+)-(S)-MND.     

Molecular characterization and genetic origin of the Brassica napus acetohydroxyacid synthase multigene family

Summary The Brassica napus rapeseed cultivar Topas contains an acetohydroxyacid synthase (AHAS) multigene family consisting of five members (AHAS 1–5). DNA sequence analysis indicate that AHAS1 and AHAS3 share extensive homology. They probably encode the AHAS enzymes essential for plant growth and development. AHAS2 has diverged significantly from AHAS1 and AHAS3 and has unique features in the coding region of the mature polypeptide, transit peptide and upstream non-coding DNA, which raises the possibility that it has a distinct function. AHAS4 and AHAS5 have interrupted coding regions and may be defective. The complexity of the AHAS multigene family in the allotetraploid species B. napus is much greater than reported for Arabidopsis thaliana and Nicotiana tabacum. Analysis of the presumptive progenitor diploid species B. campestris and B. oleracea indicated that AHAS2, AHAS3 and AHAS4 originate from the A genome, whereas AHAS1 and AHAS5 originate from the C genome. Further variation within each of the AHAS genes in these species was found.     

Differential accumulation of hydroxyproline-rich glycoproteins in bean root nodule cells infected with a wild-type strain or a C4-dicarboxylic acid mutant of Rhizobium leguminosarum bv. phaseoli

An antiserum raised against deglycosylated hydroxyproline-rich glycoproteins (HPGPs) from melon (Cucumis melo L.) was used to study the relationship between Rhizobium infection and induction of HRGPs in bean (Phaseolus vulgaris L.) root nodule cells infected with either the wild-type or a C₄-dicarboxylic acid mutant strain of Rhizobium leguminosarum bv. phaseoli. In effective nodules, where fixation of atmospheric dinitrogen is taking place, HRGPs were found to accumulate mainly in the walls of infected cells and in peribacteroid membranes surrounding groups of bacteroids. Internal ramifications of the peribacteroid membrane were also enriched in HRGPs whereas the peribacteroid space as well as the bacteroids themselves were free of these glycoproteins. In mutant-induced root nodules, HRGPs were specifically associated with the electron-dense, laminated structures formed in plastids as a reaction to infection by this mutant. The presence of HRGPs was also detected in the host cytoplasm. The aberrant distribution of HRGPs in infected cells of mutant-induced nodules likely reflects one aspect of the altered host metabolism in relation to peribacteroid-membrane breakdown. The possibility that the antiserum used for HRGP localization may have cross-reacted with ENOD 2 gene products is discussed in relation to amino-acid sequences and sites of accumulation.     

Mass-spectrometric determination of O2 and CO2 gas exchange in illuminated higher-plant cells

In order to estimate photosynthetic and respiratory rates in illuminated photoautotrophic cells of carnation (Dianthus caryophyllus L.), simultaneous measurements of CO₂ and O₂ gas exchange were performed using ¹⁸O₂, ¹³CO₂ and a mass-spectrometry technique. This method allowed the determination, and thus the comparison, of unidirectional fluxes of O₂ and CO₂. In optimum photosynthetic conditions (i.e. in the presence of high light and a saturating level of CO₂), the rate of CO₂ influx represented 75±5% of the rate of gross O₂ evolution. After a dark-to-light transition, the rate of CO₂ efflux was inhibited by 50% whereas the O₂-uptake rate was little affected. The effect of a recycling of respiratory CO₂ through photosynthesis on the exchange of CO₂ gas was investigated using a mathematical model. The confliction of the experimental data with the simulated gas-exchange rates strongly supported the view that CO₂ recycling was a minor event in these cells and could not be responsible for the observed inhibition of CO₂ efflux. On the basis of this assumption it was concluded that illumination of carnation cells resulted in a decrease of substrate decarboxylations, and that CO₂ efflux and O₂ uptake were not as tightly coupled in the light as in the dark. Furthermore, it could be calculated from the rate of gross photosynthesis that the chloroplastic electron-transport chain produced enough ATP in the light to account for the measured CO₂-uptake rate without involving cyclic transfer of electrons around PS I or mitochondrial supplementation.Abbreviations Chl chlorophyll - K_d permeability coefficient The authors thank Drs A. Vermeglio and P. Thibault, Dépt. de Biologie, CEN-Cadarache, St. Paul Lez Durance, France, for helpful discussions.     

Regulation of the maizerab17 gene promoter in transgenic heterologous systems

The maizerab17 gene is expressed in different plant parts in response to ABA and osmotic stress (J. Vilardellet al., Plant Mol Biol 14 (1990) 423–432). Here we demonstrate that 5 upstream sequences of therab17 gene confer the appropriate patterns of expression on the chloramphenicol acetyl transferase (CAT) reporter gene in transgenic tobacco plants, as well as in protoplasts derived from cultured rice cells. Specifically, a CAT construct containing a large 5 upstream fragment ofrab17 (–1330/+29) results in high levels of CAT activity in embryos, leaves and roots of transgenic plants subjected to water stress or ABA treatment. Transient expression assays in rice protoplasts transfected with CAT genes fused torab17 promoter deletions indicate that a 300 bp DNA fragment (–351/–102) is sufficient to confer ABA responsiveness upon the reporter gene. Furthermore, a 100 bp sequence (–219/–102) is capable of conferring ABA responsiveness upon a minimal promoter derived from the 35S CaMV promoter. Gel retardation experiments indicate that maize nuclear proteins bind to this fragment. This region of 100 bp contains a sequence (ACGTGGC) which has been identified as an abscisic acid response element in studies of other ABA-responsive plant genes.     

Anther-specific,developmentally regulated expression of genes encoding a new class of proline-rich proteins in sunflower

We have used RNA gel blot analysis to demonstrate the anther-specific expression of three genes in sunflower. Expression of these genes was first detected shortly before flower opening, which occurs sequentially on the sunflower inflorescence, and continues during pollination. In contrast, these genes are not expressed (or only weakly expressed) in a male-sterile line in which anther development aborts. In situ hybridization experiments showed that these genes are only expressed in the single cell layer of the sunflower anther epidermis. In the case of one of these genes, which codes for an abundant mRNA, we report the peptide sequences deduced from the sequence of two similar but non identical cDNAs. These proteins contain a potential signal peptide and are characterized by the presence of a proline-rich region which reads KPSTPAPPPPPP(PP)K. Our results also suggest that several proline-rich proteins of unknown functions are specifically synthesized during the maturation of anthers in sunflower.     

Use of ars18 based vectors to increase protein production in Yarrowia lipolytica.

The isolation of ars sequence from the yeast Yarrowia lipolytica has recently been reported (Fournier et al., 1991). Vectors containing ars18 have been used to increase homologous and heterologous protein production. Examples presented are the Yarrowia lipolytica alkaline extracellular protease (AEP), the porcine alpha 1-interferon and the bovine prochymosin. A 2- to 6-fold increase in the corresponding protein production was observed and in several cases it was established that it corresponded to the copy number of plasmid in the cell.