Reversion of autonomously replicating sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic replication origin within procaryotic vector DNA.

Kipling, David; Kearsey, Stephen

doi:10.1128/mcb.10.1.265

Cited by 50 publications

(30 citation statements)

References 28 publications

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“…The rate of loss of the circular minichromosome, CM3112, was too high to use the above method. Another method was employed (Kipling and Kearsey 1990}. Briefly, an Ade + colony was picked, resuspended in a small volume of medium, and the cell density was measured.…”

Section: Mitotic Chromosome Stability Assaysmentioning

confidence: 99%

See 1 more Smart Citation

Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation.

Allshire¹,

Nimmo²,

Ekwall³

et al. 1995

Genes Dev.

434

393

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The ura4 + gene displays phenotypes consistent with variegated expression when inserted at 11 sites throughout fission yeast centromere 1. An abrupt transition occurs between the zone of centromeric repression and two adjacent expressed sites. Mutations in six genes alleviate repression of the silent-mating type loci and of ura4 + expressed from a site adjacent to the silent locus, mat3-M. Defects at all six loci affect repression of the ara4 + gene adjacent to telomeres and at the three centromeric sites tested. The clr4-$5 and rikl-304 mutations cause the most dramatic derepression at two out of three sites within cenl. All six mutations had only slight or intermediate effects on a third site in the center of cenl or on telomeric repression. Strains with lesions at the clr4, rikl, and swi6 loci have highly elevated rates of chromosome loss. We propose that the products of these genes are integral in the assembly of a heterochromatin-like structure, with distinct domains, enclosing the entire centromeric region that reduces or excludes access to transcription factors. The formation of this heterochromatic structure may be an absolute requirement for the formation of a fully functional centromere.[Key Words: S. pombe; PEV; heterochromatin; telomere; mating type] Received October 7, 1994; revised version accepted December 13, 1994.The centromeres of the fission yeast Schizosaccharomyces pombe are complex structures composed of large repetitive arrays symmetrically arranged around a nonrepetitive central domain (Hahnenberger et al. 1991;Murakami et al. 1991;Takahashi et al. 1992;Steiner et al. 1993; cenl is shown in Fig. 1A, below). Long arrays of tandem satellite repeats are frequently associated with the centromeres of many eukaryotes (Miklos and Cotsell 1990;Rattner 1991;Tyler-Smith and Willard 1993). These centromeric satellites are packaged into a highly compacted structure known as constitutive heterochromatin, but it is not known if this contributes to centromere function. It is likely that mammalian satellite repeats contribute to kinetochore formation, but alone, they may not be sufficient to form a functional centromere (Earnshaw et al. 1989;Haaf et al. 1992;Larin et al. 1994).Genes placed in the vicinity of centromeric heterochromatin in mouse cells can exhibit variable states of expression (Butner and Lo 1986). This phenomenon, termed position effect variegation (PEV), was first described in Drosophila, where it was found that genes placed adjacent to centromeric heterochromatin were unstably repressed (Eissenberg 1989;Henikoff 1990;Karpen 1994). In Drosophila, mouse, and human cells, some 1Corresponding author. studies indicate that heterochromatin is often associated with the nuclear periphery (Hochstrasser et al. 1986;Ferguson and Ward 1992;Vourc'h et al. 1993). It is possible that this and other associations serve to lock these regions of chromosomes in an inactive state (Karpen 1994}. In unicellular organisms, such as yeasts, there are chromosomal regions with features characteristic of heterochro...

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Section: Mitotic Chromosome Stability Assaysmentioning

confidence: 99%

“…GENES & DEVELOPMENT other m e t h o d , m o r e suited to e s t i m a t i n g high loss rates, was e m p l o y e d (Kipling and Kearsey 1990). Table 1C shows t h a t in strains w i t h clr4-$5, rikl-304, or swi6-115 m u t a t i o n s the m i n i c h r o m o s o m e CM3112 is lost in 18%, 17%, and 21% of divisions, respectively.…”

mentioning

confidence: 99%

Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation.

Allshire¹,

Nimmo²,

Ekwall³

et al. 1995

Genes Dev.

434

393

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“…Several reports of autonomously replicating sequence elements that function in mammalian cells and cell extracts have been documented in detail and shown to correspond to initiation sites for DNA replication in mammalian chromosomes (17)(18)(19)(20). This does not exclude the possibility that, under appropriate conditions, other sequences may promote autonomous plasmid DNA replication in mammals (21) as they do in Xenopus eggs (22) and in yeast (23).…”

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DNA Methylation at Mammalian Replication Origins

Rein

Kobayashi²,

Malott³

et al. 1999

Journal of Biological Chemistry

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In Escherichia coli, DNA methylation regulates both origin usage and the time required to reassemble prereplication complexes at replication origins. In mammals, at least three replication origins are associated with a high density cluster of methylated CpG dinucleotides, and others whose methylation status has not yet been characterized have the potential to exhibit a similar DNA methylation pattern. One of these origins is found within the ϳ2-kilobase pair region upstream of the human c-myc gene that contains 86 CpGs. Application of the bisulfite method for detecting 5-methylcytosines at specific DNA sequences revealed that this region was not methylated in either total genomic DNA or newly synthesized DNA. Therefore, DNA methylation is not a universal component of mammalian replication origins. To determine whether or not DNA methylation plays a role in regulating the activity of origins that are methylated, the rate of remethylation and the effect of hypomethylation were determined at origin ␤ (ori-␤), downstream of the hamster DHFR gene. Remethylation at ori-␤ did not begin until ϳ500 base pairs of DNA was synthesized, but it was then completed by the time that 4 kilobase pairs of DNA was synthesized (<3 min after release into S phase). Thus, DNA methylation cannot play a significant role in regulating reassembly of prereplication complexes in mammalian cells, as it does in E. coli. To determine whether or not DNA methylation plays any role in origin activity, hypomethylated hamster cells were examined for ori-␤ activity. Cells that were >50% reduced in methylation at ori-␤ no longer selectively activated ori-␤. Therefore, at some loci, DNA methylation either directly or indirectly determines where replication begins.In early embryos undergoing rapid cell cleavage (e.g. frogs, flies, sea urchin, fish), initiation of DNA replication appears neither to require specific DNA sequences nor to occur at specific DNA sites. However, as development progresses past the blastula stage, initiation of DNA replication begins to occur at specific sites (1, 2). Thus, it is not surprising that initiation sites for DNA replication in cultured mammalian cells occur at specific genomic loci (3). For example, a 200-kb 1 region at the human ␤-globin gene (4, 5) and a 500-kb region at the mouse IgH gene (6) are both replicated from a single initiation locus. Moreover, what previously had been viewed as random initiation events distributed throughout "initiation zones" in Schizosaccharomyces pombe and hamster cells more likely reflects the presence of several strongly preferred initiation sites (7,8).Nevertheless, the precise size and composition of these initiation loci, as well as the parameters that define them remain the subject of intense investigation.The fact that specific sites for initiation of DNA replication can be developmentally acquired makes it clear that initiation sites in the metazoa are determined at least in part by epigenetic parameters. These parameters include nuclear structure, chromatin structure, and even...

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“…Specifically, two nonoverlapping restriction fragments encompassing the left and right halves of HMR-E, respectively, are each sufficient to confer ARS activity on plasmids (7). This redundancy of ARS information may be due to multiple near matches to the 11-bp ARS consensus sequence found at HMR-E, since multiple near matches to this consensus sequence have been shown to be sufficient to confer ARS activity (26,51,53).…”

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A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae.

McNally¹,

Rine²

1991

Mol. Cell. Biol.

100

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Copies of the mating-type genes are present at three loci on chromosome III of the yeast Saccharomyces cerevisiae. The genes at the MAT locus are transcribed, whereas the identical genes at the silent loci, HML and HMR, are not transcribed. Several genes, including the four SIR genes, and two sites, HMR-E and HMR-I, are required for repression of transcription at the HMR locus. Three elements have been implicated in the function of the HMR-E silencer: a binding site for the RAPI protein, a binding site for the ABF1 protein, and an ll-bp consensus sequence common to nearly all autonomously replicating sequence (ARS) elements (putative origins of DNA replication). RAP1 and ABF1 binding sites of different sequence than those found at HMR-E were joined with an ll-bp ARS consensus sequence to form a synthetic silencer. The synthetic silencer was able to repress transcription of the HMRal gene, confirming that binding sites for RAP1 and ABF1 and the ll-bp ARS consensus sequence were the functional components of the silencer in vivo. Mutations in the ABF1 binding site or in the ARS consensus sequence of the synthetic silencer caused nearly complete derepression of transcription at HMR. The ARS consensus sequence mutation also eliminated the ARS activity of the synthetic silencer. These data suggested that replication initiation at the HMR-E silencer was required for establishment of the repressed state at the HMR locus.In the yeast Saccharomyces cerevisiae, mating type and sporulation are controlled by the MATa allele (a] and a2 genes) and the MATa allele (al and a2 genes) of the mating-type locus (MAI) mutations has been observed only in cells under starvation conditions (1,22). Repression of HMRa is also dependent on the products of the SIR], SIR2, SIR3, and SIR4 genes (37) and on the product of at least one wild-type histone H4 gene (23).The HMR-E site has been termed a silencer because its properties appear to be opposite those of a mammalian enhancer. Specifically, a 530-bp DNA fragment containing the E site is able to maintain repression of HMRa in an orientation-independent manner when positioned at a variety of locations near the HMRa locus (6). In addition, a number of different RNA polymerase II and RNA polymerase III-transcribed genes are repressed in a SIR-dependent manner when these genes are inserted adjacent to the silencer in the HMR locus (6,42). Whereas mammalian enhancers act as independent elements that can function when moved to heterologous contexts (3), there is limited information as to whether the HMR-E silencer can act as an independent element. In the only relevant case, a 490-bp DNA fragment containing the E site, when placed upstream of a chimeric promoter on a plasmid, was able to repress transcription in an orientation-independent manner (10). The generality of this observation has not yet been established.In addition to transcriptional repression, three other properties have been associated with the 490-bp fragment of the HMR-E silencer. This fragment, which contains an 11-bp autonomous...

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Reversion of autonomously replicating sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic replication origin within procaryotic vector DNA.

Cited by 50 publications

References 28 publications

Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation.

Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation.

DNA Methylation at Mammalian Replication Origins

A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae.

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