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

1992 ◽  
Vol 3 (9) ◽  
pp. 999-1013 ◽  
Author(s):  
S A Greenfeder ◽  
C S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Ring Chromosome ◽  
Replication Initiation ◽  
Cis Acting ◽  
Dna Replication Initiation ◽  
Ars Elements ◽  
Highly Active ◽  
Origin Function ◽  
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.

scholarly journals DNA Sequence and Functional Analysis of Homologous ARS Elements of Saccharomyces cerevisiae and S. carlsbergensis

Genetics ◽  
1999 ◽  
Vol 152 (3) ◽  
pp. 943-952
Author(s):  
James F Theis ◽  
Chen Yang ◽  
Christopher B Schaefer ◽  
Carol S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
Chromosomal Dna ◽  
Functional Elements ◽  
Sequence Comparisons ◽  
Cis Acting ◽  
Homologous Sequences ◽  
Ars Elements

Abstract ARS elements of Saccharomyces cerevisiae are the cis-acting sequences required for the initiation of chromosomal DNA replication. Comparisons of the DNA sequences of unrelated ARS elements from different regions of the genome have revealed no significant DNA sequence conservation. We have compared the sequences of seven pairs of homologous ARS elements from two Saccharomyces species, S. cerevisiae and S. carlsbergensis. In all but one case, the ARS308-ARS308carl pair, significant blocks of homology were detected. In the cases of ARS305, ARS307, and ARS309, previously identified functional elements were found to be conserved in their S. carlsbergensis homologs. Mutation of the conserved sequences in the S. carlsbergensis ARS elements revealed that the homologous sequences are required for function. These observations suggested that the sequences important for ARS function would be conserved in other ARS elements. Sequence comparisons aided in the identification of the essential matches to the ARS consensus sequence (ACS) of ARS304, ARS306, and ARS310carl, though not of ARS310.

scholarly journals Completion of Replication Map of Saccharomyces cerevisiae Chromosome III

2001 ◽  
Vol 12 (11) ◽  
pp. 3317-3327 ◽  
Author(s):  
Arkadi Poloumienko ◽  
Ann Dershowitz ◽  
Jitakshi De ◽  
Carol S. Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Complete Analysis ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Eukaryotic Chromosome ◽  
Ars Elements ◽  
Chromosome Iii

In Saccharomyces cerevisiae chromosomal DNA replication initiates at intervals of ∼40 kb and depends upon the activity of autonomously replicating sequence (ARS) elements. The identification of ARS elements and analysis of their function as chromosomal replication origins requires the use of functional assays because they are not sufficiently similar to identify by DNA sequence analysis. To complete the systematic identification of ARS elements onS. cerevisiae chromosome III, overlapping clones covering 140 kb of the right arm were tested for their ability to promote extrachromosomal maintenance of plasmids. Examination of chromosomal replication intermediates of each of the seven ARS elements identified revealed that their efficiencies of use as chromosomal replication origins varied widely, with four ARS elements active in ≤10% of cells in the population and two ARS elements active in ≥90% of the population. Together with our previous analysis of a 200-kb region of chromosome III, these data provide the first complete analysis of ARS elements and DNA replication origins on an entire eukaryotic chromosome.

Effects of excess centromeres and excess telomeres on chromosome loss rates

1991 ◽  
Vol 11 (6) ◽  
pp. 2919-2928
Author(s):  
K W Runge ◽  
R J Wellinger ◽  
V A Zakian
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Dna Sequences ◽  
Fluctuation Analysis ◽  
Chromosome Stability ◽  
Chromosome Loss ◽  
Division Iii ◽  
Daughter Cells ◽  
Chromosome Iii ◽  
Linear Chromosomes

The linear chromosomes of eukaryotes contain specialized structures to ensure their faithful replication and segregation to daughter cells. Two of these structures, centromeres and telomeres, are limited, respectively, to one and two copies per chromosome. It is possible that the proteins that interact with centromere and telomere DNA sequences are present in limiting amounts and could be competed away from the chromosomal copies of these elements by additional copies introduced on plasmids. We have introduced excess centromeres and telomeres into Saccharomyces cerevisiae and quantitated their effects on the rates of loss of chromosome III and chromosome VII by fluctuation analysis. We show that (i) 600 new telomeres have no effect on chromosome loss; (ii) an average of 25 extra centromere DNA sequences increase the rate of chromosome III loss from 0.4 x 10(-4) events per cell division to 1.3 x 10(-3) events per cell division; (iii) centromere DNA (CEN) sequences on circular vectors destabilize chromosomes more effectively than do CEN sequences on 15-kb linear vectors, and transcribed CEN sequences have no effect on chromosome stability. We discuss the different effects of extra centromere and telomere DNA sequences on chromosome stability in terms of how the cell recognizes these two chromosomal structures.

Domain B of ARS307 contains two functional elements and contributes to chromosomal replication origin function

1994 ◽  
Vol 14 (11) ◽  
pp. 7652-7659
Author(s):  
J F Theis ◽  
C S Newlon
Keyword(s):  
Replication Origin ◽  
Consensus Sequence ◽  
Functional Elements ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Chromosomal Origin ◽  
Highly Active ◽  
Origin Function ◽  
Chromosome Iii ◽  
Replication Activity

ARS307 is highly active as a replication origin in its native location on chromosome III of Saccharomyces cerevisiae. Its ability to confer autonomous replication activity on plasmids requires the presence of an 11-bp autonomously replicating sequence (ARS) consensus sequence (ACS), which is also required for chromosomal origin function, as well as approximately 100 bp of sequence flanking the ACS called domain B. To further define the sequences required for ARS function, a linker substitution mutagenesis of domain B was carried out. The mutations defined two sequences, B1 and B2, that contribute to ARS activity. Therefore, like ARS1, domain B of ARS307 is composed of functional subdomains. Constructs carrying mutations in the B1 element were used to replace the chromosomal copy of ARS307. These mutations caused a reduction in chromosomal origin activity, demonstrating that the B1 element is required for efficient chromosomal origin function.

The HML mating-type cassette of Saccharomyces cerevisiae is regulated by two separate but functionally equivalent silencers

1989 ◽  
Vol 9 (11) ◽  
pp. 4621-4630
Author(s):  
D J Mahoney ◽  
J R Broach
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Binding Sites ◽  
The Other ◽  
Functional Domains ◽  
Gene Products ◽  
Cis Acting ◽  
Full Expression ◽  
Mating Type Genes ◽  
Chromosome Iii

Mating-type genes resident in the silent cassette HML at the left arm of chromosome III are repressed by the action of four SIR gene products, most likely mediated through two cis-acting sites located on opposite sides of the locus. We showed that deletion of either of these two cis-acting sites from the chromosome did not yield any detectable derepression of HML, while deletion of both sites yielded full expression of the locus. In addition, each of these sites was capable of exerting repression of heterologous genes inserted in their vicinity. Thus, HML expression is regulated by two independent silencers, each fully competent for maintaining repression. This situation was distinct from the organization of the other silent locus, HMR, at which a single silencer served as the predominant repressor of expression. Examination of identifiable domains and binding sites within the HML silencers suggested that silencing activity can be achieved by a variety of combinations of various functional domains.

The effect on chromosome stability of deleting replication origins

1993 ◽  
Vol 13 (1) ◽  
pp. 391-398
Author(s):  
A Dershowitz ◽  
C S Newlon
Keyword(s):  
Ring Chromosome ◽  
High Rate ◽  
Chromosome Stability ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Circular Chromosome ◽  
Cell Cycles ◽  
Highly Active ◽  
Dna Replication Origins ◽  
Chromosome Iii

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.

The replication of yeast chromosomes: lessons fromSaccharomyces cerevisiaechromosome III

1995 ◽  
Vol 73 (S1) ◽  
pp. 208-214 ◽  
Author(s):  
Carol S. Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Replication Origins ◽  
Chromosomal Replication ◽  
Position Effects ◽  
Autonomously Replicating Sequence ◽  
Systematic Analysis ◽  
Eukaryotic Chromosome ◽  
Ars Elements ◽  
Constant Rate ◽  
Chromosome Iii

To understand how a eukaryotic chromosome is replicated, a systematic analysis of chromosome III of Saccharomyces cerevisiae has been undertaken. Replication origins are specified by autonomously replicating sequence (ARS) elements, whose sequences can be dissected using a simple plasmid assay. Only a subset of ARS elements are active as chromosomal replication origins. Replication origins are required for normal chromosome transmission, but they appear to be redundant; several origins can be deleted without affecting chromosome stability. Replication origin position has been conserved on chromosome III in diverged strains, suggesting that origin position is important for chromosome function. The inability of some ARS elements to function as chromosomal replication origins appears likely to result from chromosomal context or position effects. Replication termination occurs over broad regions between active replication origins. The position of termination can be altered by deleting origins, suggesting that no specific replication termination elements are required. Replication forks appear to move at a relatively constant rate through the chromosome. A replication pause site associated with the centromere results from the kinetochore protein complex that binds the centromere to mediate chromosome segregation. Key words: Saccharomyces cerevisiae, ARS elements, replication origins, replication termination, DNA replication intermediates.

scholarly journals The effect on chromosome stability of deleting replication origins.

1993 ◽  
Vol 13 (1) ◽  
pp. 391-398 ◽  
Author(s):  
A Dershowitz ◽  
C S Newlon
Keyword(s):  
Ring Chromosome ◽  
High Rate ◽  
Chromosome Stability ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Circular Chromosome ◽  
Cell Cycles ◽  
Highly Active ◽  
Dna Replication Origins ◽  
Chromosome Iii

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.

Analysis of Replication Origin Function on Chromosome III of Saccharomyces cerevisiae

1993 ◽  
Vol 58 (0) ◽  
pp. 415-423 ◽  
Author(s):  
C.S. Newlon ◽  
I. Collins ◽  
A. Dershowitz ◽  
A.M. Deshpande ◽  
S.A. Greenfeder ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Replication Origin ◽  
Origin Function ◽  
Chromosome Iii
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