In the yeast Saccharomyces cerevisiae, origins of replication (autonomously replicating sequences; ARSs), centromeres, and telomeres have been isolated and characterized. The identification of these structures allows the construction of artificial chromosomes in which the architecture of eukaryotic chromosomes may be studied. A common feature of most, and possibly all, natural yeast chromosomes is that they have an ARS within 2 kilobases of their physical ends. To study the effects of such telomeric ARSs on chromosome maintenance, we introduced artificial chromosomes of approximately 15 and 60 kilobases into yeast cells and analyzed the requirements for telomeric ARSs and the effects of ARS-free chromosomal arms on the stability of these molecules. We find that terminal blocks of telomeric repeats are sufficient to be recognized as telomeres. Moreover, artificial chromosomes containing telomere-associated Y' sequences and telomeric ARSs were no more stable during both mitosis and meiosis than artificial chromosomes lacking terminal ARSs, indicating that yeast-specific blocks of telomeric sequences are the only cis-acting requirement for a functional telomere during both mitotic growth and meiosis. The results also show that there is no requirement for an origin of replication on each arm of the artificial chromosomes, indicating that a replication fork may efficiently move through a functional centromere region.
Short stretches of cloned telomeric sequences are necessary and sufficient for telomere formation in yeast as long as the sequences are present in the same orientation as they are found in vivo. During telomere formation, DNA termini usually undergo RAD52-independent recombination with other DNA termini as would be predicted by models of recombination-mediated telomere replication.
The nib 1 allele of yeast confers a sensitivity to an endogenous plasmid, 2 mu DNA, in that nib 1 strains bearing 2 mu DNA (cir+) exhibit a reduction in division potential. In the present study, the reduction in division potential characteristic of nib 1 cir+ strains is shown to be dependent on the simultaneous presence of both the A and the D open reading frames of 2 mu DNA as well as on the presence of an unidentified extrachromosomal element other than 2 mu DNA. Furthermore, in nib 1 strains, an uncharacterized extrachromosomal element can cause a less severe reduction of division potential in the absence of intact 2 mu DNA. Thus, the nib 1 allele may confer a generalized sensitivity to extrachromosomal elements.
Pulsed-field gel electrophoresis was used to examine the distribution of telomere-associated sequences on individual chromosomes in four strains of Saccharomyces cerevisiae. The pattern of X and Y' distribution was different for each strain. At least one chromosome in each strain lacked Y', and in some strains, chromosome I, the smallest yeast chromosome, lacked detectable amounts of both X and Y'.
Acentric yeast plasmids are mitotically unstable, apparently because they cannot freely diffuse after replicating and therefore are not included in the daughter nucleus. This behavior could result if plasmids remain attached to structural elements of the nucleus after replicating. Since DNA replication is believed to take place on the nuclear matrix, we tested whether there was a correlation between the mitotic stability of a given plasmid and the extent to which it was found associated with residual nuclear structures. Residual nuclei were prepared from yeast nuclei by extraction with either high salt, 2 M NaCl, or low salt, 10 mM lithium diiodosalicylate (LIS). Hybridization analysis was used to estimate the fraction of plasmid molecules remaining after nuclei were extracted. We examined the extent of matrix association of three ARS1 plasmids, Trp1-RI circle (1.45 kb), YRp7 (5.7 kb) and p lambda BAT (45.1 kb) with mitotic loss rates ranging from 3-25%. In addition we examined the matrix binding of the endogenous 2 micron plasmid and the 2 micron-derived YEp13 which is relatively stable in the presence of 2 micron and less stable in cir degree strains. Among the ARS1 plasmids we observed a negative correlation between stability and matrix association, consistent with models in which binding to the nuclear matrix prevents passive segregation of ARS1 plasmid molecules. No such correlation was observed among the 2 micron plasmids. Among all plasmids examined there is a positive correlation between size and matrix association.
Two middle repetitive DNA sequences called X and Y' are found near the telomeres of many chromosomes in Saccharomyces cerevisiae. Orthogonal field gel electrophoresis (OFAGE) was used to examine the distribution of X and Y' on different yeast chromosomes. Although the distribution of X and Y' varies among different laboratory strains of yeast, most yeast chromosomes in four different strains carry both X and Y'. However, at least one chromosome in each strain lacks the Y' element. This result indicates that Y' is not essential for replication or segregation of at least some yeast chromosomes.
A 9-kilobase pair CEN4 linear minichromosome constructed in vitro transformed Saccharomyces cerevisiae with high frequency but duplicated or segregated inefficiently in most cells. Stable transformants were only produced by events which fundamentally altered the structure of the minichromosome: elimination of telomeres, alteration of the centromere, or an increase of fivefold or greater in its size. Half of the stable transformants arose via homologous recombination between an intact chromosome IV and the CEN4 minichromosome. This event generated a new chromosome from each arm of chromosome IV. The other "arm" of each new chromosome was identical to one "arm" of the unstable minichromosome. Unlike natural yeast chromosomes, these new chromosomes were telocentric: their centromeres were either 3.9 or 5.4 kilobases from one end of the chromosome. The mitotic stability of the telocentric chromosome derived from the right arm of chromosome IV was determined by a visual assay and found to be comparable to that of natural yeast chromosomes. Both new chromosomes duplicated, paired, and segregated properly in meiosis. Moreover, their structure, as deduced from mobilities in orthogonal field gels, did not change with continued mitotic growth or after passage through meiosis, indicating that they did not give rise to isochromosomes or suffer large deletions or additions. Thus, in S. cerevisiae the close spacing of centromeres and telomeres on a DNA molecule of chromosomal size does not markedly alter the efficiency with which it is maintained. Taken together these data suggest that there is a size threshold below which stable propagation of linear chromosomes is no longer possible.
The macronuclear DNA in the ciliated protozoan O. nova consists of integral of 10(7) gene-sized DNA molecules, all of which terminate with 20 bp of C4A4 repeats followed by a 3' (G4T4)2 single-stranded tail. Two immunologically distinct proteins of 55 and 26 kd, which are tenaciously, but noncovalently associated with Oxytricha macronuclear DNA termini, have been purified. These proteins protect DNA termini from degradation by the exonuclease Bal31. They also facilitate retention of natural and synthetic telomeric DNAs onto nitrocellulose. The Oxytricha proteins are not simply C4A4-binding proteins. Rather, their efficient binding requires both the 3' single-stranded (G4T4)2 tail and the adjacent duplex region. Thus, these proteins require both the sequence and the structure of natural DNA termini for efficient binding. As such they represent the first described example of telomeric-specific proteins.
Natural termini from macronuclear DNA of the ciliated protozoans Tetrahymena thermophila and Oxytricha fallax can support telomere formation yeast. However, plasmids carrying these ciliate termini are modified by the addition of DNA which hybridizes to the synthetic oligonucleotide poly [d(C-A]), a sequence which also hybridizes to terminal restriction fragments from yeast chromosomes but not to Tetrahymena or Oxytricha macronuclear DNAs. Thus, in yeast, the creation of new telomeres on ciliate termini involves the acquisition of yeast-specific terminal sequences presumably by either recombination or non-templated DNA synthesis. The RAD52 gene is required for the majority of yeast mitotic and meiotic recombination events. Moreover, the absence of an active RAD52 gene product results in high rates of chromosome loss. Here we demonstrate that terminal restriction fragments from Tetrahymena macronuclear ribosomal DNA (rDNA) support the formation of modified telomeres in a yeast strain carrying a defect in the RAD52 gene. Moreover, linear plasmids bearing these modified ciliate termini are stably propagated in rad52- cells.
The termini of macronuclear DNA molecules from the protozoan Oxytricha fallax share a common sequence and structure, both of which differ markedly from those deduced for yeast telomeres. Despite these differences, terminal restriction fragments from O. fallax macronuclear DNA can support telomere formation in yeasts. Two linear plasmids (LYX-1 and LYX-2) constructed by ligating BamHI-digested total Oxytricha macronuclear DNA to a yeast vector were analyzed. One end of LYX-1 and both ends of LYX-2 are derived from the Oxytricha DNA that encodes rRNA (rDNA) whereas the other end of LYX-1 is from an Oxytricha fragment other than rDNA. After propagation in yeast, both ends of LYX-1 and LYX-2 retain the C4A4 repeat characteristic of the O. fallax terminal sequence. In addition, both ends of both plasmids acquire 300-1000 base pairs of DNA containing the sequence (C-A)n, a sequence found near the termini of yeast chromosomes. Thus, at least two different Oxytricha termini display distinctive properties in yeast cells in that linear plasmids containing them are not degraded nor are they integrated into chromosomal DNA. These Oxytricha termini may act directly as telomeres in yeast; alternatively, the Oxytricha DNA may serve as a signal that results in the elaboration of a yeast telomere on the ciliate DNA.
Fragments of chromosomal DNA from a variety of eucaryotes can act as ARSs (autonomously replicating sequence) in yeasts. ARSs enable plasmids to be maintained in extrachromosomal form, presumably because they function as initiation sites for DNA replication. We isolated eight different sequences from mouse chromosomal DNA which function as ARSs in Saccharomyces cerevisiae (bakers' yeast). Although the replication efficiency of the different mouse ARSs in yeasts appears to vary widely, about one-half of them functions as well as the yeast chromosomal sequence ARS1. Moreover, five of the ARSs also promote self replication of plasmids in Schizosaccharomyces pombe (fission yeast). Each of the ARSs was cloned into plasmids suitable for transformation of mouse tissue culture cells. Plasmids were introduced into thymidine kinase (TK)-deficient mouse L cells by the calcium phosphate precipitation technique in the absence of carrier DNA. In some experiments, the ARS plasmid contained the herpes simplex virus type 1 TK gene; in other experiments (cotransformations), the TK gene was carried on a separate plasmid used in the same transformation. In contrast to their behavior in yeasts, none of the ARS plasmids displayed a significant increase in transformation frequency in mouse cells compared with control plasmids. Moreover, only 1 of over 100 cell lines contained the original plasmid in extrachromosomal form. The majority of cell lines produced by transformation with an ARS TK plasmid contained multiple copies of plasmid integrated into chromosomal DNA. In most cases, results with plasmids used in cotransformations were similar to those for plasmids carrying TK. However, cell lines produced by cotransformations with plasmids containing any one of three of the ARSs (m24, m25, or m26) often contained extrachromosomal DNAs.
Circular recombinant DNA plasmids that contain autonomously replicating sequences (ARSs) are maintained in extrachromosomal form in transformed yeast cells. However, these plasmids are unstable, being rapidly lost from cells growing without selection. Although the stability of such a plasmid can be increased by the presence of yeast centromere DNA (CEN), even CEN plasmids are lost at a high rate compared to a bona fide yeast chromosome. Natural yeast chromosomes are linear molecules; therefore, we have asked if linearization can improve the stability of recombinant DNA plasmids. Linear plasmids with and without yeast CENs were constructed in vitro by using termini from the extrachromosomal ribosomal DNA (rDNA) of the ciliated protozoan Tetrahymena thermophila as "telomeres." These linear plasmids transformed yeast at high frequency and were maintained as linear extrachromosomal molecules during mitotic growth. Moreover, linear plasmids containing CENs were also transmitted through meiosis: these plasmids segregate predominantly 2+:2- at the first meiotic division, indicating that Tetrahymena rDNA termini can provide telomere function during yeast meiosis. Linear plasmids without CENs were about as stable in mitosis as the comparable circular plasmid. Thus, the Tetrahymena rDNA termini have no marked positive or negative effect on the mitotic stability of ARS plasmids. However, linear plasmids containing CENs are three to four times less stable in mitotic cells than circular CEN plasmids. This decrease in stability is not due to a functional change in the centromere itself; rather, linearization of a CEN plasmid has a direct detrimental effect on its mitotic stability. These results may reflect the existence of spatial constraints on the positions of centromeres and telomeres, constraints which must be satisfied to achieve stable segregation of chromosomes during mitosis.
Transformation studies with Saccharomyces cerevisiae (bakers' yeast) have identified DNA sequences which permit extrachromosomal maintenance of recombinant DNA plasmids in transformed cells. It has been hypothesized that such sequences (called ARS for autonomously replicating sequence) serve as initiation sites for DNA replication in recombinant DNA plasmids and that they represent the normal sites for initiation of replication in yeast chromosomal DNA. We have constructed a novel plasmid called TRP1 R1 Circle which consists solely of 1,453 base pairs of yeast chromosomal DNA. TRP1 RI Circle contains both the TRP1 gene and a sequence called ARS1. This plasmid is found in 100 to 200 copies per cell and is relatively stable during both mitotic and meiotic cell cycles. Replication of TRP1 RI Circle requires the products of the same genes (CDC28, CDC4, CDC7, and CDC8) required for replication of chromosomaL DNA. Like chromosomal DNA, its replication does not occur in cells arrested in the B1 phase of the cell cycle by incubation with the yeast pheromone alpha-factor. In addition, TRP1 RI Circle DNA is organized into nucleosomes whose size and spacing are indistinguishable from that of bulk yeast chromatin. These results indicate that TRP1 RI Circle has the replicative and structural properties expected for an origin of replication from yeast chromosomal DNA. Thus, this plasmid is a suitable model for further studies of yeast DNA replication in both cells and cell-free extracts.