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1.
Bending the rules: the 2-mu plasmid of yeast 总被引:2,自引:0,他引:2
J A Murray 《Molecular microbiology》1987,1(1):1-4
The replication of eukaryotic DNA is normally initiated at each origin only once per cell cycle. Yet, in spite of this restriction, the 2-mu plasmid of yeast has evolved an elegant mechanism which can allow it to rapidly amplify its copy number without initiating multiple rounds of replication. It achieves this by exploiting a plasmid-encoded site-specific recombination system in a way that is apparently unique to this plasmid. The 2-mu plasmid has also evolved a mechanism that allows effective partition of itself between mother and daughter cells. Together these processes ensure the persistence of the 2-mu plasmid within a population, even though retention of the plasmid is of no advantage to the host cell and causes a slightly slower growth rate. The success of this survival strategy is illustrated by the near ubiquity of the 2-mu plasmid in both wild-type and laboratory strains of yeast. 相似文献
2.
The yeast plasmid 2μ and certain hybrid plasmids constructed from it are maintained stably and at high copy number in yeast cells. By examining various mutant hybrid 2μ plasmids, we show that these properties require the integrity of four plasmid loci. Two of these, designated REPI and REP2, are active in trans and correspond to two open coding regions of 2μ. The other two loci are active only in cis and correspond to the origin of replication and to a region, designated REP3, located several hundred bp away from the origin and consisting of direct repeats of a 62 bp sequence. We propose that the REP loci constitute a copy control system that overrides normal cellular restriction on plasmid replication and amplifies the plasmid when copy number is low. 相似文献
3.
By examining both the transformation efficiency of yeast of various plasmids containing defined regions of the 2μ circle genome and the characteristics of the resultant transformants, we have identified several regions of the 2μ circle genome which are involved in 2μ circle replication or recombination. First, by identifying those DNA fragments from the molecule which promote high frequency transformation of yeast, we have localized the origin of replication to a sequence partially within the large unique region, which, as determined by subsequent deletion analysis, extends from the middle of the inverted repeat region into the contiguous unique region. Second, by examining the relative efficiency of replication in yeast of hybrid plasmids containing either the entire 2μ circle genome or a fragment of 2μ circle encompassing the origin of replication, we have determined that efficient use of the 2μ circle origin requires some function or functions encoded in the molecule at a site away from the origin. Third, by examining the ability of a mutant 2μ circle molecule to undergo intramolecular recombination in yeast, we have identified a 2μ circle gene which codes for a product required for this process. 相似文献
4.
Bergero R 《Fungal genetics and biology : FG & B》2006,43(5):337-342
Autonomous replicating sequences are DNA elements that trigger DNA replication and are widely used in the development of episomal transformation vectors for fungi. In this paper, a genomic library from the mycorrhizal fungus Gigaspora rosea was constructed in the integrative plasmid YIp5 and screened in the budding yeast Saccharomyces cerevisiae for sequences that act as ARS and trigger plasmid replication. Two genetic elements (GrARS2, GrARS6) promoted high-rates of yeast transformation. Sequence analysis of these elements shows them to be AT-rich (72-80%) and to contain multiple near-matches to the yeast autonomous consensus sequences ACS and EACS. GrARS2 contained a putative miniature inverted-repeat transposable element (MITE) delimited by 28-bp terminal inverted repeats (TIRs). Disruption of this element and removal of one TIR increased plasmid stability several fold. The potential for palindromes to affect DNA replication is discussed. 相似文献
5.
The yeast 2 microns plasmid is found in the nucleus of almost all Saccharomyces cerevisiae strains. Its replication is very similar to that of chromosomal DNA. Although the plasmid does not encode essential genes it is stably maintained in the yeast population and exhibits only a small, though detectable, loss rate. This stability is achieved by a plasmid-encoded copy-number control system which ensures constant plasmid levels. For the investigation of 2 microns replication, a yeast strain that is absolutely dependent on this plasmid was constructed. This was achieved by disruption of the chromosomal CDC9 gene, coding for DNA ligase and providing this essential gene on a 2 microns-derived plasmid. This plasmid is absolutely stable under all growth conditions tested. Using the temperature-sensitive mutant allele cdc9-1 we have developed an artificial control system which allows one to change the copy number of 2 microns-derived plasmids solely by changing the incubation temperature. 相似文献
6.
Analysis of a 1.6-μm circular plasmid from the yeast Kluyveromyces drosophilarum: Structure and molecular dimorphism 总被引:4,自引:0,他引:4
A new plasmid has been found in the yeast Kluyveromyces drosophilarum. It is a double-stranded circular DNA, 1.6 micron in length (4.8 kilobase pairs). As in the case of Saccharomyces 2 mu circles, this plasmid occurs in two isomeric forms corresponding to the inversion of a segment between two 346-bp-long inverted repeats within the molecule. Each form has been separately cloned into bacterial plasmids. The new yeast plasmid, called pKD1, contains sequences that allow its replication in Saccharomyces cerevisiae. 相似文献
7.
ARS replication during the yeast S phase 总被引:43,自引:0,他引:43
A 1.45 kb circular plasmid derived from yeast chromosome IV contains the autonomous replication element called ARS1. Isotope density transfer experiments show that each plasmid molecule replicates once each S phase, with initiation depending on two genetically defined steps required for nuclear DNA replication. A density transfer experiment with synchronized cells demonstrates that the ARS1 plasmid population replicates early in the S phase. The sequences adjacent to ARS1 on chromosome IV also initiate replication early, suggesting that the ARS1 plasmid contains information which determines its time of replication. The times of replication for two other yeast chromosome sequences, ARS2 and a sequence referred to as 1OZ, indicate that the temporal order of replication is ARS1 leads to ARS2 leads to 1OZ. These experiments show directly that specific chromosome regions replicate at specific times during the yeast S phase. If ARS elements are origins of chromosome replication, then the experiment reveals times of activation for two origins. 相似文献
8.
Partitioning of the 2-microm circle plasmid of Saccharomyces cerevisiae. Functional coordination with chromosome segregation and plasmid-encoded rep protein distribution
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The efficient partitioning of the 2-microm plasmid of Saccharomyces cerevisiae at cell division is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In addition, host encoded factors are likely to contribute to plasmid segregation. Direct observation of a 2-microm-derived plasmid in live yeast cells indicates that the multiple plasmid copies are located in the nucleus, predominantly in clusters with characteristic shapes. Comparison to a single-tagged chromosome or to a yeast centromeric plasmid shows that the segregation kinetics of the 2-microm plasmid and the chromosome are quite similar during the yeast cell cycle. Immunofluorescence analysis reveals that the plasmid is colocalized with the Rep1 and Rep2 proteins within the yeast nucleus. Furthermore, the Rep proteins (and therefore the plasmid) tend to concentrate near the poles of the yeast mitotic spindle. Depolymerization of the spindle results in partial dispersion of the Rep proteins in the nucleus concomitant with a loosening in the association between plasmid molecules. In an ipl1-2 yeast strain, shifted to the nonpermissive temperature, the chromosomes and plasmid almost always missegregate in tandem. Our results suggest that, after DNA replication, plasmid distribution to the daughter cells occurs in the form of specific DNA-protein aggregates. They further indicate that the plasmid partitioning mechanism may exploit at least some of the components of the cellular machinery required for chromosomal segregation. 相似文献
9.
T N Kozhina O V Chepurnaia I V Fedorova 《Molekuliarnaia genetika, mikrobiologiia i virusologiia》1985,(5):31-36
Cotransformants of yeast cells by two partially homologous plasmids, one of which is incapable of autonomous replication, has been used to construct multiply marked recombinant plasmids. Only simultaneous elimination of three yeast markers was registered when episomal plasmid, carrying Ade2 gene, and integrative plasmid, carrying yeast genes LEU2 and URA3, were cotransformed. Transformants, in which yeast genes LEU2, URA3 and HIS3 are linked, have been isolated by analogous technique. The genetic analysis has confirmed existence of plasmid cointegrates in the transformant cells, which carry three yeast genes, bacterial DNA fragment and 2 micrometers DNA fragment, coding for replicative functions. Recombination in the region of bacterial plasmid pBR322 might have resulted in formation of such plasmids. Plasmid recombination in cotransformants has been used to construct multiply marked circular chromosomes, having included yeast genes LEU2, URA3 and TRP1, centromere of the IV yeast chromosome and the sequence coding for their replication in yeast as well as in E. coli cells. 相似文献
10.
11.
Copy number amplification of the 2 micron circle plasmid of Saccharomyces cerevisiae 总被引:27,自引:0,他引:27
A B Futcher 《Journal of theoretical biology》1986,119(2):197-204
The 2 micron circle is a small double stranded DNA plasmid that occurs at about 60 copies per cell in the nuclei of virtually all strains of Saccharomyces cerevisiae. The plasmid has no apparent phenotypic effect on host cells, and is the basis of many useful vectors for the transformation of yeast. Under certain circumstances, the plasmid is apparently able to replicate more than once per cell cycle; this over-replication allows the maintenance of the plasmid at high copy number. The plasmid has two inverted repeat sequences, and encodes a product that catalyses intra-molecular recombination between these two repeats. Models are proposed whereby recombination leads to copy number amplification. In particular, it is proposed that intra-molecular recombination during replication flips the orientation of one replication fork with respect to the other, so that both forks travel in the same direction around a circular monomer template, generating a large multimer from a monomer and a single initiation of replication. 相似文献
12.
13.
A positive selection system for intrachromosomal recombination in Saccharomyces cerevisiae has been developed. This was achieved by integration of a plasmid containing an internal fragment of the HIS3 gene into its chromosomal location. This resulted in two copies of the HIS3 gene one with a terminal deletion at the 3' end and the other with a terminal deletion at the 5' end. Reversion of the gene disruption could be brought about by plasmid excision, unequal sister chromatid exchange or sister chromatid conversion. The purpose of this study was to define the mechanisms involved in reversion of the gene disruption. The frequency of plasmid excision could be determined by placing a yeast sequence bearing an origin of replication onto the plasmid that was subsequently integrated into the yeast genome. Unequal sister chromatid exchange and conversion could be distinguished by determining the nature of the reciprocal product by Southern blotting. The results indicate that reversion might occur mainly by conversion between sister chromatids. This is because the frequency of plasmid excision was about two orders of magnitude lower than the overall frequency of reversion and no reciprocal product indicative of sister chromatid exchange was found. The findings of this presentation suggest that conversion might be an important mechanism for recombination of sister chromatids and possibly for repair of damaged DNA in S or G2. 相似文献
14.
Connie Holm 《Cell》1982,29(2):585-594
Strains of Saccharomyces that carry the nib allele of a nuclear gene exhibit a “nibbled” colony morphology if they also harbor the plasmid 2μ DNA. I have found that the expression of the nibbled phenotype is correlated with the presence of a subpopulation of abnormally large cells that give rise to mortal clones. Large cells apparently become large as a consequence of a defect in DNA replication or nuclear division. Large nib cells contain twice as much 2μ DNA per microgram of total DNA as small nib cells do, and elevated 2μ DNA copy number is the cause, not the effect, of increased cell size. It appears that the NIB allele can prevent an increase in 2μ DNA copy number, but cannot produce a decrease once the copy number has exceeded the normal level. I propose, therefore, that the NIB gene product normally represses the amplification of 2μ DNA copy number, and that the nib allele is partially defective in this function. 相似文献
15.
Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. 总被引:289,自引:0,他引:289
We have constructed a plasmid, YEp13, which when used in conjunction with transformation in yeast is a suitable vector for isolating specific yeast genes. The plasmid consists of pBR322, the LEU2 gene of yeast, and a DNA fragment containing a yeast origin of replication from 2 mu circule. We have demonstrated the utility of this cloning system by isolating the yeast gene encoding the arginine permease, CAN1, from a pool of random yeast DNA fragments inserted into YEp13. 相似文献
16.
Khan SA 《Molecular microbiology》2000,37(3):477-484
It is now well established that a large majority of small, multicopy plasmids of Gram-positive bacteria use the rolling-circle (RC) mechanism for their replication. Furthermore, the host range of RC plasmids now includes Gram-negative organisms as well as archaea. RC plasmids can be broadly classified into at least five families, individual members of which are spread among widely different bacteria. There is significant homology in the basic replicons of plasmids belonging to a particular family, and there is compelling evidence that such plasmids have evolved from common ancestors. Major advances have recently been made in our understanding of plasmid RC replication, including the characterization of the biochemical activities of the plasmid initiator proteins and their interaction with the double-strand origin, the domain structure of the initiator proteins and the molecular basis for the function of single-strand origins in plasmid lagging strand synthesis. Over the past several years, there has been a 'renaissance' in studies on RC replication as a result of the discovery that many plasmids replicate by this mechanism, and studies in the next few years are likely to reveal new and novel mechanisms used by RC plasmids for their regulated replication. 相似文献
17.
Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase. 总被引:45,自引:0,他引:45
Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2 micron DNA. Replication of this DNA was studied in two ways. The distribution of replication events among 2 micron DNA molecules was examined by density transfer experiments with asynchronous cultures. The data show that 2 micron DNA replication is similar to chromosomal DNA replication: essentially all 2 micron duplexes were of hybrid density at one cell doubling after the density transfer, with the majority having one fully dense strand and one fully light strand. The results show that replication of 2 micron DNA occurs by a semiconservative mechanism where each of the plasmid molecules replicates once each cell cycle. 2 micron DNA is the only known example of a multiple-copy, extrachromosomal DNA in which every molecule replicates in each cell cycle. Quantitative analysis of the data indicates that 2 micron DNA replication is limited to a fraction of the cell cycle. The period in the cell cycle when 2 micron DNA replicates was examined directly with synchronous cell cultures. Synchronization was accomplished by sequentially arresting cells in G1 phase using the yeast pheromone alpha-factor and incubating at the restrictive temperature for a cell cycle (cdc 7) mutant. Replication was monitored by adding 3H-uracil to cells previously labeled with 14C-uracil, and determining the 3H/14C ratio for purified DNA species. 2 micron DNA replication did not occur during the G1 arrest periods. However, the population of 2 micron DNA doubled during the synchronous S phase at the permissive temperature, with most of the replication occurring in the first third of S phase. Our results indicate that a mechanism exists which insures that the origin of replication of each 2 micron DNA molecule is activated each S phase. As with chromosomal DNA, further activation is prevented until the next cell cycle. We propose that the mechanism which controls the replication initiation of each 2 micron DNA molecule is identical to that which controls the initiation of chromosomal DNA. 相似文献
18.
It is shown in this paper that a DNA fragment of Hepatitis B virus possessing structural features of yeast replication enhancer increases the mitotic stability of yeast transformants containing hybrid plasmids of episomal and replicative types. The mitotic stability of transformants with plasmid of the replicative type and with the replication enhancer increases only in [cir+] cells. Comparison of primary sequences of HBV DNA of different subtypes revealed that only DNA has unique structural features of the yeast enhancer of replication. 相似文献
19.
20.
The sequence and organization of Ddp2, a high-copy-number nuclear plasmid of Dictyostelium discoideum 总被引:3,自引:0,他引:3
The complete nucleotide sequence of the plasmid Ddp2 found in the nucleus of the simple eukaryote Dictyostelium discoideum is reported. This 5852-bp plasmid contains a 2661-bp open reading frame (ORF), named the "Rep gene," and 501-bp imperfect inverted repeats. A 1762-bp section of Ddp2, which includes one of the 501-bp repeat sequences, could be deleted without abolishing extrachromosomal replication. Deletion of the second 501-bp repeat, or interruption of the Rep gene, removed the ability to replicate extrachromosomally. We suggest that Ddp2 encodes a protein, "REP," that positively regulates replication initiation, a regulatory mechanism different from that of the yeast 2 mu plasmid which also possesses inverted repeat sequences. Ddp2 has a structure similar to that of plasmid pDG1, found in an unidentified isolate of Dictyostelium, with a similar sized ORF and inverted repeats. A common evolutionary origin is suggested by considerable sequence homology between the ORFs of pDG1 and Ddp2. 相似文献