Gene deletion from Plasmodium falciparum using FLP and Cre recombinases: Implications for applied site-specific recombination

The ability to manipulate the genome and induce site-specific recombination using either Flippase (FLP) or Cre recombinase has been useful in many systems including Plasmodium berghei for specific deletion events or to obtain conditional gene expression. To test whether these recombinases are active in Plasmodium falciparum we constructed gene knockouts that contain sequences recognised as templates for site-specific recombination. We tested the ability of FLP and Cre recombinases, expressed conditionally in P. falciparum, to mediate deletion of the human dihydrofolate reductase (hdhfr) drug resistance gene. We show that Cre recombinase is capable of efficient removal of hdhfr by site-specific recombination. In contrast, FLP recombinase is very inefficient, even at the optimum temperature of 30 °C for this enzyme. These results demonstrate that Cre recombinase can be utilised in P. falciparum for deletion of specific sequences such as drug resistance genes. This can be exploited for recycling of drug resistance cassettes and for the design of specific recombination events in P. falciparum.     

FLP recombinase-mediated site-specific recombination in rice

The feasibility of using the FLP/ FRT site-specific recombination system in rice for genome engineering was evaluated. Transgenic rice plants expressing the FLP recombinase were crossed with plants harbouring the kanamycin resistance gene ( neomycin phosphotransferase II , nptII ) flanked by FRT sites, which also served to separate the corn ubiquitin promoter from a promoterless gusA . Hybrid progeny were tested for excision of the nptII gene and the positioning of the ubiquitin promoter proximal to gusA . While the hybrid progeny from various crosses exhibited β-glucuronidase (GUS) expression, the progeny of selfed parental rice plants did not show detectable GUS activity. Despite the variable GUS expression and incomplete recombination displayed in hybrids from some crosses, uniform GUS staining and complete recombination were observed in hybrids from other crosses. The recombined locus was shown to be stably inherited by the progeny. These data demonstrate the operation of FLP recombinase in catalysing excisional DNA recombination in rice, and confirm that the FLP/ FRT recombination system functions effectively in the cereal crop rice. Transgenic rice lines expressing active FLP recombinase generated in this study provide foundational stock material, thus facilitating the future application and development of the FLP/ FRT system in rice genetic improvement.     

Gene conversion in transgenic maize plants expressing FLP/FRT and Cre/loxP site-specific recombination systems

DNA recombination reactions (site-specific and homologous) were monitored in the progeny of transgenic maize plants by bringing together two recombination substrates (docking sites and shuttle vectors) in the zygotes. In one combination of transgenic events, the recombination marker gene (yellow fluorescent protein gene, YFP) was activated in 1%-2% of the zygotes receiving both substrates. In other crosses, chimeric embryos and plants were identified, indicative of late recombination events taking place after the first mitotic division of the zygotes. The docking site structure remained unchanged; therefore, all recovered recombination events were classified as gene conversions. The recombinant YFP-r gene segregated as a single locus in subsequent generations. The recombination products showed evidence of homologous recombination at the 5' end of the YFP marker gene and recombinational rearrangements at the other end, consistent with the conclusion that DNA replication was involved in generation of the recombination products. Here, we demonstrate that maize zygotes are efficient at generating homologous recombination products and that the homologous recombination pathways may successfully compete with other possible DNA repair/recombination mechanisms such as site-specific recombination. These results indicate that maize zygotes provide a permissive environment for homologous recombination, offering a new strategy for gene targeting in maize.     

Characterization of Holliday structures in FLP protein-promoted site-specific recombination.

Holliday structures are formed in the course of FLP protein-promoted site-specific recombination. Here, we demonstrate that Holliday structures are formed in reactions involving wild-type substrates and that they are kinetically competent with respect to the overall reaction rate. Together with a previous demonstration of chemical competence (L. Meyer-Leon, L.-C. Huang, S. W. Umlauf, M. M. Cox, and R. B. Inman, Mol. Cell. Biol. 8:3784-3796, 1988), Holliday structures therefore meet all criteria necessary to establish that they are obligate reaction intermediates in FLP-mediated site-specific recombination. In addition, kinetic evidence suggests that two distinct forms of the Holliday intermediate are present in the reaction pathway, interconverted in an isomerization process that is rate limiting at 0 degree C.     

Holliday intermediates and reaction by-products in FLP protein-promoted site-specific recombination.

Holliday structures are formed and resolved by FLP protein during site-specific recombination. These structures have been isolated and are visualized in both native and partially denatured states by electron microscopy. No single-strand breaks are found within the junction, indicating that the structure results from a reciprocal exchange of strands. These structures have properties consistent with being reaction intermediates. Double-strand cleavage products and "Y structures" are also detected and appear to be by-products of the reaction. The Y structures are three-armed branched molecules with a covalently closed junction located at the FLP recombination target site. Models are discussed, suggesting that both of these novel structures are made by aberrant cleavages during formation and resolution of the Holliday intermediate.     

A general model for site-specific recombination by the integrase family recombinases.

We present here a general model for integrase family site-specific recombination using the geometric relationships of the cleavable phosphodiester bonds and the disposition of the recombinase monomers (defined by their binding planes) with respect to them. The 'oscillation model' is based largely on the conformations of the recombinase-bound DNA duplexes and their dynamics within Holliday junctions. The duplex substrate or the Holliday junction intermediate is capable of 'oscillating' between two cleavage-competent asymmetric states with respect to corres-ponding chemically inert 'equilibrium positions'. The model accommodates several features of the Flp system and predicts two modes of DNA cleavage during a normal recombination event. It is equally applicable to other systems that mediate recombination across 6, 7 or 8 bp long strand exchange regions (or spacers). The model is consistent with approximately 0-1, 1-2 and 2-3 bp of branch migration during recombination reactions involving 6, 7 and 8 bp spacers, respectively.     

Protein-based asymmetry and protein-protein interactions in FLP recombinase-mediated site-specific recombination

When the FLP recombination target (FRT) is cut in half so that only one FLP protein-binding site is present, FLP protein forms a complex in which two such sites are linked head to head. Although held together exclusively by noncovalent interactions, this complex survives electrophoresis in an agarose gel and exhibits a half-life that can be measured in hours. Characterization of this complex indicates that a very stable, asymmetric dimeric complex of FLP protein monomers bound to the FRT is a likely early intermediate in FLP-mediated site-specific recombination. The apparent asymmetry is a property of the protein components of the complex. Even though the DNA components form a perfect palindrome, only one of the two possible DNA cleavage steps takes place in the course of complex formation. Formation of this complex does not occur with half-FRT site DNA substrates that preclude head to head monomer contact or when a FLP mutant protein is used that binds the FRT site but cannot cleave it. Trimeric and tetrameric complexes are also observed, the latter at very low frequency. These results are discussed in terms of an expanded model for early events in FLP-mediated site-specific recombination.     

High-efficiency FLP and PhiC31 site-specific recombination in mammalian cells

DNA site-specific recombinases (SSRs) such as Cre, FLPe, and phiC31, are powerful tools for analyzing gene function in vertebrates. While the availability of multiple high-efficiency SSRs would facilitate a wide array of genomic engineering possibilities, efficient recombination in mammalian cells has only been observed with Cre recombinase. Here we report the de novo synthesis of mouse codon-optimized FLP (FLPo) and PhiC31 (PhiC31o) SSRs, which result in recombination efficiencies similar to Cre.     

Interactions of the site-specific recombinases XerC and XerD with the recombination site dif.

The Xer site-specific recombination system of Escherichia coli is involved in the stable inheritance of circular replicons. Multimeric replicons, produced by homologous recombination, are converted to monomers by the action of two related recombinases XerC and XerD. Site-specific recombination at a locus, dif, within the chromosomal replication terminus region is thought to convert dimeric chromosomes to monomers, which can then be segregated prior to cell division. The recombinases XerC and XerD bind cooperatively to dif, where they catalyse recombination. Chemical modification of specific bases and the phosphate-sugar backbone within dif was used to investigate the requirements for binding of the recombinases. Site-directed mutagenesis was then used to alter bases implicated in recombinase binding. Characterization of these mutants by in vitro recombinase binding and in vivo recombination, has demonstrated that the cooperative interactions between XerC and XerD can partially overcome DNA alterations that should interfere with specific recombinase-dif interactions.     

Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells

Background  

Cell-permeant Cre DNA site-specific recombinases provide an easily controlled means to regulate gene structure and function in living cells. Since recombination provides a stable and unambiguous record of protein uptake, the enzyme may also be used for quantitative studies of cis- and trans-acting factors that influence the delivery of proteins into cells.     

Step-arrest mutants of FLP recombinase: implications for the catalytic mechanism of DNA recombination.

The site-specific recombinase (FLP) encoded by the yeast plasmid 2 micron circle belongs to the integrase (of phage lambda) family of recombinases. The sparse homology within the members of this family contrasts with the invariance of three residues, His-396, Arg-399, and Tyr-433 (the numbers correspond to the family alignment positions), among them. We report here results on substrate recognition and catalysis by FLP proteins altered at these residues. Mutations of the conserved His and Tyr that aborted the reaction at specific steps of catalysis permitted genetic dissection of the possible biochemical steps of recombination. We provide indirect evidence that recombination by FLP proceeds through a Holliday junction intermediate.     

Bacteriophage P1 Cre-loxP site-specific recombination. Site-specific DNA topoisomerase activity of the Cre recombination protein

Site-specific recombination in bacteriophage P1 occurs between two loxP sites in the presence of the Cre recombination protein. The structure of the 34-base pair loxP site consists of two 13-base pair inverted repeats separated by an 8-base pair spacer region. A mutation in the loxP site has been constructed which deletes one of the internal bases of the spacer region at the axis of dyad symmetry. This mutant loxP site shows a 10-fold reduction in recombination activity with a wild-type site both in vivo and in vitro. This low level of intramolecular recombination between a wild-type loxP site and the mutant loxP501 site is observed in vitro only when the DNA substrate is supercoiled. The majority of the supercoiled substrate is relaxed by the Cre protein, and on longer incubations, single-stranded nicks accumulate in the DNA. We have determined that these nicks occur in both the wild-type and the mutant sites. The positions of these nicks correspond to the positions of cleavage found during recombination of two wild-type sites, suggesting that the Cre protein is attempting to carry out recombination with the mutant site but most of the time this reaction is abortive. We have determined that the Cre protein relaxes a supercoiled topoisomer of a DNA substrate containing one wild-type site and one mutant site to yield a distribution of topoisomers whose linking numbers differ by steps of one, indicating that Cre can act as a type I topoisomerase.     

Reactions between half- and full-FLP recombination target sites. A model system for analyzing early steps in FLP protein-mediated site-specific recombination.

The FLP recombination target (FRT) can be cut in half so that only one FLP protein binding site is present (a "half site"). FLP protein binds the half sites and joins them into dimeric, asymmetric head-to-head complexes held together chiefly by strong noncovalent interactions. These complexes react with full (normal) FRT sites to generate a variety of products. Analysis of these DNA species reveals that the reaction follows a well-defined reaction pathway that generally parallels the normal reaction pathway. The system is useful in analyzing early steps in recombination, since the identity of the products in a given recombination event unambiguously pinpoints the order in which the cleavage and strand exchange reactions occur. Two conclusions are derived from the present study: (i) Formation of the dimeric head-to-head complex of half sites is a prerequisite to further steps in recombination. (ii) The identity of the base pairs at positions 6 and -6 within the FRT site has a subtle effect in directing the first strand exchange event in the reaction to predominantly one of two possible cleavage sites. In addition, results are presented that suggest that a DNA-DNA pairing intermediate involving only two base pairs of the core sequence is formed prior to the first cleavage and strand exchange. DNA-DNA interactions may therefore not be limited to the isomerization step that follows the first strand exchange.     

The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome

We have transferred the site-specific recombination system of the yeast 2 micron plasmid, the FLP recombinase and its recombination targets (FRTs), into the genome of Drosophila. Flies were transformed with an FLP gene under the control of hsp70 regulatory sequences and with a white gene flanked by FRTs. The heat-induced recombinase catalyzes recombination between FRTs, causing loss of white (seen somatically as white patches in the eye) and, less frequently, gain of white (seen as dark-red patches). Loss and gain frequencies vary with the severity of the heat shock, and patterns of mosaicism vary with the developmental stage at which the heat shock is applied. The recombinase is also active in the germline, producing white-eyed and dark-red-eyed progeny.     

Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein

Bacteriophage P1 encodes a site-specific recombination system that consists of a site (loxP) at which recombination occurs and a gene, cre, whose protein product is essential for recombination. The loxP-Cre recombination event can be studied in greater detail by the use of an in vitro system that efficiently carries out recombination between two loxP sites. This paper presents a purification and characterization of the Cre protein (Mr = 35,000), which is the only protein required for the in vitro reaction. No high energy cofactors are needed. The purified Cre protein binds to loxP-containing DNA and makes complexes that are resistant to heparin. Cre efficiently converts 70% of the DNA substrate to products and appears to act stoichiometrically. The action of Cre on a loxP2 supercoiled substrate containing two directly repeated loxP sites results in product molecules that are topologically unlinked. Several models to account for the ability of Cre to produce free supercoiled products are discussed.     

Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination

Cre and Flp site-specific recombinase variants harboring point mutations at their conserved catalytic pentad positions were characterized using single molecule tethered particle motion (TPM) analysis. The findings reveal contributions of these amino acids to the pre-chemical steps of recombination. They suggest functional differences between positionally conserved residues in how they influence recombinase-target site association and formation of ‘non-productive’, ‘pre-synaptic’ and ‘synaptic’ complexes. The most striking difference between the two systems is noted for the single conserved lysine. The pentad residues in Cre enhance commitment to recombination by kinetically favoring the formation of pre-synaptic complexes. These residues in Flp serve a similar function by promoting Flp binding to target sites, reducing non-productive binding and/or enhancing the rate of assembly of synaptic complexes. Kinetic comparisons between Cre and Flp, and between their derivatives lacking the tyrosine nucleophile, are consistent with a stronger commitment to recombination in the Flp system. The effect of target site orientation (head-to-head or head-to-tail) on the TPM behavior of synapsed DNA molecules supports the selection of anti-parallel target site alignment prior to the chemical steps. The integrity of the synapse, whose establishment/stability is fostered by strand cleavage in the case of Flp but not Cre, appears to be compromised by the pentad mutations.     

Seed-specific gene activation mediated by the Cre/lox site-specific recombination system.

The Cre/lox site-specific recombination system was used to activate a transgene in a tissue-specific manner. Cre-mediated activation of a beta-glucuronidase marker gene, by removal of a lox-bounded blocking fragment, allowed the visualization of the activation process. By using seed-specific promoters, the timing and efficiency of gene activation could be followed within the developing tobacco (Nicotiana tabacum) embryo. To serve as a basis for analyzing gene expression after-Cre-mediated activation, the timing and patterns of expression of the promoters of the genes encoding French bean (Phaseolus vulgaris) beta-phaseolin and the alpha' subunit of soybean (Glycine max) beta-conglycinin, as well as the cauliflower mosaic virus 35S promoter, were studied in developing transgenic tobacco embryos using the same visual marker. These seed-specific promoters were expressed earlier than anticipated. The 35S promoter was expressed earlier than the seed-specific promoters, but not in globular-stage embryos. Cre-mediated gene activation occurred approximately 1 d after promoter activation, based on developmental staging, and spread progressively throughout the embryo. The timing of gene activation was varied by altering Cre expression. Efficient Cre expression ultimately directed gene activation throughout the model tissue, whereas inefficient Cre expression resulted in mosaic tissue. Limited gene activation provides a system for cell lineage and developmental analyses.     

A novel suicide substrate for DNA topoisomerases and site-specific recombinases.

DNA topoisomerases and DNA site-specific recombinases are biologically important enzymes involved in a diverse set of cellular processes. We show that replacement of a phosphodiester linkage by a 5'-bridging phosphorothioate linkage creates an efficient suicide substrate for calf thymus topoisomerase I and lambda integrase protein (Int). Although the bridging phosphorothioate linkage is cleaved by these enzymes, the 5'-sulfhydryl which is generated is not competent for subsequent ligation reactions. We use the irreversibility of Int-promoted cleavage to explore conditions and factors that contribute to various steps of lambda integrative recombination. The phosphorothioate substrates offer advantages over conventional suicide substrates, may be potent tools for inhibition of the relevant cellular enzymes and represent a unique tool for the study of many other phosphoryl transfer reactions.     

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.     

The minimal duplex DNA sequence required for site-specific recombination promoted by the FLP protein of yeast in vitro.

The 2-micron plasmid of the yeast Saccharomyces cerevisiae codes for a site-specific recombinase ('FLP') that efficiently catalyses recombination across the plasmid's two 599 bp repeats both in vivo and in vitro. We have used the partially purified FLP protein to define the minimal duplex DNA sequence required for intra- and intermolecular recombination in vitro. Previous DNase footprinting experiments had shown that FLP protected 50 bp of DNA around the recombination site. We made BAL31 deletions and synthetic FLP sites to show that the minimal length of the site that was able to recombine with a wild-type site was 22 bp. The site consists of two 7 bp inverted repeats surrounding an 8 bp core region. We also showed that the deleted sites recombined with themselves and that one of three 13 bp repeated elements within the FLP target sequence was not necessary for efficient recombination in vitro. Mutants lacking this redundant 13 bp element required a lower amount of FLP recombinase to achieve maximal yield of recombination than the wild type site. Finally, we discuss the structure of the FLP site in relation to the proposed function of FLP recombination in copy number amplification of the 2-micron plasmid in vivo.