Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.6.3.1 (Mg2+-ATPase)
1,484 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A set of vectors which facilitates the sequential integration of new functions into the Escherichia coli chromosome by homologous recombination has been developed. These vectors are based on plasmids described by Posfai et al. (J. Bacteriol. 179:4426-4428, 1997) which contain conditional replicons (pSC101 or R6K), a choice of three selectable markers (ampicillin, chloramphenicol, or kanamycin), and a single FRT site. The modified vectors contain two FRT sites which bracket a modified multiple cloning region for DNA insertion. After integration, a helper plasmid expressing the flippase (FLP) recombinase allows precise in vivo excision of the replicon and the marker used for selection. Sites are also available for temporary insertion of additional functions which can be subsequently deleted with the replicon. Only the DNA inserted into the multiple cloning sites (passenger genes and homologous fragment for targeting) and a single FRT site (68 bp) remain in the chromosome after excision. The utility of these vectors was demonstrated by integrating Zymomonas mobilis genes encoding the ethanol pathway behind the native chromosomal adhE gene in strains of E. coli K-12 and E. coli B. With these vectors, a single antibiotic selection system can be used repeatedly for the successive improvement of E. coli strains with precise deletion of extraneous genes used during construction.
...
PMID:Chromosomal integration of heterologous DNA in Escherichia coli with precise removal of markers and replicons used during construction. 1055 84

Here, we document a technique to reduce the size of the genome of Pseudomonas putida by using a combinatorial mini-Tn5-targeted Flp-FRT recombination system. This method combines random insertions with the site-specific Flp-FRT recombination system to generate successive random deletions in a single strain in which parts of the genome are excised via the action of the cognate flippase. For this purpose, we have generated two mini-Tn5 transposon mutant libraries with single and double integrations of either mini-Tn5 KpF alone or mini-Tn5 KpF in parallel with mini-Tn5 TF, respectively. These mini-Tn5 transposons carry different selectable markers and each has an FRT (Flippase Recognition Target) site. Mapping of the position of both mini-Tn5 transposons in the chromosome of P. putida was conducted by Arbitrary Primed-PCR (AP-PCR). Subsequent sequencing of the PCR fragments led to the identification of the coordinates of the transposons and the orientation of both FRT sites. Under specific laboratory conditions, both FRT sites were recognized by the flippase, and the deletion of a nonessential intervening genomic segment along with the transposon backbones occurred without inheritance of any marker genes.
...
PMID:Streamlining of a Pseudomonas putida genome using a combinatorial deletion method based on minitransposon insertion and the Flp-FRT recombination system. 2208 47

The site-specific recombinases Cre and Flp can mutate genes in a spatially and temporally restricted manner in mice. Conditional recombination of the tumor suppressor gene p53 using the Cre-loxP system has led to the development of multiple genetically engineered mouse models of human cancer. However, the use of Cre recombinase to initiate tumors in mouse models limits the utilization of Cre to genetically modify other genes in tumor stromal cells in these models. To overcome this limitation, we inserted FRT (flippase recognition target) sites flanking exons 2-6 of the endogenous p53 gene in mice to generate a p53(FRT) allele that can be deleted by Flp recombinase. We show that FlpO-mediated deletion of p53 in mouse embryonic fibroblasts impairs the p53-dependent response to genotoxic stress in vitro. In addition, using FSF-Kras(G12D/+); p53(FRT/FRT) mice, we demonstrate that an adenovirus expressing FlpO recombinase can initiate primary lung cancers and sarcomas in mice. p53(FRT) mice will enable dual recombinase technology to study cancer biology because Cre is available to modify genes specifically in stromal cells to investigate their role in tumor development, progression and response to therapy.
...
PMID:Generation of primary tumors with Flp recombinase in FRT-flanked p53 mice. 2222 55

Genetic mosaic approach is commonly used in the Drosophila eye by completely abolishing or misexpressing a gene within a subset of cells to unravel its role during development. Classical genetic mosaic approach involves random clone generation in all developing fields. Consequently, a large sample size needs to be screened to generate and analyze clones in specific domains of the developing eye. To address domain specific functions of genes during axial patterning, we have developed a system for generating mosaic clones by combining Gal4/UAS and flippase (FLP)/FRT system which will allow generation of loss-of-function as well as gain-of-function clones on the dorsal and ventral eye margins. We used the bifid-Gal4 driver to drive expression of UAS-FLP. This reagent can have multiple applications in (i) studying spatio-temporal function of a gene during dorso-ventral (DV) axis specification in the eye, (ii) analyzing genetic epistasis of genes involved in DV patterning, and (iii) conducting genome wide screens in a domain specific manner.
...
PMID:Domain specific genetic mosaic system in the Drosophila eye. 2310 78

Genetic mosaics in Drosophila typically involve derivation of homozygous daughter cells from heterozygous precursors through mitotic recombination. MARCM (mosaic analysis with a repressible cell marker) couples loss of heterozygosity with derepression of a marker gene, permitting unique labeling of specific homozygous daughter cells. The generation of GAL80-minus homozygous daughter cells in otherwise heterozygous tissues allows GAL4-dependent activation of upstream activation sequence (UAS)-reporter specifically in the homozygous cells of interest. To make MARCM clones, organisms must carry at least five genetic elements (flippase [FLP], flippase recognition targets [FRTs], tubP-GAL80, GAL4, and UAS-marker) in specific configurations. One major application of MARCM, as described here, is to study cell-autonomous function(s) of a gene within single cells or a group of cells in otherwise unperturbed organisms. A mutation of interest distal to one FRT site is put in trans to a tubP-GAL80-containing chromosome arm that carries the same FRT. The resulting MARCM clones, which are negative for tubP-GAL80 and thus specifically marked, will become homozygous for the mutation in otherwise heterozygous organisms. By including a UAS-transgene, one can perform rescue experiments in the mutant MARCM clones. Conversely, if the mutation is placed on the same chromosome arm as tubP-GAL80, MARCM-labeled cells will be homozygous wild-type and may lie adjacent to sister cells that are homozygous mutant. This variant, called reverse MARCM, allows one to determine non-cell-autonomous effects of a mutation.
...
PMID:Reverse genetics by loss-of-function mosaic analysis in Drosophila. 2328 32

Genetic mosaics in Drosophila typically involve derivation of homozygous daughter cells from heterozygous precursors through mitotic recombination. MARCM (mosaic analysis with a repressible cell marker) couples loss of heterozygosity with derepression of a marker gene, permitting unique labeling of specific homozygous daughter cells. The generation of GAL80-minus homozygous daughter cells in otherwise heterozygous tissues allows GAL4-dependent activation of upstream activation sequence (UAS)-reporter specifically in the homozygous cells of interest. To make MARCM clones, organisms must carry at least five genetic elements (flippase [FLP], flippase recognition targets [FRTs], tubP-GAL80, GAL4, and UAS-marker) in specific configurations. In neurons whose progenitors can be efficiently targeted for mitotic recombination, genetic mosaic screens can be used to systematically uncover cell-autonomous genes that are required for development or function. This technique involves the generation of numerous FRT lines carrying various independent mutations, followed by derivation and phenotypic analysis of MARCM clones using these mutant FRT lines in combination with an MARCM-enabling stock that carries all the other genetic elements required for MARCM. Mutants of interest are recovered based on the MARCM phenotypes, which are imaged live using diverse fluorescent markers. Mutant genes that underlie the phenotypes of interest can then be identified by conventional genetics including derivation and analysis of series of recombinant chromosomes. Besides chemical mutagenesis, genes on a particular FRT chromosome may be randomly disrupted by P element insertion. This protocol describes procedures specifically used for genetic mosaic screens in the mushroom bodies (MBs).
...
PMID:Genetic mosaic screens in Drosophila mushroom bodies. 2328 33

Mouse embryonic fibroblasts (MEFs) are mesenchymal stem cell (MSC)-like multipotent progenitor cells and can undergo self-renewal and differentiate into to multiple lineages, including bone, cartilage and adipose. Primary MEFs have limited life span in culture, which thus hampers MEFs' basic research and translational applications. To overcome this challenge, we investigate if piggyBac transposon-mediated expression of SV40 T antigen can effectively immortalize mouse MEFs and that the immortalized MEFs can maintain long-term cell proliferation without compromising their multipotency. Using the piggyBac vector MPH86 which expresses SV40 T antigen flanked with flippase (FLP) recognition target (FRT) sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized. The immortalized MEFs (piMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by FLP recombinase. The piMEFs express most MEF markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages upon BMP9 stimulation in vitro. Stem cell implantation studies indicate that piMEFs can form bone, cartilage and adipose tissues upon BMP9 stimulation, whereas FLP-mediated removal of SV40 T antigen diminishes the ability of piMEFs to differentiate into these lineages, possibly due to the reduced expansion of progenitor populations. Our results demonstrate that piggyBac transposon-mediated expression of SV40 T can effectively immortalize MEFs and that the reversibly immortalized piMEFs not only maintain long-term cell proliferation but also retain their multipotency. Thus, the high transposition efficiency and the potential footprint-free natures may render piggyBac transposition an effective and safe strategy to immortalize progenitor cells isolated from limited tissue supplies, which is essential for basic and translational studies.
...
PMID:The piggyBac transposon-mediated expression of SV40 T antigen efficiently immortalizes mouse embryonic fibroblasts (MEFs). 2484 66

Biological processes can be elucidated by investigating complex networks of relevant factors and genes. However, this is not possible in species for which dominant selectable markers for genetic studies are unavailable. To overcome the limitation in selectable markers for the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes), we adapted the flippase (FLP) recombinase-recombination target (FRT) site-specific recombination system from the yeast Saccharomyces cerevisiae as a selectable marker recycling system for this fungus. Taking into account practical applicability, we designed FLP/FRT modules carrying two FRT sequences as well as the flp gene adapted to the pathogenic yeast Candida albicans (caflp) or a synthetic codon-optimized flp (avflp) gene with neomycin resistance (nptII) cassette for one-step marker excision. Both flp genes were under control of the Trichophyton rubrum copper-repressible promoter (PCTR4). Molecular analyses of resultant transformants showed that only the avflp-harbouring module was functional in A. vanbreuseghemii. Applying this system, we successfully produced the Ku80 recessive mutant strain devoid of any selectable markers. This strain was subsequently used as the recipient for sequential multiple disruptions of secreted metalloprotease (fungalysin) (MEP) or serine protease (SUB) genes, producing mutant strains with double MEP or triple SUB gene deletions. These results confirmed the feasibility of this system for broad-scale genetic manipulation of dermatophytes, advancing our understanding of functions and networks of individual genes in these fungi.
...
PMID:Flippase (FLP) recombinase-mediated marker recycling in the dermatophyte Arthroderma vanbreuseghemii. 2499 27

Genetically engineered mouse models (GEMMs) have dramatically improved our understanding of tumor evolution and therapeutic resistance. However, sequential genetic manipulation of gene expression and targeting of the host is almost impossible using conventional Cre-loxP-based models. We have developed an inducible dual-recombinase system by combining flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies to improve GEMMs of pancreatic cancer. This enables investigation of multistep carcinogenesis, genetic manipulation of tumor subpopulations (such as cancer stem cells), selective targeting of the tumor microenvironment and genetic validation of therapeutic targets in autochthonous tumors on a genome-wide scale. As a proof of concept, we performed tumor cell-autonomous and nonautonomous targeting, recapitulated hallmarks of human multistep carcinogenesis, validated genetic therapy by 3-phosphoinositide-dependent protein kinase inactivation as well as cancer cell depletion and show that mast cells in the tumor microenvironment, which had been thought to be key oncogenic players, are dispensable for tumor formation.
...
PMID:A next-generation dual-recombinase system for time- and host-specific targeting of pancreatic cancer. 2584 72

The Burkholderia cepacia complex (Bcc) displays a wealth of metabolic diversity with great biotechnological potential, but the utilization of these bacteria is limited by their opportunistic pathogenicity to humans. The third replicon of the Bcc, megaplasmid pC3 (0.5 to 1.4 Mb, previously chromosome 3), is important for various phenotypes, including virulence, antifungal, and proteolytic activities and the utilization of certain substrates. Approximately half of plasmid pC3 is well conserved throughout sequenced Bcc members, while the other half is not. To better locate the regions responsible for the key phenotypes, pC3 mutant derivatives of Burkholderia cenocepacia H111 carrying large deletions (up to 0.58 Mb) were constructed with the aid of the FLP-FRT (FRT, flippase recognition target) recombination system from Saccharomyces cerevisiae The conserved region was shown to confer near-full virulence in both Caenorhabditis elegans and Galleria mellonella infection models. Antifungal activity was unexpectedly independent of the part of pC3 bearing a previously identified antifungal gene cluster, while proteolytic activity was dependent on the nonconserved part of pC3, which encodes the ZmpA protease. To investigate to what degree pC3-encoded functions are dependent on chromosomally encoded functions, we transferred pC3 from Burkholderia cenocepacia K56-2 and Burkholderia lata 383 into other pC3-cured Bcc members. We found that although pC3 is highly important for virulence, it was the genetic background of the recipient that determined the pathogenicity level of the hybrid strain. Furthermore, we found that important phenotypes, such as antifungal activity, proteolytic activity, and some substrate utilization capabilities, can be transferred between Bcc members using pC3.IMPORTANCE The Burkholderia cepacia complex (Bcc) is a group of closely related bacteria with great biotechnological potential. Some strains produce potent antifungal compounds and can promote plant growth or degrade environmental pollutants. However, their agricultural potential is limited by their opportunistic pathogenicity, particularly for cystic fibrosis patients. Despite much study, their virulence remains poorly understood. The third replicon, pC3, which is present in all Bcc isolates and is important for pathogenicity, stress resistance, and the production of antifungal compounds, has recently been reclassified from a chromosome to a megaplasmid. In this study, we identified regions on pC3 important for virulence and antifungal activity and investigated the role of the chromosomal background for the function of pC3 by exchanging the megaplasmid between different Bcc members. Our results may open a new avenue for the construction of antifungal but nonpathogenic Burkholderia hybrids. Such strains may have great potential as biocontrol strains for protecting fungus-borne diseases of plant crops.
...
PMID:Use of Synthetic Hybrid Strains To Determine the Role of Replicon 3 in Virulence of the Burkholderia cepacia Complex. 2843 94


1 2 Next >>

---