Operation of an efficient site-specific recombination system ofZygosaccharomyces rouxiiin tobacco cells

Abstract

Recombinase encoded by the R gene of pSR1 of Zygosaccharomyces rouxii mediates reciprocal recombination between two specific recombination sites (RSs) to induce excision or inversion of the DNA segment that is flanked by the RSs. We report here that site-specific recombination mediated by this system takes place effeciently in tobacco cells. To monitor the recombination events in tobacco cells, we have constructed two types of cryptic beta-glucuronidase reporter gene in such a way that recombination such as inversion of the construct or excision of the intervening sequence results in their expression. When these cryptic reporter constructs were transiently introduced together with the R gene by electroporation into protoplasts of tobacco cells, beta-glucuronidase activity was detected. The cryptic reporter genes, when stably resident in the chromosome of tobacco cells, were also activated by the R gene. Structural analyses of the genomic DNA isolated from these tobacco cells showed that the R protein did in fact catalyze precise recombination between two copies of RSs in tobacco cells, with resultant activation of the cryptic reporter genes. This observation provides the basis for development of a DNA technology whereby large regions of DNA can be manipulated in plant chromosomes. Potential uses of this recombination system are discussed.

Generation of Marker- and Backbone-Free Transgenic Potatoes by Site-Specific Recombination and a Bi-Functional Marker Gene in a Non-Regular One-Border Agrobacterium Transformation Vector

“…Deletions and inversions using the Z. rouxii R/Rs recombination system have been demonstrated in tobacco, Arabidopsis and rice (Onouchi et al, 1991;Toriyama et al, 2003). Here we have shown that the R/Rs system is also active in potato.…”

Genomic Exploration of the Hemiascomycetous Yeasts: 8. Zygosaccharomyces rouxii1

“…rouxii contains a plasmid called pSR1 of 6251 bp which shows no homology with the S. cerevisiae 2W plasmid [9]. It has been extensively characterized especially for the recombinases Flp and R [10^14] and its ability to be active in heterologous backgrounds [15,16]. This paper reports the genomic analysis of strain CBS732 which contains seven chromosomes with sizes ranging from 1 to 2.75 Mb corresponding to an estimated total size of 12.8 Mb according to pulsed ¢eld gel electrophoresis determination [17].…”

Multiple new site-specific recombinases for use in manipulating animal genomes

“…PhiC31 integrase, which catalyzes unidirectional recombination between attP and attB sequences, has been used extensively for integration (27, 28) and cassette exchange (29,30), applications that take advantage of its nonreversibility. The R recombinase, a relative of FLP from the yeast Zygosaccharomyces rouxii (31, 32), has been shown to function in plants (33).In this report we describe the characterization of R and three other recombinases encoded by 2-μm circle-like plasmids of other yeasts. Although such plasmids are rare among yeasts, those characterized so far are very similar in structure to the 2-μm plasmid of S. cerevisiae (reviewed in ref.…”

“…PhiC31 integrase, which catalyzes unidirectional recombination between attP and attB sequences, has been used extensively for integration (27,28) and cassette exchange (29,30), applications that take advantage of its nonreversibility. The R recombinase, a relative of FLP from the yeast Zygosaccharomyces rouxii (31,32), has been shown to function in plants (33).…”

“…In particular, they appear to encode site-specific recombinases related to FLP and undergo high-frequency intramolecular recombination between inverted repeat domains both in their native species and in S. cerevisiae (32,(34)(35)(36)(37)(38)(39). However, only the R recombinase has previously been shown to work in a multicellular organism (33), and none has been used in an animal genome. We compare the properties of these four yeast recombinases to FLP, Cre, and Dre in Drosophila.…”