Simple and efficient generation in vitro of nested deletions and inversions: Tn5 intramolecular transposition

We have exploited the intramolecular transposition preference of the Tn 5 in vitro transposition system to test its effectiveness as a tool for generation of nested families of deletions and inversions. A synthetic transposon was constructed containing an ori, an ampicillin resistance (Ampr) gene, a multi-cloning site (MCS) and two hyperactive end sequences. The donor DNA that adjoins the transposon contains a kanamycin resistance (Kanr) gene. Any Amprreplicating plasmid that has undergone a transposition event (Kans) will be targeted primarily to any insert in the MCS. Two different size targets were tested in the in vitro system. Synthetic transposon plasmids containing either target were incubated in the presence of purified transposase (Tnp) protein and transformed. Transposition frequencies (Ampr/Kans) for both targets were found to be 30-50%, of which >95% occur within the target sequence, in an apparently random manner. By a conservative estimate 10(5) or more deletions/inversions within a given segment of DNA can be expected from a single one-step 20 microl transposition reaction. These nested deletions can be used for structure-function analysis of proteins and for sequence analysis. The inversions provide nested sequencing templates of the opposite strand from the deletions.     

Escherichia coli DNA topoisomerase I and suppression of killing by Tn5 transposase overproduction: topoisomerase I modulates Tn5 transposition

Tn5 transposase (Tnp) overproduction is lethal to Escherichia coli. The overproduction causes cell filamentation and abnormal chromosome segregation. Here we present three lines of evidence strongly suggesting that Tnp overproduction killing is due to titration of topoisomerase I. First, a suppressor mutation of transposase overproduction killing, stkD10, is localized in topA (the gene for topoisomerase I). The stkD10 mutant has the following characteristics: first, it has an increased abundance of topoisomerase I protein, the topoisomerase I is defective for the DNA relaxation activity, and DNA gyrase activity is reduced; second, the suppressor phenotype of a second mutation localized in rpoH, stkA14 (H. Yigit and W. S. Reznikoff, J. Bacteriol. 179:1704-1713, 1997), can be explained by an increase in topA expression; and third, overexpression of wild-type topA partially suppresses the killing. Finally, topoisomerase I was found to enhance Tn5 transposition up to 30-fold in vivo.     

Tn10 transposition via a DNA hairpin intermediate

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, a birdlike face, growth retardation, immunodeficiency, lack of secondary sex characteristics in females, and increased incidence of lymphoid cancers. NBS cells display a phenotype similar to that of cells from ataxia-telangiectasia patients, including chromosomal instability, radiation sensitivity, and aberrant cell-cycle-checkpoint control following exposure to ionizing radiation. A recent study reported genetic linkage of NBS to human chromosome 8q21, with strong linkage disequilibrium detected at marker D8S1811 in eastern European NBS families. We collected a geographically diverse group of NBS families and tested them for linkage, using an expanded panel of markers at 8q21. In this article, we report linkage of NBS to 8q21 in 6/7 of these families, with a maximum LOD score of 3.58. Significant linkage disequilibrium was detected for 8/13 markers tested in the 8q21 region, including D8S1811. In order to further localize the gene for NBS, we generated a radiation-hybrid map of markers at 8q21 and constructed haplotypes based on this map. Examination of disease haplotypes segregating in 11 NBS pedigrees revealed recombination events that place the NBS gene between D8S1757 and D8S270. A common founder haplotype was present on 15/18 disease chromosomes from 9/11 NBS families. Inferred (ancestral) recombination events involving this common haplotype suggest that NBS can be localized further, to an interval flanked by markers D8S273 and D8S88.     

Switching from cut-and-paste to replicative Tn7 transposition

The bacterial transposon Tn7 usually moves through a cut-and-paste mechanism whereby the transposon is excised from a donor site and joined to a target site to form a simple insertion. The transposon was converted to a replicative element that generated plasmid fusions in vitro and cointegrate products in vivo. This switch was a consequence of the separation of 5'- and 3'-end processing reactions of Tn7 transposition as demonstrated by the consequences of a single amino acid alteration in an element-encoded protein essential for normal cut-and-paste transposition. The mutation specifically blocked cleavage of the 5' strand at each transposon end without disturbing the breakage and joining on the 3' strand, producing a fusion (the Shapiro Intermediate) that resulted in replicative transposition. The ability of Tn7 recombination products to serve as substrates for both the limited gap repair required to complete cut-and-paste transposition and the extensive DNA replication involved in cointegrate formation suggests a remarkable plasticity in Tn7's recruitment of host repair and replication functions.     

Tn916 transposition in Haemophilus influenzae Rd: preferential insertion into noncoding DNA

OBJECTIVES: A low level of education is associated with an increased risk of developing a dementia disorder, as well as with a higher risk of cardiovascular disease. The aim of this study was to investigate the association between education and cardiovascular risk factors, and to study the relation between these factors and cognitive function in elderly men. DESIGN: Cross-sectional population-based study. SETTING: Uppsala, Sweden. SUBJECTS: 504 men aged 69-74 years, participants in a longitudinal health survey concerning cardiovascular risk factors. MAIN OUTCOME MEASURE: Cognitive function as measured by a composite score of 13 standard psychometric tests. RESULTS: A low level of education was associated with poorer cognitive performance, as well as with obesity, smoking, diabetes, high concentrations of serum triglycerides and plasma fibrinogen. In the entire cohort, subjects with obesity, smoking, diabetes or hypertriglyceridaemia showed impaired cognitive test results, independent of socio-economic factors. When stroke cases were excluded, obesity and smoking were still related to impaired cognitive function. CONCLUSIONS: Smoking and obesity with associated metabolic disturbances are inversely related both to educational level and to cognitive function. Cognitive decline of vascular origin is potentially preventable by treatment of risk factors. The question of whether the increased vascular risk contributes to the higher prevalence of cognitive disorders in individuals with low socio-economic status, needs to be further evaluated in longitudinal population-based studies.     

Tn552 transposase catalyzes concerted strand transfer in vitro

MDC-15 (ADAM-15, metargidin), a membrane-anchored metalloprotease/disintegrin/cysteine-rich protein, is expressed on the surface of a wide range of cells and has an RGD tripeptide in its disintegrin-like domain. MDC-15 is potentially involved in cell-cell interactions through its interaction with integrins. We expressed a recombinant MDC-15 disintegrin-like domain as a fusion protein with glutathione S-transferase (designated D-15) in bacteria and examined its binding function to integrins using mammalian cells expressing different recombinant integrins. We found that D-15 specifically interacts with alphavbeta3 but not with the other integrins tested (alpha2beta1, alpha3beta1, alpha4beta1, alpha5beta1, alpha6beta1, alpha6beta4, alphavbeta1, alphaIIbbeta3, and alphaLbeta2). Mutation of the tripeptide RGD to SGA totally blocked binding of D-15 to alphavbeta3, suggesting that D-15-alphavbeta3 interaction is RGD-dependent. When the sequence RPTRGD is mutated to NWKRGD, D-15 is recognized by both alphaIIbbeta3 and alphavbeta3, suggesting that the receptor binding specificity is mediated by the sequence flanking the RGD tripeptide, as in snake venom disintegrins. These results indicate that the disintegrin-like domain of MDC-15 functions as an adhesion molecule and may be involved n alphavbeta3-mediated cell-cell interactions.     

Conversion of pBR322-based plasmids into broad-host-range vectors by using the Tn3 transposition mechanism

We constructed a series of transposon vectors which allow efficient in vitro gene manipulation and subsequent introduction of cloned DNA into a variety of gram-negative bacteria. Transfer of the cloned fragment from these multicopy plasmids into self-transmissible broad-host-range vectors is achieved in vivo, using the Tn3 transposition mechanism. Transposition into a variety of broad-host-range plasmids proceeds efficiently, and the resulting recombinant plasmids can be readily transferred and maintained in a variety of gram-negative bacteria. The utility of the transposable vectors was demonstrated by the introduction and expression of the lacIPOZY sequences of Escherichia coli into Pseudomonas putida strains, allowing them to utilize lactose as a sole source of carbon and energy.     

Functional characterization of the Tn5 transposase by limited proteolysis

The 476 amino acid Tn5 transposase catalyzes DNA cutting and joining reactions that cleave the Tn5 transposon from donor DNA and integrate it into a target site. Protein-DNA and protein-protein interactions are important for this tranposition process. A truncated transposase variant, the inhibitor, decreases transposition rates via the formation of nonproductive complexes with transposase. Here, the inhibitor and the transposase are shown to have similar secondary and tertiary folding. Using limited proteolysis, the transposase has been examined structurally and functionally. A DNA binding region was localized to the N-terminal 113 amino acids. Generally, the N terminus of transposase is sensitive to proteolysis but can be protected by DNA. Two regions are predicted to contain determinants for protein-protein interactions, encompassing residues 114-314 and 441-476. The dimerization regions appear to be distinct and may have separate functions, one involved in synaptic complex formation and one involved in nonproductive multimerization. Furthermore, predicted catalytic regions are shown to lie between major areas of proteolysis.     

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.     

High and low UV-dose responses in SOS-induction of the precise excision of transposons tn1, Tn5 and Tn10 in Escherichia coli

UV-inducible precise excision of transposons is a specific SOS-mutagenesis process. It deals with the deletion formation which has previously been demonstrated to involve direct or inverted IS-sequences of transposons. The process was used for revisiting the targeted and untargeted SOS-mutability and its relationship to the key genes for SOS-mutagenesis: the recA, lexA and umuDC. The precise excision of transposons Tn5 and Tn10 from the chromosomal insertion sites ade128 and cyc750 is induced in Escherichia coli K-12 and B cells, wild-type for DNA-repair, both by the low doses of UV-light ranging from 0.25 J m-2 to 2.5 J m-2 and the high doses within the range 5.0-40.0 J m-2. Precise excision of these transposons induced by the range of low doses incapable to induce targeted point mutations reveals its mostly untargeted nature. This process for the transposon Tn1 is not induced by UV-light within the range of doses 0.25-2.5 J m-2 while its induction is possible by UV-fluences ranging from 5.0 to 40.0 J m-2. A dose-response of the precise excision of Tn1 is similar to that of the UV-induced reversion of trpUAA point mutation that is targeted by nature and contrasts to the UV-inducible precise excision of Tn5 and Tn10. Both types of UV-inducible precise excision, demonstrated either by Tn1 or Tn5 and Tn10, are eliminated by mutations in the lexA, recA and umuDC genes indispensable for UV-induced SOS-mutability. The palindromic structures different for the transposons Tn1, Tn5 and Tn10 are discussed to be involved and affect the targeted and untargeted precise excision of transposons induced by UV-light.     

IHF modulation of Tn10 transposition: sensory transduction of supercoiling status via a proposed protein/DNA molecular spring

Architectural protein IHF modulates Tn10 transposition in vitro. IHF stimulates transposon excision. Also, separately, IHF forces transposon end/target DNA interactions into a constrained pathway, "channeling," that yields only unknotted intratransposon inversion circles. Negative supercoiling influences both effects, differently. We infer that IHF is an architectural catalyst: it promotes initial transpososome assembly and is then ejected from the transpososome. IHF then rebinds, altering transpososome conformation to promote channeling. We also infer that the developing transpososome is a molecular spring: DNA provides basic elasticity; a conformational change in transposase provides force; and IHF and/or supercoiling provide conformational inputs. In vivo, IHF is a sensory transducer of chromosomal supercoiling status: with supercoiling absent, IHF is "supercoiling relief factor"; with supercoiling present, stimulation and channeling comprise a homeostatic pair such that modest changes in chromosome condition strongly influence transpositional outcome.     

Transposase is the only nematode protein required for in vitro transposition of Tc1

The Tc1 element of Caenorhabditis elegans is a member of the most widespread class of DNA transposons known in nature. Here, we describe efficient and precise transposition of Tc1 in a cell-free system. Tc1 appears to jump by a cut-and-paste mechanism of transposition. The terminal 26 bp of the Tc1 terminal repeats together with the flanking TA sequence are sufficient for transposition. The target site choice in vitro is similar to that in vivo. Transposition is achieved with an extract prepared from nuclei of transgenic nematodes that overexpress Tc1 transposase but also by recombinant transposase purified from Escherichia coli. The simple reaction requirements explain why horizontal spread of Tc1/mariner transposons can occur. They also suggest that Tcl may be a good vector for transgenesis of diverse animal species.     

Host proteins can stimulate Tn7 transposition: a novel role for the ribosomal protein L29 and the acyl carrier protein

The bacterial transposon Tn7 is distinguished by its ability to insert at a high frequency into a specific site in the Escherichia coli chromosome called attTn7. Tn7 insertion into attTn7 requires four Tn7-encoded transposition proteins: TnsA, TnsB, TnsC and TnsD. The selection of attTn7 is determined by TnsD, a sequence-specific DNA-binding protein. TnsD binds attTn7 and interacts with TnsABC, the core transposition machinery, which facilitates the insertion of Tn7 into attTn7. In this work, we report the identification of two host proteins, the ribosomal protein L29 and the acyl carrier protein (ACP), which together stimulate the binding of TnsD to attTn7. The combination of L29 and ACP also stimulates Tn7 transposition in vitro. Interestingly, mutations in L29 drastically decrease Tn7 transposition in vivo, and this effect of L29 on Tn7 transposition is specific for TnsABC+D reactions.     

Evidence that Tn5565, which includes the enterotoxin gene in Clostridium perfringens, can have a circular form which may be a transposition intermediate

The Clostridium perfringens enterotoxin gene is on a transposon-like element, Tn5565, integrated in the chromosome in human food poisoning strains. The flanking IS elements, IS1470 A and B, are related to IS30. The IS element found in the transposon, IS1469, is related to IS200 and has been found upstream of cpe in all Type A strains. PCR and sequencing studies from cell extracts and plasmid isolations of C. perfringens indicate that Tn5565 can form a circular form with the tandem repeat (IS1470)2, similar to the transposition intermediates described for a number of IS elements.     

Synthesis of Tn and sialyl Tn antigen-lipid A analog conjugates for synthetic vaccines

Conjugates of Tn and sialyl Tn antigen with N-teradecanoyl L-seryl-beta-alanine-containing D-glucosamine derivatives structurally related to lipid A as an immunoadjuvant were synthesized for the development of totally synthetic vaccines against cancers or HIV.     

副猪嗜血杆菌EZ-Tn5转座子插入突变体库的构建及减毒株的筛选

[目的]获得副猪嗜血杆菌减毒株.[方法]应用转座子技术构建转座子插入突变体库,卡那抗性筛选阳性菌株,PCR扩增卡那特异片段去除假阳性,小鼠感染试验检测突变株毒力,并对获得的减毒株进行生物学特性检测.[结果]所获得减毒突变菌株具有与野毒株相似的增殖能力,传代后毒力稳定,遗传学特性稳定.[结论]该研究结果为进一步探讨HPS毒力因子、致病机制奠定了基础.     

Two classes of Tn10 transposase mutants that suppress mutations in the Tn10 terminal inverted repeat

Tn10 transposition requires IS10 transposase and essential sequences at the two ends of the element. Mutations in terminal basepairs 6-13 confer particularly strong transposition defects. We describe here the identification of transposase mutations that suppress the transposition defects of such terminus mutations. These mutations are named "SEM" for suppression of ends mutations. All of the SEM mutations suppress more than a single terminus mutation and thus are not simple alterations of transposase/end recognition specificity. The mutations identified fall into two classes on the basis of genetic tests, location within the protein and nature of the amino acid substitution. Class I mutations, which are somewhat allele specific, appear to define a small structural and functional domain of transposase in which hydrophobic interactions are important at an intermediate stage of the transposition reaction, after an effective interaction between the ends but before transposon excision. Class II mutations, which are more general in their effects, occur at a single residue in a small noncritical amino-terminal proteolytic domain of transposase and exert their affects by altering a charge interaction; these mutations may affect act early in the reaction, before or during establishment of an effective interaction between the ends.     

Isolation of Sinorhizobium meliloti Tn5 mutants with altered cytochrome terminal oxidase expression and improved symbiotic performance

OBJECTIVES: We sought to investigate whether, in humans, the timing and incidence of a relapse of atrial fibrillation (AF) during the first month after cardioversion indicates the presence of electrical remodeling and whether this could be influenced by prevention of intracellular calcium overload during AF. BACKGROUND: Animal experiments have shown that AF induces shortening of the atrial refractory period, resulting in an increased vulnerability for reinduction of AF. This electrical remodeling was completely reversible within 1 week after cardioversion of AF and was presumably related to intracellular calcium overload. METHODS: Using transtelephonic monitoring in 61 patients cardioverted for chronic AF, we evaluated the daily incidence of recurrence of AF and determined, by Cox regression analysis, the influence of patient characteristics and medication on relapse of AF. RESULTS: During 1 month of follow-up, 35 patients (57%) had a relapse of AF, with a peak incidence during the first 5 days after cardioversion. Furthermore, in patients with a recurrence of AF, there was a positive correlation between the duration of the shortest coupling interval of the premature atrial beats after cardioversion and the timing of the recurrence of AF (p = 0.0013). Multivariate analysis revealed that the use of intracellular calcium-lowering drugs during AF was the only significant variable related to maintenance of sinus rhythm after cardioversion (p = 0.03). CONCLUSIONS: The daily distribution of recurrences of AF suggests a temporary vulnerable electrophysiologic state of the atria. Use of intracellular calcium-lowering medications during AF appeared to reduce recurrences, possibly due to a reduction of electrical remodeling during AF.     

Clinical experiences in hyoid arch transposition

Autogenous hyoid bone grafts for the repair of subglottic stenosis have been used successfully in canine experiments. This technique was subsequently modified for human use, and the experience in six patients over the past year at Washington University is reported. In five of the six patients, satisfactory results were obtained. Several case reports are included, and the surgical procedure is described in detail.