FRT Sequences Research Articles

Modular and Excisable Molecular Switch for the Induction of Gene Expression by the Yeast FLP Recombinase

Site-specific recombinases (SSRs) and their cognate target sequences have become instrumental in biological research (1,2). By varying the number, type, and orientation of SSR recognition sites, one can induce predictable genomic manipulations, like inversions, deletions, and insertions, in cells of virtually any organism. Prominent applications include: (i) conditional gene activation or inactivation to study gene function in cells and in whole organisms; (ii) generation of cell lines that encode cytotoxic products or synthesize constant amounts of proteins after DNA integration into predefined loci; and (iii) selection marker removal. The FLP/FRT system of Saccharomyces cerevisiae consists of an enzyme (FLP) that mediates highfidelity recombination between two 48-bp-long recognition sequences (FRT sites) (1,3). Plasmids containing one FRT motif can be inserted into a separate template containing another FRT site. In addition, FLP catalyzes the excision and inversion of DNA flanked by FRT sequences in a direct or inverted repeat configuration, respectively. In the former case, the excision product consists of a circularized molecule. Normally, recombinaseinducible gene expression relies on constructs containing upstream of the open reading frame (ORF), a promoter linked to a direct repeat of FRT sites framing a transcriptional terminator. Recombinase-mediated excision of the latter element leads to the synthesis of the desired product. Here, we generated and tested constructs harboring a FLPresponsive molecular switch with a different arrangement (i.e., with the promoter located downstream of the ORF) (Figure 1A). In this configuModular and excisable molecular switch for the induction of gene expression by the yeast FLP recombinase

Site-specific recombination in Zea mays

The elimination of marker genes after selection is recommended for the commercial use of genetically modified plants. We compared the applicability of the two site-specific recombination systems Cre/lox and Flp/FRT for marker gene elimination in maize plants. The selection marker gene pat surrounded by two identically directed lox or FRT sites was introduced into maize. Sexual crossing with plants harboring the corresponding constitutively expressed recombinase led to the precise and complete excision of the lox-flanked marker gene in the F1 progeny, whereas Flp-mediated recombination of FRT sequences occurred rarely. Further examination of site-specific integration was done by biolistic bombardment of immature embryos harboring only one lox site with a lox.uidA sequence with results indicating directed integration.

Nucleosome positioning on yeast plasmids is determined only by the internal signal of DNA sequence occupied by the nucleosome.

The possible role of border factors in determining the nucleosome positioning on a DNA sequence was investigated. To this end a family of recombinant plasmids based on Gal10Cyc1 promoter and neomycin phosphotransferase gene NPTII were created. A DNA sequence adjoining the GalCyc promoter was varied in these plasmids. Three nearly equally represented nucleosome positions on the GalCyc promoter were found. In the basal plasmid an FRT sequence adjoins the GalCyc promoter at the right. It contains an internal signal of multiple positioning. Its replacement with different DNA sequences does not affect nucleosome positioning on the GalCyc promoter. The nucleosome positioning on the GalCyc promoter does not depend on nucleosome positioning (or its absence) on adjoining sequences. The same is true for nucleosome positioning on FRT sequence. It was found also that nucleosomes' positioning on the NPTII gene and their mutual disposition, namely the spacing between neighboring nucleosomes (linker length) are determined by the location of positioning signals only. Generally the nucleosome positioning in our experimental model is determined solely by internal DNA sequence occupied by nucleosome. On the other hand, the action of this internal positioning signal does not extend to neighboring DNA sequences.

A GAL4-HP1 fusion protein targeted near heterochromatin promotes gene silencing.

We have constructed a new reporter transgene, Winkelried, equipped with a synthetic binding site for the yeast GAL4 transcriptional activator. The binding site is inserted between the white and lacZ reporter genes, and is flanked by FRT sequences. These elements allow excision of the GAL4 binding site by crossing the transgenic line with an FLP recombinase producing strain. We have generated by X-ray irradiation two independent chromosomal rearrangements, Heidi and Tell, relocating Winkelried next to pericentromeric heterochromatin. These rearrangements induce variegation of both white and lacZ. Variegation of Winkelried in the rearranged transgenic lines responds to the loss and excess of doses of the dominant suppressors of position-effect variegation (PEV) Su(var)3-7 and Su(var)2-5. Winkelried therefore constitutes a unique tool to test the effect on variegation in cis of any factor fused to the GAL4 DNA binding domain. Indeed, a chimeric protein, made of the DNA binding site of GAL4 and of HP1, the modifier of PEV encoded by Su(var)2-5, is shown to enhance variegation of Heidi and Tell. Excision of the binding sites for GAL4 in the variegating rearrangements Heidi and Tell abolishes the modifier effect of the GAL4-HP1 chimera. Therefore, in the Heidi and Tell rearrangements, enhancement of position-effect variegation depends strictly both on the concentration of GAL4-HP1 and on the presence of its binding site in the vicinity of the reporter genes.

Protein phosphorylation in plants: enzymes, substrates and regulators

IOMMENT construction of stocks and markers by Xu and Rubin 13. They first con- structed genes encoding chimeric proteins that serve as gratuitous cell- autonomous markers for all tissues. The 7tM marker encodes a fusion between a peptide (MYC) that derives from the human C-MYCgene and part of the Drosophila P-element transposase. The marker is targeted to nuclei, and cells expressing xM can be recognized by a commercially available monoclonal antibody. A membrane marker was also con- structed that encodes the MYC pep- tide and the signal peptide and trans- membrane domain of the Notch protein. These genetic markers have been placed on all major chromo- some arms together with FRT sequences that are located near the centromere. All that remains for the individual is to meiotically recombine his or her favourite mutation onto the distal part of the FRT-carrying chromosome arm (the chromosomes are cleverly constructed to facilitate screening o~ such recombinants) and to perfom~ appropriate crosses to introduce the FLP onto a different chromosome (Fig. 1). Xu and Rubin 13 also demon- strated that the frequency of mosaicism is sufficiently high to permit screening for new mutations that affect deve!opmen.t and function of various tissues. One can thus look at the phenotype of homozygous clones in heterozygous flies and hence isolate mutations in first- generation screens, a device that saves a considerable amount of time. The stocks have generously been made available to the entire com- munity and so we can now look forward to the undoubtedly success- ful application of this superb new tool.