Naturally occurring variation in copia expression is due to both element (cis) and host (trans) regulatory variation.

Significant differences in levels of copia [Drosophila long terminal repeat (LTR) retrotransposon] expression exist among six species representing the Drosophila melanogaster species complex (D. melanogaster, Drosophila mauritiana, Drosophila simulans, Drosophila sechellia, Drosophila yakuba, and Drosophila erecta) and a more distantly related species (Drosophila willistoni). These differences in expression are correlated with major size variation mapping to putative regulatory regions of the copia 5' LTR and adjacent untranslated leader region (ULR). Sequence analysis indicates that these size variants were derived from a series of regional duplication events. The ability of the copia LTR-ULR size variants to drive expression of a bacterial chloramphenicol acetyltransferase reporter gene was tested in each of the seven species. The results indicate that both element-encoded (cis) and host-genome-encoded (trans) genetic differences are responsible for the variability in copia expression within and between Drosophila species. This finding indicates that models purporting to explain the dynamics and distribution of retrotransposons in natural populations must consider the potential impact of both element-encoded and host-genome-encoded regulatory variation to be valid. We propose that interelement selection among retrotransposons may provide a molecular drive mechanism for the evolution of eukaryotic enhancers which can be subsequently distributed throughout the genome by retrotransposition.

A rigid trans-spanning dinitrile ligand.

A rigid dinitrile ligand was synthesized from two xanthene units condensed to a naphthalene-1,4,5,8-diimide spacer. The rigidity and C shape of the ligand gave exclusively trans complexes with Pd(II), Ag(I), and Au(I). Evidence for complexation, coordination geometry, and stoichiometry was provided by a combination of 1H NMR, 19F NMR, and IR spectroscopy. The AuBF4 and PdCl2 complexes were shown to have a 1:1 (metal-to-ligand) stoichiometry and the AgBF4 complex was shown to have a 1:2 stoichiometry in solution. The preorganization of the dinitrile ligand resulted in complexes much more stable than their monodentate counterparts.

In vitro trans-splicing in Saccharomyces cerevisiae.

The interactions established at the 5'-splice site during spliceosome assembly are likely to be important for both precise recognition of the upstream intron boundary and for positioning this site in the active center of the spliceosome. Definition of the RNA-RNA and the RNA-protein interactions at the 5' splice site would be facilitated by the use of a small substrate amenable to modification during chemical synthesis. We describe a trans-splicing reaction performed in Saccharomyces cerevisiae extracts in which the 5' splice site and the 3' splice site are on separate molecules. The RNA contributing the 5' splice site is only 20 nucleotides long and was synthesized chemically. The trans-splicing reaction is accurate and has the same sequence, ATP, and Mg2+ requirements as cis-splicing. We also report how deoxy substitutions around the 5'-splice site affect trans-splicing efficiency.

Trans-dominant inhibitory human immunodeficiency virus type 1 protease monomers prevent protease activation and virion maturation.

Production of infectious human immunodeficiency virus (HIV) requires proper polyprotein processing by the dimeric viral protease. The trans-dominant inhibitory activity of a defective protease monomer with the active site Asp-25 changed to Asn was measured by transient transfection. A proviral plasmid that included the drug-selectable Escherichia coli gpt gene was used to deliver the wild-type (wt) or mutant proteases to cultured cells. Coexpression of the wt proviral DNA (HIV-gpt) with increasing amounts of the mutant proviral DNA (HIV-gpt D25N) results in a concomitant decrease in proteolytic activity monitored by in vivo viral polyprotein processing. The viral particles resulting from inactivation of the protease were mostly immature, consisting predominantly of unprocessed p55gag and p160gag-pol polyproteins. In the presence of HIV-1 gp160 env, the number of secreted noninfectious particles correlated with the presence of increasing amounts of the defective protease. Greater than 97% reduction in infectivity was observed at a 1:6 ratio of wt to defective protease DNA. This provides an estimate of the level of inhibition required for effectively preventing virion processing. Stable expression of the defective protease in monkey cells reduced the yield of infectious particles from these cells by 90% upon transfection with the wt proviral DNA. These results show that defective subunits of the viral protease exert a trans-dominant inhibitory effect resulting from the formation of catalytically compromised heterodimers in vivo, ultimately yielding noninfectious viral particles.

The hepatitis C virus NS3 serine proteinase and NS4A cofactor: establishment of a cell-free trans-processing assay.

The hepatitis C virus RNA genome encodes a long polyprotein that is proteolytically processed into at least 10 products. The order of these cleavage products in the polyprotein is NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. A serine proteinase domain located in the N-terminal one-third of nonstructural protein NS3 mediates cleavage at four downstream sites (the 3/4A, 4A/4B, 4B/5A, and 5A/5B sites). In addition to the proteinase catalytic domain, the NS4A protein is required for processing at the 4B/5A site but not at the 5A/5B site. These cleavage events are likely to be essential for virus replication, making the serine proteinase an attractive antiviral target. Here we describe an in vitro assay where the NS3-4A polyprotein, NS3, the serine proteinase domain (the N-terminal 181 residues of NS3), and the NS4A cofactor were produced by cell-free translation and tested for trans-processing of radiolabeled substrates. Polyprotein substrates, NS4A-4B or truncated NS5A-5B, were cleaved in trans by all forms of the proteinase, whereas NS4A was also required for NS4B-5A processing. Proteolysis was abolished by substitution mutations previously shown to inactivate the proteinase or block cleavage at specific sites in vivo. Furthermore, N-terminal sequence analysis established that cleavage in vitro occurred at the authentic 4A/4B site. Translation in the presence of microsomal membranes enhanced processing for some, but not all, proteinase-substrate combinations. Trans-processing was both time and temperature dependent and was eliminated by treatment with a variety of detergents above their critical micelle concentrations. Among many common proteinase inhibitors tested, only high (millimolar) concentrations of serine proteinase inhibitors tosyllysyl chloromethyl ketone and 4-(2-aminoethyl)benzenesulfonyl fluoride inactivated the NS3 proteinase. This in vitro assay should facilitate purification and further characterization of the viral serine proteinase and identification of molecules which selectively inhibit its activity.

Specific binding of RNA polymerase II to the human immunodeficiency virus trans-activating region RNA is regulated by cellular cofactors and Tat.

The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression in response to Tat is dependent on an element downstream of the HIV-1 transcriptional initiation site designated the trans-activating region (TAR). TAR forms a stable stem-loop RNA structure in which a 3-nt bulge structure and a 6-nt loop structure are important for Tat activation. In the absence of Tat, the HIV-1 promoter generates so-called short or nonprocessive transcripts terminating at +60, while in the presence of Tat the synthesis of these short transcripts is markedly decreased and transcripts that extend through the 9.0-kb HIV-1 genome are synthesized. Tat effects on transcriptional elongation are likely due to alterations in the elongation properties of RNA polymerase II. In this study we demonstrated that a set of cellular cofactors that modulate the binding of the cellular protein TRP-185 to the TAR RNA loop sequences also functioned to markedly stimulate the specific binding of hypophosphorylated (IIa) and hyperphosphorylated (IIo) RNA polymerase II to TAR RNA. The concentrations of RNA polymerase II required for this interaction with TAR RNA were similar to those required to initiate in vitro transcription from the HIV-1 long terminal repeat. RNA gel retardation analysis with wild-type and mutant TAR RNAs indicated that the TAR RNA loop and bulge sequences were critical for the binding of RNA polymerase II. The addition of wild-type but not mutant Tat protein to gel retardation analysis with TAR RNA and RNA polymerase II resulted in the loss of binding of RNA polymerase II binding to TAR RNA. These results suggest that Tat may function to alter RNA polymerase II, which is paused due to its binding to HIV-1 TAR RNA with resultant stimulation of its transcriptional elongation properties.

Resistance to human immunodeficiency virus type 1 infection conferred by transduction of human peripheral blood lymphocytes with ribozyme, antisense, or polymeric trans-activation response element constructs.

Human peripheral blood lymphocytes (PBLs) were transduced with a number of recombinant retroviruses including RRz2, an LNL6-based virus with a ribozyme targeted to the human immunodeficiency virus (HIV) tat gene transcript inserted within the 3' region of the neomycin-resistance gene; RASH5, and LNHL-based virus containing an antisense sequence to the 5' leader region of HIV-1 downstream of the human cytomegalovirus promoter; and R20TAR, an LXSN-based virus with 20 tandem copies of the HIV-1 trans-activation response element sequence driven by the Moloney murine leukemia virus long terminal repeat. After G418 selection, transduced PBLs were challenged with the HIV-1 laboratory strain IIIB and a primary clinical isolate of HIV-1, 82H. Results showed that PBLs from different donors could be transduced and that this conferred resistance to HIV-1 infection. For each of the constructs, a reduction of approximately 70% in p24 antigen level relative to the corresponding control-vector-transduced PBLs was observed. Molecular analyses showed constitutive expression of all the transduced genes from the retroviral long terminal repeat, but no detectable transcript was seen from the internal human cytomegalovirus transcript was seen from the internal human cytomegalovirus promoter for the antisense construct. Transduction of, and consequent transgene expression in, PBLs did not impact on the surface expression of either CD4+/CD8+ (measured by flow cytometry) or on cell doubling time (examined by [3H]thymidine uptake). These results indicate the potential utility of these anti-HIV-1 gene therapeutic agents and show the preclinical value of this PBL assay system.

Enhancer-dependent interaction between 5' and 3' splice sites in trans.

Splice-site selection and alternative splicing of nuclear pre-mRNAs can be controlled by splicing enhancers that act by promoting the activity of upstream splice sites. Here we show that RNA molecules containing a 3' splice site and enhancer sequence are efficiently spliced in trans to RNA molecules containing normally cis-spliced 5' splice sites or to normally trans-spliced spliced leader RNAs from lower eukaryotes. In addition, we show that this reaction is stimulated by (Ser + Arg)-rich splicing factors that are known to promote protein-protein interactions in the cis-splicing reaction. Thus, splicing enhancers facilitate the assembly of protein complexes on RNAs containing a 3' splice site, and this complex is sufficiently stable to functionally interact with 5' splice sites located on separate RNAs. This trans-splicing is mediated by interactions between (Ser + Arg)-rich splicing factors bound to the enhancer and general splicing factors bound to the 5' and 3' splice sites. These same interactions are likely to play a crucial role in alternative splicing and splice-site selection in cis.

The bend in RNA created by the trans-activation response element bulge of human immunodeficiency virus is straightened by arginine and by Tat-derived peptide.

The trans-activation response element (TAR) found near the 5' end of the viral RNA of the human immunodeficiency virus contains a 3-nt bulge that is recognized by the virally encoded trans-activator protein (Tat), an important mediator of transcriptional activation. Insertion of the TAR bulge into double-stranded RNA is known to result in reduced electrophoretic mobility, suggestive of a bulge-induced bend. Furthermore, NMR studies indicate that Arg causes a change in the structure of the TAR bulge, possibly reducing the bulge angle. However, neither of these effects has been quantified, nor have they been compared with the effects of the TAR-Tat interaction. Recently, an approach for the quantification of bulge-induced bends has been described in which hydrodynamic measurements, employing the method of transient electric birefringence, have yielded precise estimates for the angles of a series of RNA bulges, with the angles ranging from 7 degrees to 93 degrees. In the current study, transient electric birefringence measurements indicate that the TAR bulge introduces a bend of 50 degrees +/- 5 degrees in the absence of Mg2+. Addition of Arg leads to essentially complete straightening of the helix (to < 10 degrees) with a transition midpoint in the 1 mM range. This transition demonstrates specificity for the TAR bulge: no comparable transition was observed for U3 or A3 (control) bulges with differing flanking sequences. An essentially identical structural transition is observed for the Tat-derived peptide, although the transition midpoint for the latter is near 1 microM. Finally, low concentrations of Mg2+ alone reduce the bend angle by approximately 50%, consistent with the effects of Mg2+ on other pyrimidine bulges. This last observation is important in view of the fact that most previous structural/binding studies were performed in the absence of Mg2+.

Coenzyme Q reductase from liver plasma membrane: purification and role in trans-plasma-membrane electron transport.

A specific requirement for coenzyme Q in the maintenance of trans-plasma-membrane redox activity is demonstrated. Extraction of coenzyme Q from membranes resulted in inhibition of NADH-ascorbate free radical reductase (trans electron transport), and addition of coenzyme Q10 restored the activity. NADH-cytochrome c oxidoreductase (cis electron transport) did not respond to the coenzyme Q status. Quinone analogs inhibited trans-plasma-membrane redox activity, and the inhibition was reversed by coenzyme Q. A 34-kDa coenzyme Q reductase (p34) has been purified from pig-liver plasma membranes. The isolated enzyme was sensitive to quinone-site inhibitors. p34 catalyzed the NADH-dependent reduction of coenzyme Q10 after reconstitution in phospholipid liposomes. When plasma membranes were supplemented with extra p34, NADH-ascorbate free radical reductase was activated but NADH-cytochrome c oxidoreductase was not. These results support the involvement of p34 as a source of electrons for the trans-plasma-membrane redox system oxidizing NADH and support coenzyme Q as an intermediate electron carrier between NADH and the external acceptor ascorbate free radical.

Trans-Pacific migrations of the loggerhead turtle (Caretta caretta) demonstrated with mitochondrial DNA markers.

Juvenile loggerhead turtles (Caretta caretta) have recently been documented in the vicinity of Baja California and thousands of these animals have been captured in oceanic fisheries of the North Pacific. The presence of loggerhead turtles in the central and eastern North Pacific is a prominent enigma in marine turtle distribution because the nearest documented nesting concentrations for this species are in Australia and Japan, over 10,000 km from Baja California. To determine the origin of the Baja California feeding aggregate and North Pacific fishery mortalities, samples from nesting areas and pelagic feeding aggregates were compared with genetic markers derived from mtDNA control region sequences. Overall, 57 of 60 pelagic samples (95%) match haplotypes seen only in Japanese nesting areas, implicating Japan as the primary source of turtles in the North Pacific Current and around Baja California. Australian nesting colonies may contribute the remaining 5% of these pelagic feeding aggregates. Juvenile loggerhead turtles apparently traverse the entire Pacific Ocean, approximately one-third of the planet, in the course of developmental migrations, but mortality in high-seas fisheries raises concern over the future of this migratory population.

Inactivation of ethanol-inducible cytochrome P450 and other microsomal P450 isozymes by trans-4-hydroxy-2-nonenal, a major product of membrane lipid peroxidation.

Of the microsomal P450 cytochromes, the ethanol-inducible isoform, P450 2E1, is believed to be predominant in leading to oxidative damage, including the generation of radical species that contribute to lipid peroxidation, and in the reductive beta-scission of lipid hydroperoxides to give hydrocarbons and aldehydes. In the present study, the sensitivity of a series of P450s to trans-4-hydroxy-2-nonenal (HNE), a known toxic product of membrane lipid peroxidation, was determined. After incubation of a purified cytochrome with HNE, the other components of the reconstituted system (NADPH-cytochrome P450 reductase, phosphatidylcholine, and NADPH) were added, and the rate of oxygenation of 1-phenylethanol to yield acetophenone was assayed. Inactivation occurs in a time-dependent and HNE concentration-dependent manner, with P450s 2E1 and 1A1 being the most sensitive, followed by isoforms 1A2, 3A6, and 2B4. At an HNE concentration of 0.24 microM, which was close to the micromolar concentration of the enzyme, four of the isoforms were significantly inhibited, but not P450 2B4. In other experiments, the reductase was shown to be only relatively weakly inactivated by HNE. P450s 2E1 and 2B4 in microsomal membranes from animals induced with acetone or phenobarbital, respectively, are as readily inhibited as the purified forms. Evidence was obtained that the P450 heme is apparently not altered and the sulfur ligand is not displaced, that substrate protects against HNE, and that the inactivation is reversed upon dialysis. Higher levels of reductase or substrate do not restore the activity of inhibited P450 in the catalytic assay. Our results suggest that the observed inhibition of the various P450s is of sufficient magnitude to cause significant changes in the metabolism of foreign compounds such as drugs and chemical carcinogens by the P450 oxygenase system at HNE concentrations that occur in biological membranes. In view of the known activities of P450 2E1 in generating lipid hydroperoxides and in their beta-scission, its inhibition by this product of membrane peroxidation may provide a negative regulatory function.

The representation of the world in national Arab news agencies: an exploration of (trans)national networks in the official Arab media

The purpose of this paper is to draw a map of the representation of the world and of Arab states as reflected by the countries of the region. To do so, we have analysed the news (4,093 news randomly collected on February and August 2005) produced by the governments of the Arab states through their national news agencies. Several regional and world maps had been constructed to show the official Arab representation of the World, the Arab countries conflict agenda, the persistence of colonial ties (with the European metropolis) and the emergence of new relationships (Asian countries). The representation of the world that appeared in the analysis focuses its interest on the USA, the war in Iraq, the Israel-Palestine conflict, the United Kingdom, France, and Iran. The Arab regional powers organise the flow of information (Saudi Arabia and Egypt) and the colonial past determines the current structure of communication (French-speaking bloc and English-speaking bloc).

Bifunctional primary amine 2-aminobenzimidazole organocatalyst anchored to trans-cyclohexane-1,2-diamine in enantioselective conjugate additions of aldehydes

Bifunctional chiral primary amine 8 containing an (S,S)-trans-cyclohexane-1,2-diamine scaffold and a 2-benzimidazole unit is used as a general organocatalyst for the Michael addition of α,α-branched aldehydes to nitroalkenes and maleimides. The reactions take place, with 20 mol % of catalyst in dichloromethane at rt for nitroalkenes and with 15 mol % catalyst loading in toluene at 10 °C for maleimides, in good yields and enantioselectivities. DFT calculations demonstrate the bifunctional character of this organocatalyst activating the aldehyde by enamine formation and the Michael acceptor by double hydrogen bonding.