Regulation of the extracellular antioxidant selenoprotein plasma glutathione peroxidase (GPx-3) in mammalian cells

Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing extracellular antioxidant protein that catalyzes the reduction of hydrogen peroxide and lipid hydroperoxides. Selenoprotein expression involves the alternate recognition of a UGA codon as a selenocysteine codon and requires signals in the 3′-untranslated region (UTR), including a selenocysteine insertion sequence (SECIS), as well as specific translational cofactors. To ascertain regulatory determinants of GPx-3 expression and function, we generated recombinant GPx-3 (rGPX-3) constructs with various 3′-UTR, as well as a Sec73Cys mutant. In transfected Cos7 cells, the Sec73Cys mutant was expressed at higher levels than the wild type rGPx-3, although the wild type rGPx-3 had higher specific activity, similar to plasma purified GPx-3. A 3′-UTR with only the SECIS was insufficient for wild type rGPx-3 protein expression. Selenocompound supplementation and co-transfection with SECIS binding protein 2 increased wild type rGPx-3 expression. These results demonstrate the importance of translational mechanisms in GPx-3 expression.     

PHYLOGENY OF FOUR DINOPHYSIACEAN GENERA (DINOPHYCEAE,DINOPHYSIALES) BASED ON rDNA SEQUENCES FROM SINGLE CELLS AND ENVIRONMENTAL SAMPLES1

Dinoflagellates are a highly diverse and environmentally important group of protists with relatively poor resolution of phylogenetic relationships, particularly among heterotrophic species. We examined the phylogeny of several dinophysiacean dinoflagellates using samples collected from four Atlantic sites. As a rule, 3.5 kb of sequence including the nuclear ribosomal genes SSU, 5.8S, LSU, plus their internal transcribed spacer (ITS) 1 and 2 regions were determined for 26 individuals, including representatives of two genera for which molecular data were previously unavailable, Ornithocercus F. Stein and Histioneis F. Stein. In addition, a clone library targeting the dinophysiacean ITS2 and LSU sequences was constructed from bulk environmental DNA from three sites. Three phylogenetic trees were inferred from the data, one using data from this study for cells identified to genus or species (3.5 kb, 28 taxa); another containing dinoflagellate SSU submissions from GenBank and the 12 new dinophysiacean sequences (1.9 kb, 56 taxa) from this study; and the third tree combing data from identified taxa, dinophysiacean GenBank submissions, and the clone libraries from this study (2.1 kb, 136 taxa). All trees were congruent and indicated a distinct division between the genera Phalacroma F. Stein and Dinophysis Ehrenb. The cyanobionts containing genera Histioneis and Ornithocercus were also monophyletic. This was the largest molecular phylogeny of dinophysoid taxa performed to date and was consistent with the view that the genus Phalacroma may not be synonymous with Dinophysis.     

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.     

Immobilization of Burkholderia cepacia in polyurethane-based foams: embedding efficiency and effect on bacterial activity

Immobilization of the trichloroethylene-degrading bacterium Burkholderia cepacia was evaluated using hydrophilic polyurethane foam. The influence of several foam formulation parameters upon cell retention was examined. Surfactant type was a major determinant of retention; a lecithin-based compound retained more cells than pluronic- or silicone-based surfactants. Excessive amounts of surfactant led to increased washout of bacteria. Increasing the biomass concentration in the foam from 4.8 to 10.5% dry weight per wet weight of foam resulted in fewer cells being washed out. Embedding at reduced temperature did not significantly affect retention, while the use of a silane binding agent gave inconsistent results. The optimal formulation retained all but 0.2% of total embedded cells during passage of 2 L of water through columns containing 2 g of foam. All foam formulations tested reduced the culturability of embedded cells by several orders of magnitude, but O₂ consumption and CO₂ evolution rates of embedded cells were never less than 50% of those of free cells. Nutrient amendments stimulated an increase in cell volume and ribosomal activity in immobilized cells as indicated by hybridization studies using fluorescently labeled ribosomal probes. These results indicate that, although immobilized cells were mostly nonculturable, they were metabolically active and thus could be used for biodegradation of toxic compounds. Received 23 December 1996/ Accepted in revised form 13 March 1997     

Restriction-map variation associated with the G6PD polymorphism in natural populations of Drosophila melanogaster

Restriction-map variation was studied in 126 copies of the G6pd region in X chromosome lines of Drosophila melanogaster from North America, Europe, and Africa. Special attention was focused on the distribution of variation relative to the geographically variable polymorphism for two electrophoretic variants. Nucleotide heterozygosity as determined by eight six-cutter restriction enzymes for the 13-kb region is estimated, on the basis of the worldwide sample, to be 0.065%, which is the lowest value reported for any comparable region in the D. melanogaster genome. Significant linkage disequilibrium between electrophoretic alleles and restriction-site variation is observed for several sites. In contrast to published studies of other genetic regions, there are large insertions that reach significant frequencies and are found across considerable geographic distances. There is a clustering of this variation inside the first large intervening sequence of the G6PD gene.      

Molecular evolution of the MAGUK family in metazoan genomes

Background  

Development, differentiation and physiology of metazoans all depend on cell to cell communication and subsequent intracellular signal transduction. Often, these processes are orchestrated via sites of specialized cell-cell contact and involve receptors, adhesion molecules and scaffolding proteins. Several of these scaffolding proteins important for synaptic and cellular junctions belong to the large family of membrane-associated guanylate kinases (MAGUK). In order to elucidate the origin and the evolutionary history of the MAGUKs we investigated full-length cDNA, EST and genomic sequences of species in major phyla.     

Inhibition of Werner syndrome helicase activity by benzo[a]pyrene diol epoxide adducts can be overcome by replication protein A

RecQ helicases are believed to function in repairing replication forks stalled by DNA damage and may also play a role in the intra-S-phase checkpoint, which delays the replication of damaged DNA, thus permitting repair to occur. Since little is known regarding the effects of DNA damage on RecQ helicases, and because the replication and recombination defects in Werner syndrome cells may reflect abnormal processing of damaged DNA associated with the replication fork, we examined the effects of specific bulky, covalent adducts at N(6) of deoxyadenosine (dA) or N(2) of deoxyguanosine (dG) on Werner (WRN) syndrome helicase activity. The adducts are derived from the optically active 7,8-diol 9,10-epoxide (DE) metabolites of the carcinogen benzo[a]pyrene (BaP). The results demonstrate that WRN helicase activity is inhibited in a strand-specific manner by BaP DE-dG adducts only when on the translocating strand. These adducts either occupy the minor groove without significant perturbation of DNA structure (trans adducts) or cause base displacement at the adduct site (cis adducts). In contrast, helicase activity is only mildly affected by intercalating BaP DE-dA adducts that locally perturb DNA double helical structure. This differs from our previous observation that intercalating dA adducts derived from benzo[c]phenanthrene (BcPh) DEs inhibit WRN activity in a strand- and stereospecific manner. Partial unwinding of the DNA helix at BaP DE-dA adduct sites may make such adducted DNAs more susceptible to the action of helicase than DNA containing the corresponding BcPh DE-dA adducts, which cause little or no destabilization of duplex DNA. The single-stranded DNA binding protein RPA, an auxiliary factor for WRN helicase, enabled the DNA unwinding enzyme to overcome inhibition by either the trans-R or cis-R BaP DE-dG adduct, suggesting that WRN and RPA may function together to unwind duplex DNA harboring specific covalent adducts that otherwise block WRN helicase acting alone.