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PubMed1998

Differential selection of cells with proviral c-myc and c-erbB integrations after avian leukosis virus infection.

Avian leukosis virus (ALV) infection induces bursal lymphomas in chickens after proviral integration within the c-myc proto-oncogene and induces erythroblastosis after integration within the c-erbB proto-oncogene. A nested PCR assay was used to analyze the appearance of these integrations at an early stage of tumor induction after infection of embryos. Five to eight distinct proviral c-myc integration events were amplified from bursas of infected 35-day-old birds, in good agreement with the number of transformed bursal follicles arising with these integrations. Cells containing these integrations are remarkably common, with an estimated 1 in 350 bursal cells having proviral c-myc integrations. These integrations were clustered within the 3' half of c-myc intron 1, in a pattern similar to that observed in bursal lymphomas. Bone marrow and spleen showed a similar number and pattern of integrations clustered within 3' c-myc intron 1, indicating that this region is a common integration target whether or not that tissue undergoes tumor induction. While all tissues showed equivalent levels of viral infection, cells with c-myc integrations were much more abundant in the bursa than in other tissues, indicating that cells with proviral c-myc integrations are preferentially expanded within the bursal environment. Proviral integration within the c-erbB gene was also analyzed, to detect clustered c-erbB intron 14 integrations associated with erythroblastosis. Proviral c-erbB integrations were equally abundant in the bone marrow, spleen, and bursa. These integrations were randomly situated upstream of c-erbB exon 15, indicating that cells carrying 3' intron 14 integrations must be selected during induction of erythroblastosis.

Animals↗
PubMed2005

Symmetrical base preferences surrounding HIV-1, avian sarcoma/leukosis virus, and murine leukemia virus integration sites.

To investigate retroviral integration targeting on a nucleotide scale, we examined the base frequencies directly surrounding cloned in vivo HIV-1, murine leukemia virus, and avian sarcoma/leukosis virus integrations. Base preferences of up to 2-fold the expected frequencies were found for three viruses, representing P values down to <10(-100) and defining what appear to be preferred integration sequences. Offset symmetry reflecting the topology of the integration reaction was found for HIV-1 and avian sarcoma/leukosis virus but not murine leukemia virus, suggesting fundamental differences in the way different retroviral integration complexes interact with host-cell DNA.

Alpharetrovirus↗
PubMed1996

Site-specific integration by adeno-associated virus.

Adeno-associated virus (AAV) has attracted considerable interest as a potential vector for gene delivery. Wild-type virus is notable for the lack of association with any human disease and the ability to stably integrate its genome in a site-specific manner in a locus on human chromosome 19 (AAVS1). Use of a functional model system for AAV DNA integration into AAVS1 has allowed us to conclude that the recombination event is directed by cellular DNA sequences. Recombinant junctions isolated from our integration assay were analyzed and showed characteristics similar to those found in latently infected cell lines. The minimal DNA signals within AAVS1 required for targeted integration were identified and shown to contain functional motifs of the viral origin of replication. A replication mediated model of AAV DNA integration is proposed.

Base Sequence↗
PubMed2004

Gene targeting in yeast is initiated by two independent strand invasions.

To study the mechanism of gene targeting, we examined heteroduplex DNA (hDNA) formation during targeting of two separate chromosomal locations in Saccharomyces cerevisiae. We examined both replacement of the entire gene with a heterologous selectable marker and correction of a single base pair insertion mutation by gene targeting, and in all cases our results were consistent with separate strand invasion/resolution at the two ends of the targeting fragment as the dominant mechanism in wild-type cells. A small subset of transformants was consistent with assimilation of a single strand of targeting DNA encompassing both flanking homology regions and the marker into hDNA. hDNA formation during correction of a point mutation by targeted integration was conspicuously altered in a mismatch repair-deficient background and was consistent with single-strand invasion/assimilation without mismatch correction, confirming that gene targeting by this pathway is actively impeded in wild-type yeast. Finally, inversion of one targeted locus and mutation of an active origin of DNA replication at the other locus affected hDNA formation significantly, suggesting that formation of productive interactions between the targeting DNA and the targeted site in the chromosome is sensitive to local DNA dynamics.

Carrier Proteins↗
PubMed2005

In vitro accuracy of a novel registration and targeting technique for image-guided template production.

OBJECTIVES: The objective of this study was to evaluate the accuracy of a novel registration and targeting technique for image-guided template production (IGTP) in a preliminary phantom study. MATERIAL AND METHODS: Registration of four standard dental stone casts with integrated target pellets to the corresponding computed tomography (CT) data was performed via a vacuum mouthpiece and an external reference frame (Medical Intelligence GmbH, Germany). Using the Treon navigation system (Medtronic Inc., Minneapolis, MN, USA) a surgical path with the entry in the centre of the dental crown and the target in the centre of the target pellet was planned on the CT data. An aiming device was adjusted according to the planned trajectory and guided drillings into the dental stone casts. The accuracy was evaluated on postoperative 3D-CT data. RESULTS: The mean fiducial registration error as given by the registration software was 0.4 mm. One hundred and twelve navigated drillings showed a mean accuracy [xy] of 0.42+/-0.26 mm (maximum 1 mm). For the z-axis, a mean accuracy [z] of 0.25+/-0.12 mm (maximum 0.6 mm) was found. CONCLUSIONS: Comparing the presented registration technique to existing registration methods in IGTP and burr tracking, no radiographic and registration templates are needed. The procedure is easy and requires only minimal effort. Navigation-controlled drillings could be performed with an accuracy that approaches the intrinsic navigation system's accuracy, a fact that warrants its use for surgical template production. Further accuracy studies of template-guided drillings are necessary before the presented registration technique can be implemented for patient treatment.

Calcium Sulfate↗
PubMed2005

Division of labor within human immunodeficiency virus integrase complexes: determinants of catalysis and target DNA capture.

Following the completion of reverse transcription, the human immunodeficiency virus integrase (IN) enzyme covalently links the viral cDNA to a host cell chromosome. An IN multimer carries out this reaction, but the roles of individual monomers within the complex are mostly unknown. Here we analyzed the distribution of functions for target DNA capture and catalysis within the IN multimer. We used forced complementation between pairs of IN deletion derivatives in vitro as a tool for probing cis-trans relationships and analyzed amino acid substitutions affecting either catalysis or target site selection within these complementing complexes. This allowed the demonstration that the IN variant contributing the active catalytic domain was also responsible for recognition of the integration target DNA. We were further able to establish that a single monomer is responsible for both functions by use of assay mixtures containing three different IN genotypes. These data specify the ligands bound at the catalytically relevant IN monomer and allow more-specific modeling of the mechanism of inhibitors that also bind this surface of IN.

Binding Sites↗
PubMed1993

A V(D)J recombinase-inducible B-cell line: role of transcriptional enhancer elements in directing V(D)J recombination.

Rapid analysis of mechanisms that regulate V(D)J recombination has been hampered by the lack of appropriate cell systems that reproduce aspects of normal prelymphocyte physiology in which the recombinase is activated, accessible antigen receptor loci are rearranged, and rearrangement status is fixed by termination of recombinase expression. To generate such a system, we introduced heat shock-inducible V(D)J recombination-activating genes (RAG) 1 and 2 into a recombinationally inert B-cell line. Heat shock treatment of these cells rapidly induced high levels of RAG transcripts and RAG proteins that were accompanied by a parallel induction of V(D)J recombinase activity, strongly suggesting that RAG proteins have a primary role in V(D)J recombination. Within hours after induction, these cells began to rearrange chromosomally integrated V(D)J recombination substrates but only if the substrates contained an active transcriptional enhancer; substrates lacking an enhancer were not efficiently rearranged. Activities necessary to target integrated substrates for rearrangement were provided by two separate lymphoid-specific transcriptional enhancers, as well as an active nonlymphoid enhancer, unequivocally demonstrating that such elements enhance both transcription and V(D)J recombinational accessibility.

Animals↗
PubMed1997

Convenient and reversible site-specific targeting of exogenous DNA into a bacterial chromosome by use of the FLP recombinase: the FLIRT system.

We have created a system that utilizes the FLP recombinase of yeast to introduce exogenous cloned DNA reversibly at defined locations in the Escherichia coli chromosome. Recombination target (FRT) sites can be introduced permanently at random locations in the chromosome on a modified Tn5 transposon, now designed so that the inserted FRT can be detected and its location mapped with base pair resolution. FLP recombinase is provided as needed through the regulated expression of its gene on a plasmid. Exogenous DNA is introduced on a cloning vector that contains an FRT, selectable markers, and a replication origin designed to be deleted prior to electroporation for targeting purposes. High yields of targeted integrants are obtained, even in a recA background. This system permits rapid and precise excision of the introduced DNA when needed, without destroying the cells. The efficiency of targeting appears to be affected only modestly by transcription initiation upstream of the chromosomal FRT site. With rare exceptions, FRTs introduced to the bacterial chromosome are targeted with high efficiency regardless of their location. The system should facilitate studies of bacterial genome structure and function, simplify a wide range of chromosomal cloning applications, and generally enhance the utility of E. coli as an experimental organism in biotechnology.

Blotting, Southern↗
PubMed2004

Epigenetic control of CACTA transposon mobility in Arabidopsis thaliana.

Epigenetic mutation, heritable developmental variation not based on a change in nucleotide sequence, is widely reported in plants. However, the developmental and evolutionary significance of such mutations remains enigmatic. On the basis of our studies of the endogenous Arabidopsis transposon CACTA, we propose that the inheritance of epigenetic gene silencing over generations can function as a transgenerational genome defense mechanism against deleterious movement of transposons. We previously reported that silent CACTA1 is mobilized by the DNA hypomethylation mutation ddm1 (decrease in DNA methylation). In this study, we report that CACTA activated by the ddm1 mutation remains mobile in the presence of the wild-type DDM1 gene, suggesting that de novo silencing is not efficient for the defense of the genome against CACTA movement. The defense depends on maintenance of transposon silencing over generations. In addition, we show that the activated CACTA1 element transposes throughout the genome in DDM1 plants, as reported previously for ddm1 backgrounds. Furthermore, the CACTA1 element integrated into both the ddm1-derived and the DDM1-derived chromosomal regions in the DDM1 wild-type plants, demonstrating that this class of transposons does not exhibit targeted integration into heterochromatin, despite its accumulation in the pericentromeric regions in natural populations. The possible contribution of natural selection as a mechanism for the accumulation of transposons and evolution of heterochromatin is discussed.

Arabidopsis↗
PubMed2003

Targeting combinatorial transcriptional complex assembly at specific modules within the interleukin-2 promoter by the immunosuppressant SB203580.

The proximal promoter sequence of the interleukin-2 (IL-2) gene contains a series of composite sites or modules that controls much of its responsiveness to environmental stimuli. The integrated targeting of these modules is therefore a major mode of regulation. This report describes how multiple functional hierarchies, required for the recruitment of the p300 co-activator to the CD28RE/AP1 (TRE) module of the IL-2 promoter, are selectively disrupted in human T-cells by the immunosuppressive and anti-inflammatory actions of the p38 mitogen-activated protein kinase inhibitor (MAPK), SB203580. The molecular hierarchies targeted by SB203580 include the combinatorial interaction of NF-kappaB and CREB at the CD28RE/AP1 element coupled with the subsequent dynamic co-assembly and activation of p300. Several aspects of this targeting are linked to the ability of SB203580 to inhibit p38 MAPK-controlled pathways. Together, these results provide the molecular basis through which the combinatorial structure and context of the composite elements of the IL-2 promoter dictates mitogen responsiveness and drug susceptibility that are quantitatively and qualitatively distinct from the isolated action of single consensus sequences and/or transcriptional motifs.

Amino Acid Motifs↗
PubMed2006

Prescriptions for optimizing intensities of short-lived RIBs at ISOL based research facilities.

In order to generate useful intensities of short-lived radioactive species at Isotope Separator On-Line (ISOL) based research facilities, species of interest must be diffused from the interior of the production target-material, effusively transported to an ion source, ionized, extracted, mass-analyzed, and accelerated to research energies in times commensurate with their lifetimes. The intensities at such facilities are principally limited by decay losses associated with times required for diffusion-release from target materials and transport from the target to the ion source and by the maximum permissible primary beam power depositional density that can be tolerated without deleteriously affecting target integrity or ion source efficiency. Consequently, it is imperative to minimize delay times associated with the independent diffusion and effusive-flow processes by choosing small dimensioned (short-diffusion length) highly refractory target materials and formatting them in highly permeable mechanically and thermally robust structures that can withstand high-power beam irradiation for extended periods of time. This article provides basic information on methods, procedures and principles for selecting target materials; for designing fast diffusion-release targets; for designing fast effusive-flow vapor transport systems; and introduces methods for controlling target temperatures as required for optimizing intensities of short-lived radioactive ion beams at ISOL based research facilities.

Journal Article↗
PubMed1988

Regulated expression of genes inserted at the human chromosomal beta-globin locus by homologous recombination.

We have examined the effect of the site of integration on the expression of cloned genes introduced into cultured erythroid cells. Smithies et al. [Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A. & Kucherlapati, R.S. (1985) Nature (London) 317, 230-234] reported the targeted integration of DNA into the human beta-globin locus on chromosome 11 in a mouse erythroleukemia-human cell hybrid. These hybrid cells can undergo erythroid differentiation leading to greatly increased mouse and human beta-globin synthesis. By transfection of these hybrid cells with a plasmid carrying a modified human beta-globin gene and a foreign gene composed of the coding sequence of the bacterial neomycin-resistance gene linked to simian virus 40 transcription signals (SVneo), cells were obtained in which the two genes are integrated at the beta-globin locus on human chromosome 11 or at random sites. When we examined the response of the integrated genes to cell differentiation, we found that the genes inserted at the beta-globin locus were induced during differentiation, whereas randomly positioned copies were not induced. Even the foreign SVneo gene was inducible when it had been integrated at the beta-globin locus. The results show that genes introduced at the beta-globin locus acquire some of the regulatory properties of globin genes during erythroid differentiation.

Animals↗
PubMed1991

Provirus integration at the 3' region of N-myc in cell lines established from thymic lymphomas spontaneously formed in AKR mice and a [(BALB/c x B6)F1----AKR] bone marrow chimera.

Among 18 thymic leukemia cell lines which have been established from spontaneous thymic lymphomas in AKR mice as well as in bone marrow chimeras which were constructed by transplanting allogeneic bone marrow cells into irradiated AKR mice, three proviral integration sites were identified; near c-myc, N-myc and pim-1 loci. No integration site specific for chimeric leukemia cell lines was found. In three thymic leukemia cell lines which contained rearranged N-myc genes, insertions of long terminal repeats (LTRs) of murine leukemia viruses were detected at 18 or 20 bp downstream of the translational termination codon. These results demonstrate that the 3' region of the N-myc gene is one of the integration targets for murine leukemia viruses in spontaneous thymic lymphomas. In these three cell lines, N-myc mRNA was stably transcribed and transcription of c-myc mRNA was down-regulated. The integrated murine leukemia viruses in AKR thymic leukemia were most likely AKV, though the DNA sequence of the LTR inserted in the genome of a leukemic cell line from [(BALB/c x B6)F1----AKR], CAK20, was different from LTRs of murine leukemia viruses so far reported.

Antigens, Surface↗
PubMed1997

Construction of chicken x human microcell hybrids for human gene targeting.

Human chromosomes 1, 2, 3, and 11 were tagged with pSV2 neo and transferred via microcell fusion from mouse A9 human monochromosomal hybrids to a chicken pre-B cell line, DT40, proficient for homologous recombination. Hybrids containing two copies of human chromosome 11 were transfected with targeting vectors containing a mammalian selectable gene with either the D11S16 or HRAS genomic sequences corresponding to two different chromosome 11 loci. Analysis of stable transfectants showed a high frequency (approximately 80%) of targeted integration of these constructs into each of the homologous loci of human chromosome 11 in DT40 hybrids. The results suggest that any human genomic sequences on human chromosomes transferred into DT40 cells could be targeted at high frequency, thereby allowing for subsequent modification of human genes and chromosomes.

Animals↗
PubMed2006

Levels of integration and specialisation within professional community teams for people with dementia.

BACKGROUND: Delivering integrated and specialist mental health services for the growing population of older people with dementia in Britain is a key concern of the present government. AIMS: To consider the nature of current practice among multi-disciplinary and single discipline health and social care teams providing a service to people with dementia and compare the quality of service offered. METHODS: A postal survey of professional community teams in North West England, providing services to people with dementia. Responses were analysed according to a number of standards measuring service quality, developed from research and policy documents. A response rate of 59% yielded a final sample of 52 teams. RESULTS: Multi-disciplinary teams scored more highly than single discipline teams on many of the measures used. Single discipline teams achieved a higher score on just one measure, culturally sensitive services. Generally teams were found to provide a more integrated, targeted and person-centred service, as measured in this study, compared with earlier findings. Teams performed less well on measures of flexibility and culturally sensitive provision. CONCLUSIONS: These findings indicate some potential gains from integration, and highlight the level of work still needed to achieve it. Further research is needed to build on the structural and process measures used in this research in order that the costs and outcomes consequent upon these practices can be measured.

Aged↗
PubMedDate not supplied

Proteolysis and mass spectrometric analysis of an integral membrane: aquaporin 0.

Due to hydrophobicity, structural analysis of integral membrane proteins poses a formidable challenge for current mass spectrometry-based proteomics approaches. Herein, we demonstrate results from optimized sample preparation and enzymatic proteolysis procedures for the complete primary structure determination of a targeted integral membrane protein, lens aquaporin 0 (AQP0). Plasma membrane from bovine lens tissue was alkali treated and tryptic digestion was performed in optimized acetonitrile-ammonium bicarbonate solution. Full sequence coverage of AQP0 was observed as tryptic peptides using both matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and capillary liquid chromatography tandem mass spectrometry (cLC/MS/MS). An amino acid mutation of Thr to Ile/Leu at residue 199 was deduced based on MS/MS results. In a complementary effort to fully sequence the protein, peptic digestion was developed to take advantage of hydrophobic protein solubility in organic acid as well as the decreased activity of pepsin at low pH. Peptic digestion in 10% formic acid (pH 1.2) generated peptides of 500 to 3000 Da and gave 100% sequence coverage by cLC/MS/MS. In addition to post-translational modifications reported previously, a new phosphorylation site at serine 229 and two oxidation sites at tryptophan 202 and 205 were detected on the protein. These methodologies provide complementary detergent- and CNBr-free procedures for detailed analysis of this important membrane channel protein and offer promise for analysis of the integral membrane proteome.

Amino Acid Sequence↗
PubMed1999

Therapeutic innovation.

The small but very focused meeting of 60 delegates from academia, small biopharmaceutical companies and large pharmaceutical companies aimed to give an overview of current R&D approaches in the cardiovascular disease area. Most of the new therapeutic approaches were presented by small biopharmaceutical companies and addressed disease targets in the vasculature for the following indications: thrombosis; atherosclerosis; restenosis; hyperlipidemia; and hypertension. Cardiac disease targets were addressed in the following indications: congestive heart failure (CHF); chronic angina; reperfusion injury; and atrial arrhythmia. The technologies presented included small molecule drugs, protein drugs and gene therapeutics as well as vaccines and an integrated target definition platform. The therapeutic approaches were complemented by the presentation of two innovative diagnostic products for thrombosis and stroke related brain injury.

Journal Article↗
PubMed1994

Integration of vectors by homologous recombination in the plant pathogen Glomerella cingulata.

An homologous transformation system has been developed for the plant pathogenic fungus Glomerella cingulata (Colletotrichum gloeosporioides). A transformation vector containing the G. cingulata gpdA promoter fused to the hygromycin phosphotransferase gene was constructed. Southern analyses indicated that this vector integrated at single sites in most transformants. A novel method of PCR amplification across the recombination junction point indicated that the integration event occurred by homologous recombination in more than 95% of the transformants. Deletion studies demonstrated that 505 bp (the minimum length of homologous promoter DNA analysed which was still capable of promoter function) was sufficient to target integration events. Homologous integration of the vector resulted in duplication of the gdpA promoter region. When transformants were grown without selective pressure, a high incidence of vector excision by recombination between the duplicated regions was evident. The significance of these recombination characteristics is discussed with reference to the feasibility of performing gene disruption experiments.

Base Sequence↗