Anatomy of a preferred target site for the bacterial insertion sequence IS903

Like many transposons the bacterial insertion sequence IS903 was thought to insert randomly. However, using both genetic and statistical approaches, we have derived a target site for IS903 that is used 84% of the time. Computational and genetic analyses of multiple IS903 insertion sites predicted a preferred target consisting of a 21 bp palindromic pattern centered on the 9 bp target duplication generated during transposition. Here we show that targeting can be dissected into four components: the 5 bp flanking sequences, the most important sequences required for site-specific insertion; the 7 bp palindromic core within the target duplication; the dinucleotide pair at the transposon-target junction; and the local DNA context. Finally, using a substrate with multiple target sites we show that a target site is more likely found by a local bind-and-slide model and not by extended DNA tracking.     

Origination of Ds elements from Ac elements in maize: evidence for rare repair synthesis at the site of Ac excision.

Although it has been known for some time that the maize transposon Ac can mutate to Ds by undergoing internal deletions, the mechanism by which these mutations arise has remained conjectural. To gain further insight into this mechanism in maize we have studied a series of Ds elements that originated de novo from Ac elements at known locations in the genome. We present evidence that new, internally deleted Ds elements can arise at the Ac donor site when Ac transposes to another site in the genome. However, internal deletions are rare relative to Ac excision footprints, the predominant products of Ac transposition. We have characterized the deletion junctions in five new Ds elements. Short direct repeats of variable length occur adjacent to the deletion junction in three of the five Ds derivatives. In the remaining two, extra sequences or filler DNA is inserted at the junction. The filler DNAs are identical to sequences found close to the junction in the Ac DNA, where they are flanked by the same sequences that flank the filler DNA in the deletion. These findings are explained most simply by a mechanism involving error-prone DNA replication as an occasional alternative to end-joining in the repair of Ac-generated double-strand breaks.     

The interleukin-6 gene locus seems to be a preferred target site for retrotransposon integration

Recently, we have observed the insertion of a retrotransposon into the interleukin-6 (II-6) locus of a mouse somatic cell line. Here we report the characterization of Il-6 genomic regions from both mouse and rat. Restriction site analysis, DNA sequence analysis, and computer-assisted search revealed eight retrotransposon-like elements distributed over a 25 kilobase (kB) mouse Il-6 region. In the rat, five different retrotransposons have been identified within a 17 kb Il-6 region. The retrotransposons belong to the LINE-, Alu I or Alu II families, or to a rat specific class of retrotransposons. Some of the retrotransposons class of retrotransposons. Some of the retrotransposons exhibit characteristic features such as target site duplication and a poly A-tract. Remarkably, several retrotransposons map to different chromosomal locations in the mouse and rat. A genealogical tree of mouse, rat, and human Il-6 loci demonstrates a series of retrotranspositions that recently occurred in evolution. These results suggest that the Il-6 locus serves as a preferred target site for retrotransposon integration during evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M36993-4 (L.1.R3), M36995 (L1.R2), and M36996 (L1.M1/L1.M2).This work contains part of the doctoral thesis of Z. Qin and I. Schuller.     

Inversion of the phosphate chirality at the target site of Mu DNA strand transfer: evidence for a one-step transesterification mechanism.

Central to transposition of phage Mu are two reactions mediated by the MuA protein. First, MuA introduces single-stranded cuts at the ends of the Mu DNA to generate 3' OH termini. In the subsequent strand-transfer step, the MuA-Mu DNA end complex cuts a target DNA and joins the Mu 3' ends to the 5' ends of the target. DNA containing chiral phosphorothioates was used to demonstrate inversion of the chirality during the course of strand transfer. This result strongly supports a one-step transesterification mechanism in which the 3' OH of the cleaved donor DNA is the attacking nucleophile. Furthermore, this donor 3' OH group was essential for target DNA cleavage. In contrast, during lambda integration the phosphate chirality was retained, as expected for a two-step transesterification involving a covalent protein-DNA intermediate.     

Triplex mediated delivery of a platinum complex to a specific DNA target site

Tethering an ethylene diamine linker to the 5' terminus of an oligothymidine sequence provides a site for complexation with K(2)PtCl(4). Due to the low reactivity of dT toward a platinum source, we chose dT(8) and dT(15) as our initial synthetic targets for platination. Post-synthetic reaction of the platinum reagent with the diamino oligothymidine generates the diamino dichloro platinum-DNA conjugate that can be used for DNA duplex targeting by oligodeoxyncleotide-mediated triplex formation. The dT(8) sequence is not sufficiently long to facilitate triplex formation and Pt-cross-linking, whereas with a dT(15) sequence cross-linking between the third strand and the duplex occurs exclusively with the duplex target strand directly involved in triplex formation. No examples of cross-linking to the complementary target strand, or of cross-linking to both target strands are observed. Most efficient cross-linking occurs when the dinucleotide d(GpG) is present in the target strand and no cross-linking occurs with the corresponding 7-deazaG dinucleotide target. Cross-linking is also observed when dC or dA residues are present in the target strand, or even with a single dG residue, but it is not observed in any cases to dT residues. Triplex formation provides the ability to target specific sequences of double-stranded DNA and the orientational control arising from triplex formation is sufficient to alter the binding preferences of platinum. Conjugates of the type described here offer the potential of delivering a platinum complex to a specific DNA site.     

Structural cell-wall proteins in protoxylem development: evidence for a repair process mediated by a glycine-rich protein

Polyclonal antibodies were used to localize structural cell-wall proteins in differentiating protoxylem elements in etiolated bean and soybean hypocotyls at the light- and electron-microscopic level. A proline-rich protein was localized in the lignified secondary walls, but not in the primary walls of protoxylem elements, which remain unlignified, as shown with lignin-specific antibodies. Secretion of the proline-rich protein was observed during lignification in different cell types. A glycine-rich protein (GRP1.8) was specifically localized in the modified primary walls of mature protoxylem elements and in cell corners between xylem elements and xylem parenchyma cells. The protein was secreted by Golgi bodies both in protoxylem cells after the lignification of their secondary walls and in the surrounding xylem parenchyma cells. The modified primary walls of protoxylem elements were visualized under the light microscope as filaments or sheets staining distinctly with the protein stain Coomassie blue. Electron micrographs of these walls show that they are composed of an amorphous material of moderate electron-density and of polysaccharide microfibrils. These materials form a three-dimensional network, interconnecting the ring- or spiral-shaped secondary wall thickenings of protoxylem elements and xylem parenchyma cells. The results demonstrate that the modified primary walls of protoxylem cells are not simply breakdown products due to partial hydrolysis and passive elongation, as believed until now. Extensive repair processes produce cell walls with unique staining properties. It is concluded that these walls are unusually rich in protein and therefore have special chemical and physical properties.     

Genetic evidence for adenylyl cyclase 1 as a target for preventing neuronal excitotoxicity mediated by N-methyl-D-aspartate receptors

The excessive activation of N-methyl-D-aspartate (NMDA) receptors by glutamate results in neuronal excitotoxicity. cAMP is a key second messenger and contributes to NMDA receptor-dependent synaptic plasticity. Adenylyl cyclases 1 (AC1) and 8 (AC8) are the two major calcium-stimulated ACs in the central nervous system. Previous studies demonstrate AC1 and AC8 play important roles in synaptic plasticity, memory, and persistent pain. However, little is known about the possible roles of these two ACs in glutamate-induced neuronal excitotoxicity. Here, we report that genetic deletion of AC1 significantly attenuated neuronal death induced by glutamate in primary cultures of cortical neurons, whereas AC8 deletion did not produce a significant effect. AC1, but not AC8, contributes to intracellular cAMP production following NMDA receptor activation by glutamate in cultured cortical neurons. AC1 is involved in the dynamic modulation of cAMP-response element-binding protein activity in neuronal excitotoxicity. To explore the possible roles of AC1 in cell death in vivo, we studied neuronal excitotoxicity induced by an intracortical injection of NMDA. Cortical lesions induced by NMDA were significantly reduced in AC1 but not in AC8 knock-out mice. Our findings provide direct evidence that AC1 plays an important role in neuronal excitotoxicity and may serve as a therapeutic target for preventing excitotoxicity in stroke and neurodegenerative diseases.     

Human hemoglobin cross-linked through the polyphosphate-binding site. Functional properties and evidence for conformers

The properties of human hemoglobin reacted with 2-nor-2-formylpyridoxal 5'-phosphate, a bifunctional derivative of pyridoxal 5'-phosphate, have been investigated both from an equilibrium and kinetic point of view. The experimental data, interpreted in terms of the two-state allosteric model, indicate that a perturbed R state is characteristic of this modified low ligand affinity hemoglobin. In flash photolysis experiments, a quickly reacting component is always observed, in spite of the lack of dissociation into free dimers; this kinetic behavior is thought to reflect the presence of functionally independent alpha beta dimers, still connected by the flexible cross-link but forming an open hemoglobin tetramer. Two possible models for the interpretation of the kinetics of CO and/or haptoglobin binding are presented and discussed.     

Human placental estradiol 17 beta-dehydrogenase: evidence for inverted substrate orientation ("wrong-way" binding) at the active site

Human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was affinity labeled with 17 alpha-estradiol 17-(bromo[2-14C]acetate) (10 microM) or 17 beta-estradiol 17-(bromo[2-14C]acetate) (10 microM). The steroid bromoacetates competitively inhibit the enzyme (against 17 beta-estradiol) with Ki values of 90 microM (17 alpha bromoacetate) and 134 microM (17 beta bromoacetate). Inactivation of the enzyme followed pseudo-first-order kinetics with a t1/2 = 110 min (17 alpha bromoacetate) and t1/2 = 220 min (17 beta bromoacetate). Amino acid analysis of the affinity radioalkylated enzyme samples from the two bromoacetates revealed that N pi-(carboxy[14C]methyl)histidine was the modified amino acid labeled in each case. Digestion with trypsin produced peptides that were isolated by reverse-phase high-performance liquid chromatography and found to contain N pi-(carboxy[14C]methyl)histidine. Both the 17 alpha bromoacetate and also the 17 beta bromoacetate modified the same histidine in the peptide Phe-Tyr-Gln-Tyr-Leu-Ala-His(pi-CM)-Ser-Lys. Previously, the same histidine had been exclusively labeled by estrone 3-(bromoacetate) and shown not to be directly involved in catalytic hydrogen transfer at the D-ring of estradiol. Therefore, this histidine was presumed to proximate the A-ring of the bound steroid substrate. The present results suggest that the 17 alpha bromoacetate and 17 beta bromoacetate D-ring analogues of estradiol react with the same active site histidine residue as estrone 3-(bromoacetate), the A-ring analogue of estrone.(ABSTRACT TRUNCATED AT 250 WORDS)     

RNA polymerase. Direct evidence for a unique topographical site for initiation

The compound 9-(3'-azido-3'-deoxy-beta-D-xylofuranosyl)adenine 5'-monophosphate is an inhibitor (Ki = 330 microM) of the initiation binding site of the DNA-dependent RNA polymerase derived from Escherichia coli. The alpha-32P derivative of this photo-labile compound is used to derivatize a site on the sigma subunit of the holoenzyme (E sigma) using either T7 delta D111 or poly[d(A-T)] as a DNA template. The incorporation of the 32P label into the sigma subunit could be prevented by the addition of either 5'-AMP or 5'-ATP. The results are suggested to support the existence of a unique initiation binding site, topographically distinct from the sites employed during the elongation phase.     

Caspase-3 mediated cleavage of HsRad51 at an unconventional site.

The human recombinase HsRad51 is cleaved during apoptosis. We have earlier observed cleavage of the 41-kDa full-length protein into a 33-kDa product in apoptotic Jurkat cells and in in vitro translated HsRad51 after treatment with activated S-100 extract. In this study, site-directed mutagenesis was used for mapping of the cleavage site to AQVD274 downward arrow G, which does not correspond to a conventional caspase cleavage site. The absence of HsRad51 cleavage in staurosporine-treated apoptotic MCF-7 cells, which lack caspase-3, indicates that caspase-3 is essential for HsRad51 cleavage in vivo. Cleavage into the 33-kDa fragment was generated by recombinant caspase-3 and -7 in in vitro translated wild type HsRad51, but not in the HsRad51 AQVE274 downward arrow G mutant. Similarly, HsRad51 of Jurkat cell extracts was cleaved into the 33-kDa product by recombinant caspase-3, whereas caspase-7 failed to cleave endogenous HsRad51. The cleavage of in vitro translated wild type and AQVE274 downward arrow G mutant HsRad51 as well as of endogenous HsRad51 also gave rise to a smaller fragment, which corresponds in size to a recently reported DVLD187 downward arrow N HsRad51 cleavage product. In Jurkat cell extracts, the AQVD274 downward arrow G and DVLD187 downward arrow N cleavage products of HsRad51 appeared at equal concentrations of caspase-3. Moreover both fragments were generated by induction of apoptosis in MDA-MB 157 cells with staurosporine and in Jurkat cells with camptothecin. Thus, two sites in the HsRad51 sequence are targets for caspase cleavage both in vitro and in vivo.     

Neuronal nicotinic acetylcholine receptors: a new target site of ethanol.

Whereas a variety of neuroreceptors and ion channels have been demonstrated to be affected by ethanol including GABAA receptors, NMDA receptors, non-NMDA glutamate receptors, 5-HT3 receptors and voltage-gated calcium channels, neuronal nicotinic acetylcholine receptors (nnAChRs) have recently emerged as a new target site of ethanol. The nnAChRs are different from the muscle type nicotinic AChRs with respect to their molecular architecture and pharmacology. This article briefly reviews the structure, distribution and function of nnAChRs for which a considerable amount of information has been rapidly accumulated during the past 5-10 years. The potent and unique action of ethanol on nnAChRs has been unveiled only during the past few years. Most recent developments along this line of ethanol action are discussed in this paper.     

Cytochrome c3 from Desulfovibrio gigas: crystal structure at 1.8 A resolution and evidence for a specific calcium-binding site.

Crystals of the tetraheme cytochrome c3 from sulfate-reducing bacteria Desulfovibrio gigas (Dg) (MW 13 kDa, 111 residues, four heme groups) were obtained and X-ray diffraction data collected to 1.8 A resolution. The structure was solved by the method of molecular replacement and the resulting model refined to a conventional R-factor of 14.9%. The three-dimensional structure shows many similarities to other known crystal structures of tetraheme c3 cytochromes, but it also shows some remarkable differences. In particular, the location of the aromatic residues around the heme groups, which may play a fundamental role in the electron transfer processes of the molecule, are well conserved in the cases of hemes I, III, and IV. However, heme II has an aromatic environment that is completely different to that found in other related cytochromes c3. Another unusual feature is the presence of a Ca2+ ion coordinated by oxygen atoms supplied by the protein within a loop near the N-terminus. It is speculated that this loop may be stabilized by the presence of this Ca2+ ion, may contribute to heme-redox perturbation, and might even be involved in the specificity of recognition with its redox partner.     

Factors responsible for target site selection in Tn10 transposition: a role for the DDE motif in target DNA capture.

Tn10, like several other transposons, exhibits a marked preference for integration into particular target sequences. Such sequences are referred to as integration hotspots and have been used to define a consensus target site in Tn10 transposition. We demonstrate that a Tn10 hotspot called HisG1, which was identified originally in vivo, also functions as an integration hotspot in vitro in a reaction where the HisG1 sequence is present on a short DNA oligomer. We use this in vitro system to define factors which are important for the capture of the HisG1 target site. We demonstrate that although divalent metal ions are not essential for HisG1 target capture, they greatly facilitate capture of a mutated HisG1 site. Analysis of catalytic transposase mutants further demonstrates that the DDE motif plays a critical role in ''divalent metal ion-dependent'' target capture. Analysis of two other classes of transposase mutants, Exc+ Int- (which carry out transposon excision but not integration) and ATS (altered target specificity), demonstrates that while a particular ATS transposase binds HisG1 mutants better than wild-type transposase, Exc+ Int- mutants are defective in HisG1 capture, further defining the properties of these classes of mutants. Possible mechanisms for the above observations are considered.     

Patterned gene programs and target remodeling following axotomy at a major site for sensory innervation

The genetic mechanisms that a target uses to reestablish the connections of regenerating axons were explored using oligonucleotide microarrays and real-time PCR. In normal and denervated mouse vibrissa follicles, patterns of genetic regulation were assessed in adjacent targets that normally receive different types of sensory and autonomic innervation. We show that a target remodeling occurs following axotomy involving reduced hair growth, altered hair follicle integrity and remodeling of the extracellular matrix. Also, we found two orphan receptors putatively involved in hair growth. Our data further demonstrate region-specific regulation of genes putatively involved in target-axon interactions. Thus, this study shows for the first time that major target remodeling occurs following denervation and suggests putative functions for several novel genes.     

Selected non-steroidal anti-inflammatory drugs and their derivatives target gamma-secretase at a novel site. Evidence for an allosteric mechanism

Gamma-secretase is a multi-component enzyme complex that performs an intramembranous cleavage, releasing amyloid-beta (Abeta) peptides from processing intermediates of the beta-amyloid precursor protein. Because Abeta peptides are thought to be causative for Alzheimer's disease, inhibiting gamma-secretase represents a potential treatment for this neurodegenerative condition. Whereas inhibitors directed at the active center of gamma-secretase inhibit the cleavage of all its substrates, certain non-steroidal antiinflammatory drugs (NSAIDs) have been shown to selectively reduce the production of the more amyloidogenic Abeta(1-42) peptide without inhibiting alternative cleavages. In contrast to the majority of previous studies, however, we demonstrate that in cell-free systems the mode of action of selected NSAIDs and their derivatives, depending on the concentrations used, can either be classified as modulatory or inhibitory. At modulatory concentrations, a selective and, with respect to the substrate, noncompetitive inhibition of Abeta(1-42) production was observed. At inhibitory concentrations, on the other hand, biochemical readouts reminiscent of a nonselective gamma-secretase inhibition were obtained. When these compounds were analyzed for their ability to displace a radiolabeled, transition-state analog inhibitor from solubilized enzyme, noncompetitive antagonism was observed. The allosteric nature of radioligand displacement suggests that NSAID-like inhibitors change the conformation of the gamma-secretase enzyme complex by binding to a novel site, which is discrete from the binding site for transition-state analogs and therefore distinct from the catalytic center. Consequently, drug discovery efforts aimed at this site may identify novel allosteric inhibitors that could benefit from a wider window for inhibition of gamma (42)-cleavage over alternative cleavages in the beta-amyloid precursor protein and, more importantly, alternative substrates.     

IS1 insertion generates duplication of a nine base pair sequence at its target site.

Three independent integrations of the E. coli insertion sequence, IS1, into the gal operon have been analyzed. DNA sequences of portions of the wild-type galT gene which act as the target sites for these insertions, as well as the corresponding gal/IS1 junctions, are reported. Two features are particularly noteworthy. First, similar sequences appearing in inverted orientation consitute the ends of IS1: 18 of the terminal 23 base pairs at each end are identical. Second, in all three insertions, a 9 base pair segment found once in the wild-type sequence at the site of insertion is duplicated and appears in the same orientation at each end of the inserted element. The sequence of this 9 base pair repeat is different for each insertion analyzed. No homology between the inverted repeat sequences at the ends of IS1 and the sequences of the target sites is observed. Models for the mechanism of IS1 insertion are proposed.