Search PubMed⌕ Search

Biomedical subjects

C S Newlon

Publications and source records attributed to C S Newlon.

At least 37 records · Page 2Linked to original sources
PubMed1993

The effect on chromosome stability of deleting replication origins.

The observed spacing between chromosomal DNA replication origins in Saccharomyces cerevisiae is at least four times shorter than should be necessary to ensure complete replication of chromosomal DNA during the S phase. To test whether all replication origins are required for normal chromosome stability, the loss rates of derivatives of chromosome III from which one or more origins had been deleted were measured. In the case of a 61-kb circular derivative of the chromosome that has two highly active origins and one origin that initiates only 10 to 20% of the time, deletion of either highly active origin increased its rate of loss two- to fourfold. Deletion of both highly active origins caused the ring chromosome to be lost in approximately 20% of cell divisions. This very high rate of loss demonstrates that there are no efficient cryptic origins on the ring chromosome that are capable of ensuring its replication in the absence of the origins that are normally used. Deletion of the same two origins from the full-length chromosome III, which contains more than six replication origins, had no effect on its rate of loss. These results suggest that the increase in the rate of loss of the small circular chromosome from which a single highly active origin was deleted was caused by the failure of the remaining highly active origin to initiate replication in a small fraction (approximately 0.003) of cell cycles.

Chromosome Mapping↗
PubMed1992

A replication map of a 61-kb circular derivative of Saccharomyces cerevisiae chromosome III.

Using two-dimensional agarose gel electrophoresis, we determined the replication map of a 61-kb circular derivative of Saccharomyces cerevisiae chromosome III. The three sites of DNA replication initiation on the ring chromosome are specific and coincide with ARS elements. The three origins are active to different degrees; two are used > 90% of the time, whereas the third is used only 10-20% of the time. The specificity of these origins is shown by the fact that only ARS elements were competent for origin function, and deletion of one of the ARS elements removed the corresponding replication origin. The activity of the least active origin was not increased by deletion of the nearby highly active origin, demonstrating that the highly active origin does not repress function of the relatively inactive origin. Replication termination on the ring chromosome does not occur at specific sites but rather occurs over stretches of DNA ranging from 3 to 10 kb. A new region of termination was created by altering the sites of initiation. The position of the new termination site indicates that termination is not controlled by specific cis-acting DNA sequences, but rather that replication termination is determined primarily by the positions at which replication initiates. In addition, two sites on the ring chromosome were found to slow the progression of replication forks through the molecule: one is at the centromere and one at the 3' end of a yeast transposable element.

Centromere↗
PubMed1992

The ARS consensus sequence is required for chromosomal origin function in Saccharomyces cerevisiae.

Replication origins have been mapped to positions that coincide, within experimental error (several hundred base pairs), with ARS elements. To determine whether the DNA sequences required for ARS function on plasmids are required for chromosomal origin function, the chromosomal copy of ARS306 was deleted and the chromosomal copy of ARS307 was replaced with mutant derivatives of ARS307 containing single point mutations in domain A within the ARS core consensus sequence. The chromosomal origin function of these derivatives was assayed by two-dimensional agarose gel electrophoresis. Deletion of ARS306 deleted the associated replication origin. The effects on chromosomal origin function of mutations in domain A paralleled their effects on ARS function, as measured by plasmid stability. These results demonstrate that chromosomal origin function is a property of the ARS element itself.

Base Sequence↗
PubMed1992

Localization of a DNA replication origin and termination zone on chromosome III of Saccharomyces cerevisiae.

Two-dimensional gel electrophoretic replicon mapping techniques were used to identify all functional DNA replication origins and termini in a 26.5-kbp stretch in the left arm of yeast chromosome III. Only one origin was detected; it coincided with an ARS element (ARS306), as have all previously mapped yeast origins. A replication termination region was identified in a 4.3-kbp stretch at the telomere-proximal end of the investigated region, between the origin identified in this paper and the neighboring, previously mapped, ARS305-associated origin (previously called the A6C origin). Termination does not occur at a specific site; instead, it appears to be the consequence of replication forks converging in a stretch of DNA of at least 4.3 kbp.

Chromosomes, Fungal↗
PubMed1992

Replication forks pause at yeast centromeres.

The 120 bp of yeast centromeric DNA is tightly complexed with protein to form a nuclease-resistant core structure 200 to 240 bp in size. We have used two-dimensional agarose gel electrophoresis to analyze the replication of the chromosomal copies of yeast CEN1, CEN3, and CEN4 and determine the fate of replication forks that encounter the protein-DNA complex at the centromere. We have shown that replication fork pause sites are coincident with each of these centromeres and therefore probably with all yeast centromeres. We have analyzed the replication of plasmids containing mutant derivatives of CEN3 to determine whether the replication fork pause site is a result of an unusual structure adopted by centromere DNA or a result of the protein-DNA complex formed at the centromere. The mutant centromere derivatives varied in function as well as the ability to form the nuclease-resistant core structure. The data obtained from analysis of these derivatives indicate that the ability to cause replication forks to pause correlates with the ability to form the nuclease-resistant core structure and not with the presence or absence of a particular DNA sequence. Our findings further suggest that the centromere protein-DNA complex is present during S phase when replication forks encounter the centromere and therefore may be present throughout the cell cycle.

Base Sequence↗
PubMed1991

Analysis of a circular derivative of Saccharomyces cerevisiae chromosome III: a physical map and identification and location of ARS elements.

DNA was isolated from a circular derivative of chromosome III to prepare a library of recombinant plasmids enriched in chromosome III sequences. An ordered set of recombinant plasmids and bacteriophages carrying the contiguous 210-kilobase region of chromosome III between the HML and MAT loci was identified, and a complete restriction map was prepared with BamHI and EcoRI. Using the high frequency transformation assay and extensive subcloning, 13 ARS elements were mapped in the cloned region. Comparison of the physical maps of chromosome III from three strains revealed that the chromosomes differ in the number and positions of Ty elements and also show restriction site polymorphisms. A comparison of the physical map with the genetic map shows that meiotic recombination rates vary at least tenfold along the length of the chromosome.

Chromosomes, Fungal↗
PubMed1991

Evidence suggesting that the ARS elements associated with silencers of the yeast mating-type locus HML do not function as chromosomal DNA replication origins.

The silent mating-type loci of Saccharomyces cerevisiae, HML and HMR, are flanked by transcriptional silencers that have ARS activity (i.e., they function as replication origins when in plasmids). To test whether these ARS elements are chromosomal origins, we mapped origins near HML (close to the left telomere of chromosome III). Our results indicate that the HML-associated ARS elements either do not function as chromosomal replication origins or do so at a frequency below our detection level, suggesting that replication from a silencer-associated origin in each S phase is not essential for the maintenance of transcriptional repression at HML. Our results also imply that the ability of a DNA fragment to function as an ARS element in a plasmid does not ensure its ability to function as an efficient chromosomal replication origin. Telomere proximity is not responsible for inactivating these ARS elements, because they are not detectably functional as chromosomal origins even in genetically modified strains in which they are far from the telomere.

Chromosome Mapping↗
PubMed1990

Mutational analysis of the consensus sequence of a replication origin from yeast chromosome III.

Yeast autonomously replicating sequence (ARS) elements contain an 11-base-pair core consensus sequence (5'-[A/T]TTTAT[A/G]TTT[A/T]-3') that is required for function. The contribution of each position within this sequence to ARS activity was tested by creating all possible single-base mutations within the core consensus sequence of ARS307 (formerly called the C2G1 ARS) and testing their effects on high-frequency transformation and on plasmid stability. Of the 33 mutations, 22 abolished ARS function as measured by high-frequency transformation, 7 caused more than twofold reductions in plasmid stability, and 4 had no effect on plasmid stability. Mutations that reduced or abolished ARS activity occurred at each position in the consensus sequence, demonstrating that each position of this sequence contributes to ARS function. Of the four mutations that had no effect on ARS activity, three created alternative perfect matches to the core consensus sequence, demonstrating that the alternate bases allowed by the consensus sequence are, indeed, interchangeable. In addition, a change from T to C at position 6 did not perturb wild-type efficiency. To test whether the essential region extends beyond the 11-base-pair consensus sequence, the effects on plasmid stability of point mutations one base 3' to the T-rich strand of the core consensus sequence (position 12) and deletion mutations that altered bases 5' to the T-rich strand of the core consensus sequence were examined. An A at position 12 or the removal of three T residues 5' to the core consensus sequence severely diminished ARS efficiency, showing that the region required for full ARS efficiency extends beyond the core consensus sequence in both directions.

Base Sequence↗
PubMed1989

Time of replication of ARS elements along yeast chromosome III.

The replication of putative replication origins (ARS elements) was examined for 200 kilobases of chromosome III of Saccharomyces cerevisiae. By using synchronous cultures and transfers from dense to light isotope medium, the temporal pattern of mitotic DNA replication of eight fragments that contain ARSs was determined. ARS elements near the telomeres replicated late in S phase, while internal ARS elements replicated in the first half of S phase. The results suggest that some ARS elements in the chromosome may be inactive as replication origins. The actively expressed mating type locus, MAT, replicated early in S phase, while the silent cassettes, HML and HMR, replicated late. Unexpectedly, chromosome III sequences were found to replicate late in G1 at the arrest induced by the temperature-sensitive cdc7 allele.

Chromosome Mapping↗
PubMed1988

A yeast replication origin consists of multiple copies of a small conserved sequence.

Autonomously replicating sequences (ARSs) of the yeast S. cerevisiae function as replication origins on plasmids and probably also on chromosomes. ARS function requires a copy of the ARS core consensus (5'-[A/T]TTTAT[A/G]TTT[A/T]-3') and additional sequences 3' to the T-rich strand of the consensus. Our analysis of an ARS from chromosome III, the C2G1 ARS, suggests that ARS function depends on the presence of an exact match to the core consensus and the presence of additional near matches in the 3' flanking region. We have demonstrated that ARS function can be mediated by multiple matches to the core consensus by constructing synthetic ARS elements from oligonucleotides containing copies of the consensus sequence. We find that two copies of the core consensus are sufficient for ARS activity and that an artificial ARS as efficient as a natural chromosomal ARS can be constructed from multiple core consensus elements in a specific orientation.

Base Sequence↗
PubMed1987

Polymorphisms on the right arm of yeast chromosome III associated with Ty transposition and recombination events.

The region of Saccharomyces cerevisiae chromosome III centromere-distal to the PGK gene is the site of frequent chromosome polymorphisms. We have sequenced this region from fragments of chromosome III isolated from three different yeast strains, GRF88, CN31C and CF4-16B. The sequence analysis demonstrates that these polymorphisms are associated with the presence of Ty and delta elements and defines a region of the chromosome which is a hot-spot for transposition events (the RAHS). The three strains can be arranged into a logical evolutionary series in which successive transposition and recombination events insert Ty elements and fuse them with consequent deletions of chromosome and of transposon sequences. The influence of such events on yeast genome evolution is discussed.

Base Sequence↗
PubMed1986

DNA sequence analysis of ARS elements from chromosome III of Saccharomyces cerevisiae: identification of a new conserved sequence.

Four fragments of Saccharomyces cerevisiae chromosome III DNA which carry ARS elements have been sequenced. Each fragment contains multiple copies of sequences that have at least 10 out of 11 bases of homology to a previously reported 11 bp core consensus sequence. A survey of these new ARS sequences and previously reported sequences revealed the presence of an additional 11 bp conserved element located on the 3' side of the T-rich strand of the core consensus. Subcloning analysis as well as deletion and transposon insertion mutagenesis of ARS fragments support a role for 3' conserved sequence in promoting ARS activity.

Base Sequence↗
PubMed1986

A 'hot-spot' for Ty transposition on the left arm of yeast chromosome III.

The small ring derivative of Saccharomyces cerevisiae chromosome III, which was formed by a cross-over between HML on the left arm and HMR on the right arm, contains three Ty elements. The class II element Ty 1-17 lies immediately centromere-distal to LEU2 on the left arm while two class I elements are tandemly arranged distal to PGK on the right arm. We have sequenced the regions of chromosome III surrounding Ty 1-17 and have defined a region where a number of transposition events have occurred. This region is flanked by the 5' ends of two tRNA genes, tRNA3Glu on the centromere distal side and tRNA3Leu immediately in front of LEU2. Close to the tRNA3Glu gene there is a region containing degenerate delta sequences organised in opposite orientations. Immediately distal to Ty 1-17 there are two complete solo delta elements, one inserted into the other. The sequence indicates that these two delta sequences were inserted into chromosome II by separate transposition events. A model is presented to explain how this structure arose and the role of solo delta elements in transposon propagation and maintenance is discussed.

Base Sequence↗
PubMed1986

The mitotic stability of deletion derivatives of chromosome III in yeast.

We have constructed a series of deletion derivatives of chromosome III in yeast. Two telocentric chromosomes, one with a deletion of about 100 kilobases (kb) from the left arm and another with a deletion of about 240 kb from the right arm, are mitotically stable, showing only a 2- to 3-fold decrease in stability compared to a normal chromosome III. Chromosomes as small as 100 kb with deletions on both the left and right arms show only slight decreases in mitotic stability. Slight decreases in size in chromosomes smaller than 100 kb produce dramatic decreases in mitotic stability. In general, deletion chromosomes of similar size but different structure display similar stabilities. We find no evidence for the existence of any new cis-acting elements [besides the centromere, autonomously replicating sequences (ARS elements) and telomeres] essential for the stabilization of chromosome III.

Chromosome Deletion↗