Physical and functional structure of a yeast plasmid, pSB3, isolated from<i>Zygosaccharomyces bisporus</i>

Toh‐e, Akio; Utatsu, Ikuyo

doi:10.1093/nar/13.12.4267

Cited by 56 publications

(26 citation statements)

References 26 publications

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“…It is noteworthy that similar 14-bp dyad symmetry was also observed by Toh-e and Utatsu (29) in the inverted repeats of another Zygosaccharomyces plasmid, pSB3, at a possible recombination site. The 14-bp dyad symmetrical sequence of pSB3 has a 60% homology with that of pSR1, whereas these regions have no significant homologies with 2,um DNA.…”

Section: Resultssupporting

confidence: 79%

Gene conversion associated with site-specific recombination in yeast plasmid pSR1.

Matsuzaki¹,

Araki²,

Oshima³

1988

Mol. Cell. Biol.

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A circular DNA plasmid, pSRl, isolated from Zygosaccharomyces rouxii has a pair of inverted repeats consisting of completely homologous 959-base pair (bp) sequences. Intramolecular recombination occurs frequently at the inverted repeats in cells of Saccharomyces cerevisiae, as well as in Z. rouxii, and is catalyzed by a protein encoded by the R gene of its own genome. The recombination is, however, independent of the RAD52 gene of the host genome. A site for initiation of the intramolecular recombination in the S. cerevisiae host was delimited into, at most, a 58-bp region in the inverted repeats by using mutant plasmids created by linker insertion. The 58-bp region contains a pair with 14-bp dyad symmetry separated by a 3-bp spacer sequence. The recombination initiated at this site was accompanied by a high frequency of gene conversion (3 to 50% of the plasmid clones examined). Heterogeneity created by the linker insertion or by a deletion (at most 153 bp so far tested) at any place on the inverted repeats was converted to a homologous combination by the gene conversion, even in the rad52-1 mutant host. A mechanism implying branch migration coupled with DNA replication is discussed.

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Section: Resultssupporting

confidence: 79%

Gene conversion associated with site-specific recombination in yeast plasmid pSR1.

Matsuzaki¹,

Araki²,

Oshima³

1988

Mol. Cell. Biol.

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“…We also show that two of the new recombinases function in mammalian cells. 40) were synthesized with Drosophila codon usage. We wanted to decrease the half-lives of the recombinases to achieve tighter temporal control of activity and reduce potential toxicity.…”

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confidence: 99%

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

Nern

Pfeiffer

Svoboda

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

159

171

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Site-specific recombinases have been used for two decades to manipulate the structure of animal genomes in highly predictable ways and have become major research tools. However, the small number of recombinases demonstrated to have distinct specificities, low toxicity, and sufficient activity to drive reactions to completion in animals has been a limitation. In this report we show that four recombinases derived from yeast-KD, B2, B3, and R-are highly active and nontoxic in Drosophila and that KD, B2, B3, and the widely used FLP recombinase have distinct target specificities. We also show that the KD and B3 recombinases are active in mice.gene expression | genetic engineering S ite-specific DNA recombinases are widely used in multicellular organisms to manipulate the structure of genomes and, in turn, to control gene expression (for reviews see refs. 1-4). These enzymes, derived from bacteria and fungi, catalyze directionally sensitive DNA exchange reactions between short (30-40 nucleotides) target site sequences that are specific to each recombinase (5). These reactions enable four basic functional modules-excision/insertion, inversion, translocation and cassette exchange-that have been used individually or combined in a wide range of configurations to control gene expression (Fig. 1A).The use of site-specific recombination to manipulate genomes has been limited by the availability of recombinases with high activity, distinct site specificity, and low toxicity. In Drosophila, the most widely used recombinase is FLP, encoded by the Saccharomyces cerevisiae 2-μm plasmid (6). FLP was first shown to work in a heterologous, multicellular organism by Golic and Lindquist in 1989 (7) who demonstrated the excision reaction on chromosomally inserted target sites (FRTs). Since that time FLP/FRT recombination has been widely used in Drosophila in applications based on excision (8) and translocation (9-11).Complex manipulations of genome structure can require the use of more than one of the modules diagrammed in Fig. 1A, or parallel independent implementations of the same module, in a single individual. To accomplish such manipulations, the modules must be implemented with different recombinases that do not recognize each other's target sites. Similarly, a number of powerful methods have been developed for using the excision and inversion reactions to control expression of a transgene specifically in cells where two independent gene expression patterns overlap (2,3,12,13). Such intersectional methods rely on pairs of orthogonal recombinases; see, for example, Fig. 1B. For these reasons, we sought to discover additional recombinases with distinct site-specificity.FLP recombinase has been mutated to recognize altered FRT sites, but some cross-reaction still remains (14, 15). Cre, encoded by the bacteriophage P1, is the most widely used recombinase in mammalian cells (16)(17)(18). Cre functions in Drosophila (19), but exhibits obvious toxicity (20), a problem also observed in mammalian cells (21, 22; reviewed in ref. 23) an...

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“…Other plasmids have a structural organization similar to that of 2,um DNA; they have an FLP gene and three to four large ORFs. In the case of pSB3, two ORFs other than the one encoding FLP enzyme were shown to participate in the stable maintenance of this plasmid (25).…”

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“…Recently, another 2,um DNA-like plasmid, pKD1, was discovered in Kluyveromyces drosophilarum (8). Of these, the nucleotide sequences of 2,um DNA (13), pSR1 (2), pSB3 (25), and pKD1 (8) have been published. These plasmids, along with 2,um DNA from Saccharomyces cerevisiae, have several structural features in common: (i) they are closed circular DNA plasmids whose size is ca.…”

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confidence: 99%

Yeast plasmids resembling 2 micron DNA: regional similarities and diversities at the molecular level

et al. 1987

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The nucleotide sequence of two Zygosaccharomyces plasmids, pSB2 (5,415 base pairs), isolated from Zygosaccharomyces bailii, and pSMl (5,416 base pairs), isolated from Zygosaccharomycesfermentati Naganishi, was determined. The predicted amino acid sequences of open reading frames among six yeast plasmids that resemble 2,um DNA indicated regional sequence similarities among FLP proteins. Greater similarities were seen among Zygosaccharomyces plasmids (pSB2, pSB3, pSRl, and pSM1) than other combinations. A putative recognition site for the FLP enzyme of a Zygosaccharomyces plasmid also showed some conservation, especially in the 4 nucleotides flanking the central spacer region. From comparative studies of the sequences of putative genes of each plasmid, we propose an apparent phylogenetic relationship among yeast plasmids resembling 2,Lm DNA. Among the Zygosaccharomyces plasmids, pSB2 and pSRl are most closely related, since not only were the FLP enzymes of these two plasmids most closely related, but also the amino acid sequence of the putative P gene of pSRl showed clear homology with that of open reading frame B of pSB2.Since the discovery of 2,um DNA in Saccharomyces species (21; see reference 6 for a review), several yeast plasmids resembling the 2Rm plasmid were isolated and characterized. We call these plasmids 2,um DNA-like plasmids. For unknown reasons, 2ptm DNA-like plasmids were found frequently in Zygosaccharomyces species. We have found five plasmids in Zygosaccharomyces yeasts: pSB1 and pSB2 from Z. bailii (23), pSR1 and pSB3 from Z. rouxii (23,24), and pSM1 from Z. fermentati (unpublished data). Recently, another 2,um DNA-like plasmid, pKD1, was discovered in Kluyveromyces drosophilarum (8). Of these, the nucleotide sequences of 2,um DNA (13), pSR1 (2), pSB3 (25), and pKD1 (8) have been published. These plasmids, along with 2,um DNA from Saccharomyces cerevisiae, have several structural features in common: (i) they are closed circular DNA plasmids whose size is ca. 6 kilobases (kb), (ii) they have a pair of inverted repeats (IRs) consisting of several hundred bases, (iii) they are cryptic and exist in high copy number, and (iv) intramolecular recombination results in two isomeric forms. In spite of resemblances in their global structure, homology at the nucleotide sequence level is poor (23).Plasmid-encoded functions have been most extensively studied in 2,um DNA. The prominent features of genes on 2,um DNA are briefly summarized as follows. It has one replication origin, which is activated once in the cell cycle by the same replication mechanism as that for replication of chromosomes (16,28). It has four open reading frames (ORFs) or genes, A, B, C, and D. The A gene (or FLP gene) encodes a recombination enzyme which functions as a site-specific recombinase (7). The B and C genes produce trans-acting factors that are needed for the stable maintenance of this plasmid. These two genes, along with the cis-acting locus STB, participate in partitioning of plasmid molecules during cell division (14,15 Our...

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Physical and functional structure of a yeast plasmid, pSB3, isolated fromZygosaccharomyces bisporus

Cited by 56 publications

References 26 publications

Gene conversion associated with site-specific recombination in yeast plasmid pSR1.

Gene conversion associated with site-specific recombination in yeast plasmid pSR1.

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

Yeast plasmids resembling 2 micron DNA: regional similarities and diversities at the molecular level

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