Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “reverse engineering”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,765 records · Page 98Linked to original sources
PubMed2001

Electrical control of spin coherence in semiconductor nanostructures.

The processing of quantum information based on the electron spin degree of freedom requires fast and coherent manipulation of local spins. One approach is to provide spatially selective tuning of the spin splitting--which depends on the g-factor--by using magnetic fields, but this requires their precise control at reduced length scales. Alternative proposals employ electrical gating and spin engineering in semiconductor heterostructures involving materials with different g-factors. Here we show that spin coherence can be controlled in a specially designed AlxGa1-xAs quantum well in which the Al concentration x is gradually varied across the structure. Application of an electric field leads to a displacement of the electron wavefunction within the quantum well, and because the electron g-factor varies strongly with x, the spin splitting is therefore also changed. Using time-resolved optical techniques, we demonstrate gate-voltage-mediated control of coherent spin precession over a 13-GHz frequency range in a fixed magnetic field of 6 T, including complete suppression of precession, reversal of the sign of g, and operation up to room temperature.

Journal Article↗
PubMed2006

Identification of dominant mutations that confer increased aluminium tolerance through mutagenesis of the Al-sensitive Arabidopsis mutant, als3-1.

Aluminium (Al) toxicity is a global agricultural problem that occurs in acid soil environments and severely limits root growth and crop productivity. The isolation and characterization of a gene, ALS3, which is absolutely required by Arabidopsis seedlings for growth in an Al-toxic environment was reported previously. Since the als3-1 loss-of-function mutant has extreme root growth inhibition even in the presence of very low levels of Al, it was an excellent candidate for using a mutagenesis approach to identify suppressor mutations that would increase either Al resistance or tolerance in Arabidopsis roots. EMS-mutagenized als3-1 seedlings were screened for mutants that could sustain root growth in an Al-containing environment that is highly toxic to als3-1 but not Col-0 wt. This approach resulted in identification of 12 strong suppressor mutants that reversed the als3-1 phenotype and grew as well or better than Col-0 wt in the presence of high levels of Al. Subsequent analysis of three representative suppressor mutants revealed that the phenotype of each probably arises from dominant gain-of-function mutations at the same locus. Detailed analysis of one of these, alt1-1 (Al tolerant), suggests that this mutation positively impacts Al resistance in a manner dependent on pH adjustment rather than enhanced Al exclusion. Identification of these suppressor mutations, should not only further elucidate the biochemical and molecular mechanisms underlying Al toxicity and tolerance but also will develop a collection of mutations that may be useful for engineering crop plants that can grow and thrive in Al-toxic environments.

ATP-Binding Cassette Transporters↗
PubMedDate not supplied

Endogenous ADP-ribosylation for eukaryotic elongation factor 2: evidence of two different sites and reactions.

Eukaryotic elongation factor 2 can undergo ADP-ribosylation in the absence of diphtheria toxin under the action of an endogenous transferase. The investigation which aimed to gain insight into the nature of endogenous ADP-ribosylation revealed that this reaction may be, in some cases, due to covalent binding of free ADP-ribose to elongation factor 2. Binding of free ADP-ribose, and NAD- and endogenous transferase-dependent ADP-ribosylation were suggested to be distinct reactions by different findings. Free ADP-ribose could bind to elongation factor 2 previously subjected to ADP-ribosylation by diphtheria toxin or endogenous transferase. The binding of free ADP-ribose was inhibited by neutral NH2OH, L-lysine and picrylsulfonate, whereas endogenous ADP-ribosyltransferase was inhibited by NAD glycohydrolase inhibitors and L-arginine. The ADP-ribosyl-elongation factor 2 adduct which formed upon binding of free ADP-ribose was resistant to neutral NH2OH, but decomposed almost completely upon treatment with NaOH. The product of endogenous transferase-dependent ADP- ribosylation was partially resistant to NH2OH and NaOH treatment. Moreover, this reaction was reversed in the presence of diphtheria toxin and nicotinamide. Both types of endogenous ADP-ribosylation gave rise to inhibition of polyphenylalanine synthesis. This study thus provides evidence for the presence of two different types of endogenous ADP-ribosylation of eukaryotic elongation factor 2. The respective sites involved in these reactions are distinct from one another as well as from diphthamide, the site of attack by diphtheria toxin.

ADP Ribose Transferases↗
PubMed2005

Functional expression of a multidrug P-glycoprotein transporter of Leishmania.

P-glycoprotein (Pgp) transporters play an important role in multidrug resistance in eukaryotic cells and in protozoan parasites such as Leishmania. To search for new reversal agents of the Leishmania tropica Pgp, we developed a screening assay using the Baculovirus-insect cell expression system. We demonstrated a MgATP-dependent, vanadate-sensitive transport of Hoechst 33342 in membrane preparations of Sf9 insect cells expressing Pgp. We have found that dihydro-beta-agarofuran sesquiterpenes from Maytenus cuzcoina inhibited Hoechst 33342 transport that correlates with their reversal effect in a multidrug-resistant L. tropica line overexpressing Pgp. The results suggest that Sf9 cell membrane Hoechst 33342 transport system represents an efficient tool for examining the interactions of Leishmania Pgp with pharmacological agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Improvement of neurological deficits by intracerebral transplantation of human adipose tissue-derived stromal cells after cerebral ischemia in rats.

Mesenchymal stem cells can be expanded rapidly in vitro and differentiated into multiple mesodermal cell types. In addition, their differentiation into neuron-like cells expressing markers typical for mature neurons has been reported. We isolated human adipose tissue stromal cells (hATSCs) from human liposuction tissues and induced neural differentiation with azacytidine. Following neural induction, hATSCs changed toward neural morphology and displayed expression of MAP2 and GFAP. hATSCs, which were labeled with LacZ adenovirus, were injected into the lateral ventricle of the rat brain. Transplanted cells migrated to various parts of the brain, and ischemic brain injury by middle cerebral artery occlusion (MCAo) increased their migration to the injured cortex. Some of the transplanted cells expressed MAP2 and GFAP. Transplantation of hATSCs improved functional deficits in ischemic brain injury induced by MCAo. Intracerebral grafting of BDNF-transduced hATSCs significantly improved motor recovery of functional deficits in MCAo rats. These data indicate that transplanted hATSCs survive, migrate, and improve functional recovery after stroke and that genetically engineered hATSCs can express biologically active gene products and, therefore, can function as effective vehicles for therapeutic gene transfer to the brain.

Adenoviridae↗
PubMed2004

Responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines.

BACKGROUND: In response to the emergence of severe infection capable of rapid global spread, WHO will issue a pandemic alert. Such alerts are rare; however, on Feb 19, 2003, a pandemic alert was issued in response to human infections caused by an avian H5N1 influenza virus, A/Hong Kong/213/03. H5N1 had been noted once before in human beings in 1997 and killed a third (6/18) of infected people. The 2003 variant seemed to have been transmitted directly from birds to human beings and caused fatal pneumonia in one of two infected individuals. Candidate vaccines were sought, but no avirulent viruses antigenically similar to the pathogen were available, and the isolate killed embryonated chicken eggs. Since traditional strategies of vaccine production were not viable, we sought to produce a candidate reference virus using reverse genetics. METHODS: We removed the polybasic aminoacids that are associated with high virulence from the haemagglutinin cleavage site of A/Hong Kong/213/03 using influenza reverse genetics techniques. A reference vaccine virus was then produced on an A/Puerto Rico/8/34 (PR8) backbone on WHO-approved Vero cells. We assessed this reference virus for pathogenicity in in-vivo and in-vitro assays. FINDINGS: A reference vaccine virus was produced in Good Manufacturing Practice (GMP)-grade facilities in less than 4 weeks from the time of virus isolation. This virus proved to be non-pathogenic in chickens and ferrets and was shown to be stable after multiple passages in embryonated chicken eggs. INTERPRETATION: The ability to produce a candidate reference virus in such a short period of time sets a new standard for rapid response to emerging infectious disease threats and clearly shows the usefulness of reverse genetics for influenza vaccine development. The same technologies and procedures are currently being used to create reference vaccine viruses against the 2004 H5N1 viruses circulating in Asia.

Animals↗
PubMed2004

Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord.

The Wharton's jelly of the umbilical cord contains mucoid connective tissue and fibroblast-like cells. Using flow cytometric analysis, we found that mesenchymal cells isolated from the umbilical cord express matrix receptors (CD44, CD105) and integrin markers (CD29, CD51) but not hematopoietic lineage markers (CD34, CD45). Interestingly, these cells also express significant amounts of mesenchymal stem cell markers (SH2, SH3). We therefore investigated the potential of these cells to differentiate into cardiomyocytes by treating them with 5-azacytidine or by culturing them in cardiomyocyte-conditioned medium and found that both sets of conditions resulted in the expression of cardiomyocyte markers, namely N-cadherin and cardiac troponin I. We also showed that these cells have multilineage potential and that, under suitable culture conditions, are able to differentiate into cells of the adipogenic and osteogenic lineages. These findings may have a significant impact on studies of early human cardiac differentiation, functional genomics, pharmacological testing, cell therapy, and tissue engineering by helping to eliminate worrying ethical and technical issues.

Adipocytes↗
PubMed2000

The cyclic contryphan motif CPxXPXC, a robust scaffold potentially useful as an omega-conotoxin mimic.

Contryphan-R, from venom of the cone-shell Conus radiatus, represents a novel cyclic peptide scaffold onto which residues may be grafted to mimic unrelated protein surfaces. Three substitutions were made at the x and X positions of the disulfide-bridged motif CPxXPXC, where X and x represent any L- and D-handed residues, respectively, P represents proline or hydroxyproline, and C a half-cystine. These substitutions were designed to mimic part of the pharmacophore of the unrelated globular polypeptide omega-conotoxin GVIA, which blocks N-type calcium channels. The structure of this engineered contryphan, YNK-contryphan-R ([D-Tyr4, Asn5, Lys7]contryphan-R), is shown to be similar to that of native contryphan-R (Pallaghy et al., Biochemistry, 1999, Vol. 38, pp. 13553-13559), confirming that the scaffold is robust with respect to the multiple substitutions. In particular, the alpha-beta bond vectors characterising the orientation of the side chains relative to the backbone are similar in contryphan-R, YNK-contryphan-R, and omega-conotoxin GVIA, which is the required result for a scaffold-based approach to molecular design. The solution structure of YNK-contryphan-R has an N-terminal, nonhydrogen-bonded, chain reversal centered on Hyp3-D-Trp4, and a C-terminal type I beta-turn. A minor form due to cis-trans isomerism of the Hyp2-Cys3 peptide bond is present in YNK-contryphan-R in a larger proportion than in contryphan-R. It is evident, particularly from the (3)J(HalphaHN) coupling constants, that YNK-contryphan-R is more flexible than contryphan-R, probably due to the absence in YNK-contryphan-R of the Pro-Trp packing present in the native molecule. Nevertheless, the structure confirms that cyclic peptide molecular designs can achieve the intended conformations.

Amino Acid Motifs↗
PubMed2005

Design and expression of peptide antibiotic hPAB-beta as tandem multimers in Escherichia coli.

Peptide antibiotics are small peptides encoded by organism genomic DNA. They are recognized to play important roles in the innate host defense of most living organisms. The growing resistance of bacteria to conventional antibiotics and the need for discovery of new antibiotics have stimulated great interest in the development of peptide antibiotics as human therapeutics. However, preparation of peptide antibiotics at a large scale is a great challenge in developing these commercial products. In this study, tandem repeat multimers of peptide antibiotic hPAB-beta were designed and the recombinant plasmids containing one to eight copies of hPAB-beta gene were generated. Eight genetic engineered bacteria harboring pQE-hPAB-beta1-8 recombinant were able to express the repetitive hPAB-beta multimers of interest in inclusion bodies, respectively. The expressed proteins could reach 2.6-28% of the total proteins. The hPAB-beta trimer construct was selected out for the subsequent study based on its higher expression level (27.8%), which yields in wet cell weights (3.15+/-0.45 g/l) and the fusion protein inclusion bodies was able to completely dissolve in 8 M urea. The tandem trimers could easily be captured by Ni-NTA affinity chromatography and cleaved into monomers by hydroxylamine. Then, the monomer hPAB-beta of interest was purified to 95% homogeneity by reverse phase chromatography and gel filtration. The final yield of purified recombinant monomer hPAB-beta was 680+/-12 mg/100 g wet cells. The minimum inhibitory concentrations (MICs) of the purified recombinant hPAB-beta against type or clinical strains of microorganisms were about 31-250 microg/ml and these results showed that the recombinant hPAB-beta could retain its bioactivity.

Amino Acid Sequence↗
PubMed2005

Ion channels and intracellular signaling proteins as potential targets for novel therapeutics for addictive and depressive disorders.

Modern neuroscience is placing increased emphasis on understanding how the activity of ion channels and intracellular molecules in the central nervous system affect behavior. An improved understanding of the brain and the biological bases of conditions such as addictive and depressive disorders is important because it should ultimately enable the design of innovative treatments for these conditions. The development of rational therapies that are based on knowledge of what is different about the addicted or depressed brain would be an important advance. Here, we describe how multidisciplinary studies that combine numerous approaches (behavioral analysis, physiology, molecular biology, and genetic engineering) have begun to provide important advances that have helped to establish causal relationships between the pathophysiology of these conditions and behavior. This type of work has identified classes of molecules on the outside of cells (receptors and ion channels) that receive signals from other cells and initiate cellular events that have short-term effects on the neurons. It has also identified other classes of molecules that are inside of cells (signal transduction molecules) that can have immediate effects on cell function (e.g., ion channel phosphorylation), as well longer term effects (alterations in protein expression) that affect the ways in which neurons function within circuits. Innovative treatments that block, negate, or even reverse the extracellular or intracellular neuroadaptations resulting from exposure to drugs of abuse or stress might be more effective than current therapies because they directly target the molecular processes that cause maladaptive behaviors.

Animals↗
PubMedDate not supplied

Chemical approaches to the study of protein tyrosine kinases and their implications for mechanism and inhibitor design.

Protein tyrosine kinases are critical enzymes for signal transduction. Using C-terminal Src kinase (Csk) as a model system, we discuss progress in three main areas. First, we describe our efforts to measure the transition state of the reaction using peptide substrates containing fluorotyrosine analogs. It is shown that the Brønsted nucleophile coefficient for the reaction is near zero (similar to the nonenzymatic reaction) and the required nucleophile is the neutral phenol (rather than the more chemically reactive phenoxide anion). By studying the kinase reaction in the reverse direction, a Brønsted leaving group coefficient of -0.3 was measured, indicative of protonation of the departing phenol in the transition state. Taken together, these results strongly support a dissociative transition state mechanism for the kinase. These findings set constraints on the design of transition state analog inhibitors. Second, we describe efforts toward defining the specificity of Csk for peptide and protein substrates. The main findings are that local amino acids surrounding a phosphorylated tyrosine can influence recognition, but that long-range interactions probably are more important in a physiologic protein substrate. These findings underscore the complexities in how protein kinases select protein substrates. Third, we describe a new method in protein engineering that has been applied to the study of protein kinases. The method, expressed protein ligation, allows a general approach for ligating synthetic peptides to recombinant proteins. Using expressed protein ligation, obtaining site-specifically phosphorylated proteins and proteins with the incorporation of biophysical probes becomes relatively straightforward. We have used this method to generate a tail phosphorylated, conformationally altered Csk that showed an unexpected increase in kinase activity.

Chemistry, Pharmaceutical↗
PubMed2003

Enhanced intracellular availability and survival of hammerhead ribozymes increases target ablation in a cellular model of osteogenesis imperfecta.

Antisense hammerhead ribozymes have the capability to cleave complementary RNA in a sequence-dependent manner. In osteogenesis imperfecta, a genetic disorder of connective tissue, mutant collagen type I has been shown to participate in but not sustain formation of the triple helix. Selective ablation of mutant collagen gene transcript could potentially remove the mutant gene product and reverse the dominant-negative effect exerted by the abnormal protein. In earlier studies we showed that the hammerhead ribozyme Col1A1Rz547 selectively cleaved a mutant Col1A1 gene transcript in a murine calvarial osteoblast cell line. In order to test the possible therapeutic efficacy of this approach, a dramatic downregulation of the mutant transcript must be achieved, a function directly related to high steady-state level of intracellular ribozyme. We report significantly enhanced expression of Col1A1Rz547 by vaccinia T7 polymerase following infection with an attenuated T7-pol vaccinia virus as shown both by the intracellular level of the ribozyme and the cleavage of the mutant Col1A1 gene transcript. We also describe the engineering of a multimeric ribozyme construct comprising eight subunits, which can self-cleave to monomers. These studies suggest the potential use of multimeric ribozymes expressed by a vaccinia-based system in the therapy of a variety of disorders.

Animals↗
PubMed2002

In vitro evolution of a polyhydroxybutyrate synthase by intragenic suppression-type mutagenesis.

In vitro evolution was applied to obtain highly active mutants of Ralstonia eutropha polyester synthase (PhbC(Re)), which is a key enzyme catalyzing the formation of polyhydroxybutyrate (PHB) from (R)-3-hydroxybutyryl-CoA (3HB-CoA). To search for beneficial mutations for activity improvement of this enzyme, we have conducted multi-step mutations, including activity loss and intragenic suppression-type activity reversion. Among 259 revertants, triple mutant E11S12 was obtained as the most active one via PCR-mediated secondary mutagenesis from mutant E11 with a single mutation (Ser to Pro at position 80), which exhibited reduced activity (as low as 27% of the wild-type level) but higher thermostability compared to the wild-type enzyme. Mutant E11S12 exhibited up to 79% of the wild-type enzyme activity. Mutation separation of E11S12 revealed that the replacement of Phe by Ser at position 420 (F420S), located in a highly conserved alpha/beta hydrolase fold region, of the E11S12 mutant contributes to the improvement of the enzyme activity. A purified sample of the genetically engineered mutant, termed E11S12-1, with the F420S mutation alone was found to exhibit a 2.4-fold increase in specific activity toward 3HB-CoA, compared to the wild-type.

Acyltransferases↗
PubMed2006

Regulation of cell-matrix adhesion dynamics and Rac-1 by integrin linked kinase.

Extracellular matrix (ECM) receptors of the integrin family initiate changes in cell shape and motility by recruiting signaling components that coordinate these events. Integrin-linked kinase (ILK) is one such partner of beta1 integrins that participates in dynamic rearrangement of cell-matrix adhesions and cell spreading by mechanisms that are not well understood. To further elucidate the role of ILK in these events, we engineered a chimeric molecule comprising ILK fused to a membrane-targeted green fluorescent protein (ILK-GFP-F). ILK-GFP-F is highly enriched in cell-matrix adhesions, and its expression in fibroblasts leads to an accumulation of focal adhesions (2-5 microm) and elongated adhesions (>5 microm). ILK-GFP-F enhances cell spreading on fibronectin and induces a constitutive increase in the levels of GTP-bound Rac-1. Conversely, ILK knock-down by siRNA transfection decreases active Rac-1. Endogenous ILK was found to associate with PKL (paxillin kinase linker) and the Rac/Cdc42 guanine nucleotide exchange factor betaPIX. Further, expression of a dominant negative betaPIX mutant reversed the increase in active Rac-1 levels of ILK-GFP-F-expressing cells, thus placing betaPIX in the pathway leading from ILK to Rac-1 activation. However, expression of constitutively active Rac only partially restores the spreading defects of ILK-depleted cells, suggesting that an additional ILK-dependent signal is required for cell spreading.

Animals↗
PubMed1988

A thermal surface phenomenon in the rabbit lung: possible basis for the conversion of heat into work.

1. Surfactant and fluid expressed from rabbit lungs has been used to form films on a simple Maxwell frame having a ratio of area-to-thermal capacity comparable to that existing in vivo. 2. When the area was increased by 2:1, the temperature fell by 5.6 degrees C and returned to 37 degrees C upon contraction as recorded by an infra-red thermometer with no thermal capacity. 3. The experiment was repeated upon thin lung sections when the temperature fall was 2.4 degrees C and was again reversed upon contraction. 4. When surfactant was removed from the surface of those sections, the temperature changed in the opposite direction, indicating that surfactant was responsible for the changes described above. 5. This surface energy phenomenon is discussed in relation to the common assumption that the lung operates under isothermal conditions when it may explain some of the discrepancies between studies of lung mechanics over different time scales. 6. Since the results are compatible with the inversion of hysteresis loops for surface tension versus the area of surfactant monolayers cycled to steady state in previous studies, it is speculated that heat, e.g. waste metabolic heat, might be the energy source for this surface 'engine' if, indeed, it is contributing to the work available for breathing.

Animals↗
PubMed2004

Cleavage between replicase proteins p28 and p65 of mouse hepatitis virus is not required for virus replication.

The p28 and p65 proteins of mouse hepatitis virus (MHV) are the most amino-terminal protein domains of the replicase polyprotein. Cleavage between p28 and p65 has been shown to occur in vitro at cleavage site 1 (CS1), (247)Gly downward arrow Val(248), in the polyprotein. Although critical residues for CS1 cleavage have been mapped in vitro, the requirements for cleavage have not been studied in infected cells. To define the determinants of CS1 cleavage and the role of processing at this site during MHV replication, mutations and deletions were engineered in the replicase polyprotein at CS1. Mutations predicted to allow cleavage at CS1 yielded viable virus that grew to wild-type MHV titers and showed normal expression and processing of p28 and p65. Mutant viruses containing predicted noncleaving mutations or a CS1 deletion were also viable but demonstrated delayed growth kinetics, reduced peak titers, decreased RNA synthesis, and small plaques compared to wild-type controls. No p28 or p65 was detected in cells infected with predicted noncleaving CS1 mutants or the CS1 deletion mutant; however, a new protein of 93 kDa was detected. All introduced mutations and the deletion were retained during repeated virus passages in culture, and no phenotypic reversion was observed. The results of this study demonstrate that cleavage between p28 and p65 at CS1 is not required for MHV replication. However, proteolytic separation of p28 from p65 is necessary for optimal RNA synthesis and virus growth, suggesting important roles for these proteins in the formation or function of viral replication complexes.

Base Sequence↗
PubMed2005

Heterologous co-expression of human cytochrome P450 1A2 and polymorphic forms of N-acetyltransferase 2 for studies on aromatic amines in V79 Chinese hamster cells.

V79 Chinese hamster cells were genetically engineered for the stable co-expression of human cytochrome P450 1A2 and the polymorphic N-acetyltransferase 2 alleles *4, *5B, *6A and *13, in order to generate an in vitro tool for studying the metabolism-dependent toxicity of aromatic amines. N-acetyltransferase 2*4-encoding cDNA was generated by the polymerase chain reaction (PCR) with defined primers from the genomic DNA of a human liver donor homozygous for *4, and served as a template to generate the *5B, *6A and *13 isoforms by site-directed mutagenesis. Human cytochrome P450 (CYP) 1A2-encoding cDNA was generated by the PCR from genomic DNA of the recombinant V79MZh1A2 cell line. All the cDNAs were inserted into a CMV promoter-containing plasmid in conjunction with the selectable marker genes, neomycin and hydromycin. The recombinant expression plasmids were transfected for stable integration into the genomic DNA of the V79 cells. Several cellular clones were obtained and checked for the genomic integration of intact cDNAs with the PCR on the genomic DNA of the recombinant cells. Stable expression was confirmed by the reverse transcriptase PCR (RT-PCR) on RNA preparations. Metabolic function was tested with ethoxyresorufin as a marker substrate for CYP1A2, and 2-aminofluorene and N-sulphametazine for N-acetyltransferase activity, and compared to data obtained from biological samples. 7-Ethoxyresorufin-O-deethylase activities ranged from 0.2 to 4 pmol resorufin/min/mg total protein. The N-acetylation of sulphametazine ranged from 0.07 to 1.7 nmol N-acetyl-sulphametazine/mg total protein/min. Selected clones showing activities in the range of physiological activities were submitted to metabolism dependent mutagenicity studies. In particular, the polymorphism-dependent N-acetylation of 2-aminofluorene and the role of CYP1A2 and N-acetyltransferase in the mutagenicity of 2-aminofluorene, were investigated. Surprisingly, the mutagenicity of 2-aminofluorene is dramatically reduced in V79 cells co-expressing CYP1A2 and N-acetyltransferase, compared to V79 cells expressing CYP1A2 only, pointing to a significant species-dependent difference in the metabolic activation of aromatic amines between rats and humans.

Animals↗
PubMed1998

A nickel complex cleaves uridine in folded RNA structures: application to E. coli tmRNA and related engineered molecules.

To gain more insight about Escherichia coli tmRNA structure, NiCR, a square planar macrocyclic nickel (II) complex, was used to probe guanine N7 exposure. On the basis of this additional structural information, a refined secondary structure of the molecule is proposed. In addition to its known specificity for guanine N7, we show here that the chemical probe can also cleave at specific uridine residues. In contrast to the alkaline-labile modification of guanine, the reactivity of NiCR at these uridine residues results in direct strand scission. To better characterize the uridine cleavage sites and assess the importance of the RNA structure for the reaction to occur, smaller RNA molecules derived from one pseudoknot (PK4) of E. coli tmRNA containing two uridine cleavage sites were engineered and probed. It is shown that this pseudoknot can fold by itself in solution and that the expected uridine residues are also cleaved by the nickel complex, suggesting that only a local sequence and/or structural context is required for cleavage. In E. coli tmRNA, the five uridine cleavage sites are located in double-stranded regions. These sites contain a G-U wobble base-pair and a downstream uridine which is cleaved. Using smaller RNAs derived from one stem of PK4, systematic changes in the proposed recognition motif indicate that the G-U pair is required for cleavage. Furthermore, there is no cleavage if the G-U pair is reversed. If the recognition motif is moved within the stem, the cleavage site moves accordingly. Additionally, if the recognition motif is changed such that the G-U pair is flanked by two uridine residues, the reactivity occurs only at the 3' uridine. Radical quenching studies have indicated that sulfate radical, as in the case of guanine oxidation, is involved in uridine oxidation. Although additional studies are required to better characterize the reaction, this paper reports a novel specificity for a chemical probe which may be useful for investigating structural motifs involving G-U pairs in folded RNAs.

Base Composition↗