FLP Recombination Research Articles

Urea transport in MDCK cells that are stably transfected with UT-A1.

Progress in understanding the cell biology of urea transporter proteins has been hampered by the lack of an appropriate cell culture system. The goal of this study was to create a polarized epithelial cell line that stably expresses the largest of the rat renal urea transporter UT-A isoforms, UT-A1. The gene for UT-A1 was cloned into pcDNA5/FRT and transfected into Madin-Darby canine kidney (MDCK) cells with an integrated Flp recombination target site. The cells from a single clone were grown to confluence on collagen-coated membranes until the resistance was >1,500 Omega.cm(2). Transepithelial [(14)C]urea fluxes were measured at 37 degrees C in a HCO(3)(-)/CO(2) buffer, pH 7.4, with 5 mM urea. The baseline fluxes were not different between unstimulated UT-A1-transfected MDCK cells and nontransfected or sham-transfected MDCK cells. However, only in the UT-A1-transfected cells was UT-A1 protein expressed (as measured by Western blot analysis) and urea transport stimulated by forskolin or arginine vasopressin. Forskolin and arginine vasopressin also increased the phosphorylation of UT-A1. Thionicotinamide, dimethylurea, and phloretin inhibited the forskolin-stimulated [(14)C]urea fluxes in the UT-A1-transfected MDCK cells. These characteristics mimic those seen in rat terminal inner medullary collecting ducts. This new polarized epithelial cell line stably expresses UT-A1 and reproduces several of the physiological responses observed in rat terminal inner medullary collecting ducts.

Global changes in Kaposi's sarcoma-associated virus gene expression patterns following expression of a tetracycline-inducible Rta transactivator.

An important step in the herpesvirus life cycle is the switch from latency to lytic reactivation. In order to study the life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV), we developed a gene expression system in KSHV-infected primary effusion lymphoma cells. This system uses Flp-mediated efficient recombination and tetracycline-inducible expression. The Rta transcriptional activator, which acts as a molecular switch for lytic reactivation of KSHV, was efficiently integrated downstream of the Flp recombination target site, and its expression was tightly controlled by tetracycline. Like stimulation with tetradecanoyl phorbol acetate (TPA), the ectopic expression of Rta efficiently induced a complete cycle of viral replication, including a well-ordered program of KSHV gene expression and production of infectious viral progeny. A striking feature of Rta-mediated lytic gene expression was that Rta induced KSHV gene expression in a more powerful and efficient manner than TPA stimulation, indicating that Rta plays a central, leading role in KSHV lytic gene expression. Thus, our streamlined gene expression system provides a novel means not only to study the effects of viral gene products on overall KSHV gene expression and replication, but also to understand the natural viral reactivation process.

Stepwise Manipulation of DNA Specificity in Flp Recombinase: Progressively Adapting Flp to Individual and Combinatorial Mutations in its Target Site

The Flp protein from Saccharomyces cerevisiae is one of the site-specific tyrosine family recombinases that are used widely in genomic engineering. As a first step towards mediating directed DNA rearrangements at non-native Flp recombination targets ( mFRTs), we have evolved three separate groups of Flp variants that preferentially act on mFRTs containing substitutions at the first, seventh or both positions of the Flp-binding elements. The variants that recombine the double-mutant mFRT contain a subset of the mutations present in those that are active on the single-mutant mFRTs, plus additional mutations. Specificity for and discrimination between target sites, effected primarily by amino acid residues that contact DNA, can be modulated by those that do not interact with DNA or with a DNA-contacting residue. The degree of modulation can range from relaxed DNA specificity to almost completely altered specificity. Our results suggest that combined DNA shuffling and mutagenesis of libraries of Flp variants active on distinct mFRTs can yield variants that can recombine mFRTs containing combinations of the individual mutations.

Symmetric DNA sites are functionally asymmetric within Flp and Cre site-specific DNA recombination synapses.

Flp and Cre-mediated recombination on symmetrized FRT and loxP sites, respectively, in circular plasmid substrates yield both DNA inversion and deletion. However, upon sequestering three negative supercoils outside the recombination complex using the resII-resIII synapse formed by Tn3 resolvase and the LER synapse formed by phage Mu transposase in the case of Flp and Cre, respectively, the reactions are channeled towards inversion at the expense of deletion. The inversion product is a trefoil, its unique topology being conferred by the external resolvase or LER synapse. Thus, Flp and Cre assign their symmetrized substrates a strictly antiparallel orientation with respect to strand cleavage and exchange. These conclusions are supported by the product profiles from tethered parallel and antiparallel native FRT sites in dilution and competition assays. Furthermore, the observed recombination bias favoring deletion over inversion in a nicked circular substrate containing two symmetrized FRT sites is consistent with the predictions from Monte Carlo simulations based on antiparallel synapsis of the DNA partners.

FLP and Cre recombinase function in Xenopus embryos.

The use of the site-specific DNA recombinases FLP and Cre is well-established in a broad range of organisms. Here we investigate the applicability of both recombinases to the Xenopus system where they have not been analyzed yet. We show that injection of FLP mRNA triggers the excision of an FLP recombination target (FRT)-flanked green fluorescent protein (GFP) sequence in a coinjected reporter construct inducing the expression of a downstream beta-galactosidase gene (lacZ). The FLP-mediated gene activation can be controlled in Xenopus embryos by injecting a mRNA encoding a fusion of FLP to the mutant ligand binding domain of the human estrogen receptor whose activity is dependent on 4-hydroxytamoxifen. We also demonstrate that a Cre reporter injected into fertilized eggs is fully recombined by Cre recombinase before zygotic gene transcription initiates. Our results indicate that in Xenopus embryos Cre is more effective than FLP in recombining a given quantity of reporter molecules. Finally, we present FLP-inducible double reporter systems encoding two fluorescence proteins (EYFP, ECFP, DsRed or GFP). These novel gene expression systems enable the continuous analysis of two reporter activities within living embryos and are expected to allow cell-lineage studies based on recombinase-mediated DNA rearrangement in transgenic Xenopus lines.

Establishment and application of both FLP and Cre site-specific recombination systems at the same position in the genome

Both FRT-FRT and LoxP-LoxP sites that are the target sequences of site-specific recombinases have been constructed in a vector, called C4LFY, using the recombinant DNA technique. C4LFY also contains P elements, 2 exons and 1 intron of Drosophilayellow gene,yellow promoter and enhancers, and flanking DNA. Since C4LFY made use of two pairs of FRT and LoxP sites, this vector included two site-specific recombination systems. C4LFY was then integrated into Drosophila genome by P-element-mediated germ line transformation. In the presence of the FLP or Cre recombinase, either FLP/FRT or Cre/LoxP recombination reaction was successfully created at the same position in the genome. Using this system, the molecular basis ofyellow gene expression and regulation during development have been investigated. Results indicate that the tissue-specific expression ofyellow gene is directly regulated by transcriptional enhancers. In addition, the 5′ and 3′ genomic sequences flanking theyellow gene have been preliminarily studied and their potential role is discussed.

Quantitative comparison of DNA looping in vitro and in vivo: chromatin increases effective DNA flexibility at short distances.

The probability that two sites on a linear DNA molecule will contact each other by looping depends on DNA flexibility. Although the flexibility of naked DNA in vitro is well characterized, looping in chromatin is poorly understood. By extending existing theory, we present a single equation that describes DNA looping over all distances. We also show that DNA looping in vitro can be measured accurately by FLP recombination between sites from 74 bp to 15 kb apart. In agreement with previous work, a persistence length of 50 nm was determined. FLP recombination of the same substrates in mammalian cells showed that chromatin increases the flexibility of DNA at short distances, giving an apparent persistence length of 27 nm.

DNA-sequence asymmetry directs the alignment of recombination sites in the FLP synaptic complex

The FLP recombinase promotes site-specific recombination in the 2 μm circle of Saccharomyces cerevisiae. FLP recognizes a 48 bp target site (FLP recombination target, or FRT) consisting of three 13 bp protein binding sites, or symmetry elements, flanking an 8 bp spacer region. Efficient recombination also occurs with DNA substrates that have minimal FRT sites, consisting only of the spacer and two surrounding 13 bp symmetry elements arranged in inverse orientation; thus, the wild-type spacer sequence is the main asymmetric feature of the minimal recombination site. FLP carries out recombination with many minimal target sites bearing symmetric or asymmetric mutant spacer sequences; however, the overall directionality of recombination defined in terms of inversion or excision of a DNA domain is determined by spacer-sequence asymmetry. In order to evaluate the potential influence of spacer-sequence asymmetry on structures formed during early steps in recombination, we used electron microscopy to investigate the structure of the FLP synaptic complex, which is the intermediate protein-DNA complex involved in site pairing and strand exchange. Using linear substrate DNAs that have minimal FRTs with wild-type spacer sequences, we find that 85 to 90 % of the FLP synaptic complexes examined contain the two FRTs aligned in parallel. This strong preference for parallel site alignment stands in contrast with prevailing models for λ integrase-class recombination systems, which postulate antiparallel site alignment, and results from biophysical studies on synthetic, immobile four-way DNA junctions. Our results show that the strong preference for parallel alignment can be attributed to conformational preferences of Holliday junctions present in the synaptosome.

Different positioning of the ligand-binding domain helix 12 and the F domain of the estrogen receptor accounts for functional differences between agonists and antagonists.

The estrogen receptor is capable of binding a diverse set of ligands that are broadly categorized as agonists or antagonists, depending on their abilities to induce or interfere with transcriptional responsiveness. We show, using a fusion protein assay for ligand-binding which does not rely on transcriptional responsiveness, that agonists and antagonists differently position the C-terminus of the ligand-binding domain (helix 12) and the F domain. Upon antagonist binding, the F domain interferes with the fusion protein activity. Mutational disruption of helix 12 alters the position of the F domain, imposing interference after agonist or antagonist binding. Genetically selected inversion mutations where only agonists, but not antagonists, induce interference are similarly reliant on helix 12 and F domain positioning. Our results demonstrate that agonists and antagonists differently position helix 12 and implicate the F domain in mechanisms of antagonist action.

A simple assay to determine the functionality of Cre or FLP recombination targets in genomic manipulation constructs.

We report the construction of two Escherichia coli strains (294-Cre and 294-FLP) which express either Cre- or FLP-recombinase. Plasmids containing authentic recognition targets for either recombinase (loxPs or FRTs) are recombined when propagated in the appropriate strain. 294-Cre and 294-FLP thus provide a simple test for the recombination competence of constructs that are designed for use in Cre- or FLP-mediated genomic manipulations.

A Protein Dissociation Step Limits Turnover in FLP Recombinase-mediated Site-specific Recombination

When two ongoing FLP-mediated recombination reactions are mixed, formation of cross-products is subject to a lag of several minutes, and the subsequent rate of cross-product formation is greatly reduced relative to normal reaction progress curves. The lag reflects the formation of a stable complex containing multiple FLP monomers and two FLP recombination target-containing DNA recombination products, a process completed within 5-10 min after addition of FLP recombinase to a reaction mixture. The reaction products are sequestered within this complex for an extended period of time, unavailable for further reaction. The length of the lag increases with increasing FLP protein concentration and is not affected by the introduction of unreacted (non FLP-bound) substrate. The results provide evidence that disassembly of FLP complexes from products occurs in a minimum of two steps. At least one FLP protein monomer is released from reaction complexes in a discrete step that leaves the reaction products sequestered. The recombination products are released in a form free to react with other FLP recombination target-containing DNA molecules only after at least one additional dissassembly step. One or both of these disassembly steps are rate limiting for reaction turnover under conditions often used to monitor FLP-mediated recombination in vitro.

Junction Mobility and Resolution of Holliday Structures by Flp Site-specific Recombinase: TESTING PARTNER COMPATIBILITY DURING RECOMBINATION

Absolute homology between partner substrates within the strand exchange region (spacer) is an essential requirement for recombination mediated by the yeast site-specific recombinase Flp. Recent experiments suggest that 3-base pair homology adjacent to the points of exchange at each end of the spacer is utilized in a base complementarity-dependent strand joining reaction. Homology of the central 2 base pairs of the spacer is also critical, but how homology is tested at these two positions is unknown. We have addressed the role of homology-dependent branch migration in Flp recombination by assaying strand cleavage and resolution in a set of synthetic Holliday junctions in which the branch point is freely or partially mobile through the spacer, or is immobilized at each position within the spacer or immediately flanking it. A strong bias in the direction of Holliday resolution is observed only when the branch point is located just outside the spacer (at the junction of the Flp binding element and the spacer). A significantly smaller bias is noticed when the branch point is frozen immediately adjacent to this position within the spacer. Resolution in these cases is most often mediated by exchange of the scissile phosphodiesters at the branch point or proximal to it, and rarely by exchange of the scissile phosphodiesters distal to it. In light of these and previous results, we discuss possible checkpoints for testing partner compatibility during Flp recombination.

Role of Partner Homology in DNA Recombination: COMPLEMENTARY BASE PAIRING ORIENTS THE 5′-HYDROXYL FOR STRAND JOINING DURING Flp SITE-SPECIFIC RECOMBINATION (∗)

Absolute homology between partner substrates within the strand exchange region is an essential requirement for recombination mediated by the yeast site-specific recombinase Flp. Using combinations of specially designed half- and full-site Flp substrates, we demonstrate that the strand joining step of recombination is exquisitely sensitive to spacer homology. At each exchange point, 2-3 spacer nucleotides adjacent to the nick within the cleaved strand of one substrate must base pair with the corresponding segment of the un-nicked strand from the second substrate for efficient strand joining in the recombinant mode. In accordance with the "cis-activation/trans-nucleophilic attack" model for each of the two transesterification steps of Flp recombination (strand cleavage and strand joining), we propose that the limited strand pairing orients the DNA-nucleophile (5'-hydroxyl) for attack on its target diester (3'-phosphotyrosyl-Flp). During one round of recombination, 4-6 terminal base pairs of the spacer (2-3 base pairs at each spacer end) must unpair, following strand cleavage, within a DNA substrate and pair with the partner substrate prior to strand union. In this model, the extent of branch migration of the covalently closed Holliday intermediate is limited to the central core of the spacer. The templated positioning of reactive nucleic acid groups (which is central to the model) may be utilized by other recombination systems and by RNA splicing reactions.

Functional expression of the yeast FLP/FRT site-specific recombination system in Nicotiana tabacum.

The FLP/FRT site-specific recombination system of Saccharomyces cerevisiae was expressed in stably transformed tobacco plants. The FLP protein efficiently catalyzes recombination between two directly repeated FLP recombination target (FRT) sites, deleting the sequence between them. In the constructs tested here, this deletion places the CaMV 35S promoter adjacent to a hygromycin resistance marker; transcriptional activation of the marker allows direct selection of recombination events. After crossing plants containing an integrated FLP expression construct with plants containing a FLP substrate, F1 seedlings can be selected directly for hygromycin resistance, indicating that recombination occurs at, or very early after zygote formation. Molecular analysis confirmed the expected recombination product.

Phosphoryl transfer in Flp recombination: a template for strand transfer mechanisms

The basic chemistry involved in DNA recombination, RNA splicing and DNA transposition is a phosphoryl transfer reaction. This review is an attempt to provoke a unified thinking on the reaction mechanisms in these nucleic acid transactions. Some of the recent results with the Flp site-specific recombinase that reveal how the chemical reactivity for recombination is derived from cooperative protein-subunit interactions on the DNA substrate are discussed. At least some of the features of Flp reaction are likely to have global implications in other DNA and RNa strand-transfer systems.

Chapter 33 Ectopic Expression in Drosophila

Publisher Summary There are now several different methods for ectopic expression in Drosophila , each with its merits and its limitations. The first technique is to drive expression of a gene using the transcriptional regulatory sequences from a defined promoter. Tissue-specific promoters allow transcription to be restricted to a defined subset of cells. The second method is to drive expression of a gene from a heat-shock promoter. A gene can then be turned on at a specific point in development by heat shocking the transgenic animal. A more recent inducible technique for ectopic expression relies on site-specific recombination catalyzed by the flp recombinase from Saccharomyces cereuisiae . flp can promote recombination between two flp recombination targets, or FRTs. Advantages of the flp /FRT system are that it is inducible and expression can be activated in any cell in the organism, although dividing cells may be favored. A disadvantage of the method is that, because the clones are generated randomly, ectopic expression varies from animal to animal. The chapter describes protocols for two of the techniques that are currently used to conduct ectopic expression experiments: the heat-shock method and the GAL4 system.

Simple and efficient generation of marked clones in Drosophila.

Cell lineage analysis and mosaic analysis of mutations are important techniques that are used to study the development of many organisms. Unfortunately, the methods employed for such analyses are usually inefficient, technically demanding or labor intensive. In Drosophila, the most common methodology used for the generation of mosaic animals is mitotic recombination, which is induced by X-rays. Although this technique is simple, it has the undesirable characteristics of a low efficiency and a high rate of cell death. Furthermore, although a large number of marker systems has been employed to detect mitotic recombinants, none allows easy identification of clones for all cell types. A system is described here that allows a highly efficient generation of clones with the concomitant expression of an easily detectable cellular marker. This method can be applied to cell lineage and mosaic analysis in Drosophila. The site-specific yeast FLP recombinase, under the control of a heat shock-inducible promoter, efficiently catalyses mitotic recombination specifically at the site of a FLP recombination target (FRT). In this system, recombination fuses the alpha-tubulin promoter to the lacZ gene, allowing transcription of the marker. Recombinant cells and their progeny can, therefore, be detected by standard assays for beta-galactosidase. Of particular importance is the fact that only the cells of interest stain, thus allowing their simple detection in any tissue. We demonstrate that, by intermolecular recombination, we can use FLIP recombinase to generate marked clones efficiently in embryonic, larval and adult tissues. This simple and efficient technique is well suited to cell-lineage analysis and can be easily extended to the generation and detection of mutant clones in mosaic animals.

Activity of yeast FLP recombinase in maize and rice protoplasts.

We have demonstrated that a yeast FLP/FRT site-specific recombination system functions in maize and rice protoplasts. FLP recombinase activity was monitored by reactivation of beta-glucuronidase (GUS) expression from vectors containing the gusA gene inactivated by insertion of two FRTs (FLP recombination targets) and a 1.31 kb DNA fragment. The stimulation of GUS activity in protoplasts cotransformed with vectors containing FRT inactivated gusA gene and a chimeric FLP gene depended on both the expression of the FLP recombinase and the presence and structure of the FRT sites. The FLP enzyme could mediate inter- and intramolecular recombination in plant protoplasts. These results provide evidence that a yeast recombination system can function efficiently in plant cells, and that its performance can be manipulated by structural modification of the FRT sites.

DNA cleavage in trans by the active site tyrosine during Flp recombination: Switching protein partners before exchanging strands

Each recombination event mediated by the Flp recombinase is the sum of four strand breakage and reunion reactions executed in two steps of two-strand exchanges. The reaction requires four Flp monomers. The key catalytic residue in Flp is Tyr-343. Arg-191, His-305, and Arg-308 appear to facilitate the cleavage and exchange steps of recombination. These four residues constitute the invariant tetrad of the Int family site-specific recombinases. Complementation tests between “step-arrest” mutants of Flp suggest that each Flp protomer harbors a “fractional active site.” Hybrid “half site-recombinase” complexes reveal that efficient catalysis occurs when the Arg-His-Arg triad is present on one Flp monomer and the active site Tyr on a second monomer. Strand cleavage by an Flp monomer occurs virtually exclusively on the half site to which its partner protein is bound (cleavage in trans), and almost never on the half site to which it is bound (cleavage in cis). Trans-cleavage by Flp can provide a means for functionally exchanging Flp monomers between two DNA partners. Such a mechanism would be germane to recombination, since cleavage and rejoining in cis can only restore the parental substrate configuration and cannot yield recombinants.

Mutagenesis of a conserved region of the gene encoding the FLP recombinase of Saccharomyces cerevisiae: A role for arginine 191 in binding and ligation

The FLP recombinase from the 2 μm plasmid of Saccharomyces cerevisiae contains a region from amino acid 185 to 203 that is conserved among several FLP-like proteins from different yeasts. Using site-directed mutagenesis, we have made mutations in this region of the FLP gene. Five of twelve mutations in the region yielded proteins that were unable to bind to the FLP recombination target (FRT) site. A change of arginine at position 191 to lysine resulted in a protein (FLP-R191K) that could bind to the FRT site but could not catalyze recombination. This mutant protein accumulated as a stable protein-DNA complex in which one of the two bound FLP proteins was covalently attached to the DNA. FLP-R191K was defective in strand exchange and ligation and was unable to promote protein-protein interaction with half-FRT sites. The conservation of three residues in all members of the integrase family of site-specific recombinases (His305, Arg308, Tyr343 in FLP) implies a common mechanism of recombination. The conservation of arginine 191 and the properties of the FLP-R191K mutant protein suggest that this arginine also plays an important role in the mechanism of FLP-mediated site-specific recombination.