Nucleotide sequence of the yeast plasmid

Hartley, James L.; Donelson, John E.

doi:10.1038/286860a0

Cited by 383 publications

(153 citation statements)

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“…SO(A-B-) exerts a comparable stabilizing effect on 3.2(A-C-), decreasing its rate of loss from 17 to 4% per generation. The rates of loss for NO(A-C-) and NSO(A-B-C-) were reduced to a similar extent when introduced into an isogenic cir+ strain (data not (16). The hybridization probe derived from the 245-bp EcoRlPstI fragment.…”

Section: Resultsmentioning

confidence: 99%

“…The S. cerevisiae strain 3482-16-1 (a met2 his3Al leu2-3 trpl-289 ura3-52), constructed by L. H. Hartwell and subsequently cured of 2p.m plasmid by the method of Dobson et al (9), was host to all the plasmids described in this study. The strain initially contained a 2,im plasmid with three direct repeats (16). To facilitate comparisons of native 2pLm plasmid with chimeric plasmids containing five direct repeats, a form of 2p.m plasmid with five direct repeats was introduced into the ciro strain as follows: p82-6B (16), a pMB9 derivative containing 1.5 copies of the 2,um plasmid, was modified by the addition of the LEU2 gene to the Sall site to create a plasmid selectable in S. cerevisiae (p82-6B+LEU).…”

Section: Methodsmentioning

confidence: 99%

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Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products.

Veit¹,

Fangman²

1985

Mol. Cell. Biol.

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We have used gene disruptions and nuclease probes to assess the roles of yeast 2,Im plasmid genes in plasmid chromatin organization. The chromatin structure at the replication origin is not dependent on any of the four major open reading frames, A, B, C, or D. While stable plasmid maintenance is known to depend on a cis-acting locus STB and genes B and C, we find that only gene B influences STB chromatin. Other interactions between plasmid gene products and sequences may reflect gene regulation: the chromatin organization at the 5' end of gene A, which codes for a site-specific recombinase, depends on both gene B and gene C. Since disruption of gene C results in an increase in plasmid copy number that is dependent on gene A, we propose that gene C (and probably gene B) controls copy number by regulating the level of the gene A recombinase.The 2,um plasmid is a 6,318-base-pair circular DNA found in most strains of Saccharomyces cerevisiae at 50 to 100 copies per diploid cell (for reviews, see references 4 and 5). Composing up to 2% of a relatively small nuclear genome, the plasmid offers a useful system for physical studies of eucaryotic DNA replication. Although multiple copy and episomal, the replication of the 2,um plasmid is like that of chromosomal DNA, with replication depending on the same set of chromosomally encoded genes (22) and each plasmid molecule replicating once during the S phase (37).Factors encoded by the 2,um plasmid itself are required for its stable maintenance. The plasmid contains several large open reading frames (ORFs), the five largest designated A, B, C, D, and E (16; see Fig. 1). Since the 2,um plasmid confers no easily scored phenotype on its host, investigations of the relationship between plasmid-encoded factors and plasmid maintenance have relied on 2,um derivatives that contain selectable nuclear genes. Stability studies of such chimeras, in which specific 2,um sequences are deleted or disrupted, indicate that the B and C ORFs (also referred to as REP1 and REP2, respectively) encode trans-acting factors necessary for stable plasmid maintenance (17,19). STB and ORI, two cis-acting sites defined by deletion analysis, are also essential for plasmid stability. STB, bounded by the PstI and AvaI sites (19), appears to be equivalent to the REP3 locus described by Jayaram et al. (17). ORI is defined by its ability to support the autonomous replication of plasmids (7,17). Electron microscopy studies of 2Rm plasmid replicative intermediates show a high frequency of eye forms centered about ORI, supporting the notion that ORI acts as an origin of replication (30).It is less clear whether other 2,um plasmid components are involved in plasmid maintenance. Although 2,um chimeric plasmids containing large insertions in the A or D ORFs are lost 10 to 100 times faster than is native 2,um plasmid (14, 15), it is unclear that the instability reflects the disruption of a component normally involved in plasmid stabilization. Unlike the instability resulting from the disruption of the B or C ORFs, ins...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products.

Veit¹,

Fangman²

1985

Mol. Cell. Biol.

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“…First, a series of seven S. cerevisiae data ¢les comprising: (1) the 6213 predicted protein sequences as described in [1]; (2) the intergenic and subtelomeric DNA sequences [4]; (3) the 2 Wm plasmid DNA sequence [8]; (4) the rDNA sequences extracted from the chromosome XII sequence [9]; (5) the 52 distinct tRNA gene sequences as de¢ned in [1]; (6) the Ty elements DNA sequences [10]; and (7) the mitochondrial DNA sequence [11] and its translation products.…”

Section: Databasesmentioning

confidence: 99%

Genomic Exploration of the Hemiascomycetous Yeasts: 3. Methods and strategies used for sequence analysis and annotation

et al. 2000

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The primary analysis of the sequences for our Hemiascomycete random sequence tag (RST) project was performed using a combination of classical methods for sequence comparison and contig assembly, and of specifically written scripts and computer visualization routines. Comparisons were performed first against DNA and protein sequences from Saccharomyces cerevisiae, then against protein sequences from other completely sequenced organisms and, finally, against protein sequences from all other organisms. Blast alignments were individually inspected to help recognize genes within our random genomic sequences despite the fact that only parts of them were available. For each yeast species, validated alignments were used to infer the proper genetic code, to determine codon usage preferences and to calculate their degree of sequence divergence with S. cerevisiae. The quality of each genomic library was monitored from contig analysis of the DNA sequences. Annotated sequences were submitted to the EMBL database, and the general annotation tables produced served as a basis for our comparative description of the evolution, redundancy and function of the Hemiascomycete genomes described in other articles of this issue. ß

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“…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).…”

mentioning

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.

154

170

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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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Nucleotide sequence of the yeast plasmid

Cited by 383 publications

References 41 publications

Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products.

Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products.

Genomic Exploration of the Hemiascomycetous Yeasts: 3. Methods and strategies used for sequence analysis and annotation

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

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