Saccharomyces cerevisiae linear chromosome stability (lcs) mutants increase the loss rate of artificial and natural linear chromosomes

Chromosoma ◽  
1993 ◽  
Vol 102 (3) ◽  
pp. 207-217 ◽  
Author(s):  
Kurt W. Runge ◽  
Virginia A. Zakian
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Loss Rate ◽  
Chromosome Stability ◽  
Linear Chromosome ◽  
Linear Chromosomes

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.

scholarly journals 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.

scholarly journals Deletion of Budding Yeast MAD2 Suppresses Clone-to-Clone Differences in Artificial Linear Chromosome Copy Numbers and Gives Rise to Higher Retention Rates

2020 ◽  
Vol 8 (10) ◽  
pp. 1495
Author(s):  
Scott C. Schuyler ◽  
Lin-Ing Wang ◽  
Yi-Shan Ding ◽  
Yi-Chieh Lee ◽  
Hsin-Yu Chen
Keyword(s):  
Budding Yeast ◽  
Retention Rate ◽  
Retention Rates ◽  
Yeast Cells ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Linear Chromosome ◽  
Copy Numbers ◽  
Clone Differences ◽  
Linear Chromosomes

Our goal was to investigate the changes in artificial short-linear chromosome average copy numbers per cell arising from partial or full loss of Mitotic Arrest-Deficient 2 (MAD2) spindle checkpoint function in budding yeast Saccharomyces cerevisiae. Average artificial linear chromosome copy numbers in a population of cells, as measured by quantitative polymerase chain reactions (qPCR), and retention rates, as measured by fluctuation analyses, were performed on a total of 62 individual wild type and mad2∆ mutant haploid and diploid clones. Wild type cells, both haploids and diploids, displayed phenotypically unique clone-to-clone differences: one group of 15 clones displayed low-copy numbers per cell and high retention rates, were 1 clone was found to have undergone a genomic integration event, and the second group of 15 clones displayed high copy numbers per cell and low retention rates, with the latter values being consistent with the previously published results where only a single clone had been measured. These chromosome states were observed to be unstable when propagated for 10 days under selection, where high copy-low retention rate clones evolved into low copy-high retention rate clones, but no evidence for integration events was observed. By contrast, mad2∆ haploid and mad2∆/mad2∆ diploids displayed a suppression of the clone-to-clone differences, where 20 out of 21 clones had mid-level artificial linear chromosome copy numbers per cell, but maintained elevated chromosome retention rates. The elevated levels in retention rates in mad2∆ and mad2∆/mad2∆ cells were also maintained even in the absence of selection during growth over 3 days. MAD2/mad2∆ heterozygous diploids displayed multiple clonal groups: 4 with low copy numbers, 5 with mid-level copy numbers, and 1 with a high copy number of artificial linear chromosomes, but all 10 clones uniformly displayed low retention rates. Our observations reveal that MAD2 function contributes to the ability of yeast cells to maintain a high number of artificial linear chromosomes per cell in some clones, but, counter-intuitively, mad2∆ suppresses clone-to-clone differences and leads to an improvement in artificial linear chromosome retention rates yielding a more uniform and stable clonal population with mid-level chromosome copy numbers per cell.

scholarly journals Phosphorylation of Sli15 by Ipl1 Is Important for Proper CPC Localization and Chromosome Stability in Saccharomyces cerevisiae

PLoS ONE ◽  
2014 ◽  
Vol 9 (2) ◽  
pp. e89399 ◽  
Author(s):  
Vasso Makrantoni ◽  
Stephen J. Corbishley ◽  
Najma Rachidi ◽  
Nicholas A. Morrice ◽  
David A. Robinson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Stability

scholarly journals Linear Derivatives of Saccharomyces cerevisiae Chromosome III Can Be Maintained in the Absence of Autonomously Replicating Sequence Elements

2007 ◽  
Vol 27 (13) ◽  
pp. 4652-4663 ◽  
Author(s):  
Ann Dershowitz ◽  
Marylynn Snyder ◽  
Mohammed Sbia ◽  
Joan H. Skurnick ◽  
Loke Y. Ong ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Loss Rate ◽  
Replication Initiation ◽  
Replication Origins ◽  
Additional Increase ◽  
Autonomously Replicating Sequence ◽  
Sequence Elements ◽  
Chromosome Fragments ◽  
Chromosome Iii ◽  
The Right

ABSTRACT Replication origins in Saccharomyces cerevisiae are spaced at intervals of approximately 40 kb. However, both measurements of replication fork rate and studies of hypomorphic alleles of genes encoding replication initiation proteins suggest the question of whether replication origins are more closely spaced than should be required. We approached this question by systematically deleting replicators from chromosome III. The first significant increase in loss rate detected for the 315-kb full-length chromosome occurred only after all five efficient chromosomal replicators in the left two-thirds of the chromosome (ARS305, ARS306, ARS307, ARS309, and ARS310) had been deleted. The removal of the inefficient replicator ARS308 from this originless region caused little or no additional increase in loss rate. Chromosome fragmentations that removed the normally inactive replicators on the left end of the chromosome or the replicators distal to ARS310 on the right arm showed that both groups of replicators contribute significantly to the maintenance of the originless chromosome. Surprisingly, a 142-kb derivative of chromosome III, lacking all sequences that function as autonomously replicating sequence elements in plasmids, replicated and segregated properly 97% of the time. Both the replication initiation protein ORC and telomeres or a linear topology were required for the maintenance of chromosome fragments lacking replicators.

scholarly journals Effect of ARS1 mutations on chromosome stability in Saccharomyces cerevisiae.

1985 ◽  
Vol 5 (7) ◽  
pp. 1676-1684 ◽  
Author(s):  
F Srienc ◽  
J E Bailey ◽  
J L Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Flow Cytometry ◽  
Cell Biology ◽  
Plasmid Stability ◽  
Chromosome Stability ◽  
Flanking Sequence ◽  
Large Deletions ◽  
Ability To Act ◽  
Actual Flow ◽  
Beta Galactosidase

We have used a set of deletion mutations in the ARS1 element of Saccharomyces cerevisiae to measure their effect on chromosome stability. This work establishes the previously proposed existence of three domains in ARS1. Domain C, which we have previously inferred, but not proved, to be a part of ARS1, is now established. In addition, we show that increasingly large deletions of the domain have increasingly large effects, which was not realized before. Furthermore, we have provided the first positive evidence for the central importance of a 14-base-pair core sequence containing the ARS consensus element by showing that it has the ability to act as a replicator on a plasmid containing no other ARS1 flanking sequence. The method of analyzing plasmid stability used in our study employs a novel and sensitive flow cytometry assay for beta-galactosidase. We discuss ways in which flow cytometry, based on this assay, could be generalized beyond its particular application in this work to studying other aspects of the cell biology of yeast and higher cells. The actual flow cytometry method will be described in detail elsewhere.

scholarly journals Saccharomyces cerevisiae centromere CEN11 does not induce chromosome instability when integrated into the Aspergillus nidulans genome.

1986 ◽  
Vol 6 (11) ◽  
pp. 3621-3625 ◽  
Author(s):  
M T Boylan ◽  
M J Holland ◽  
W E Timberlake
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Aspergillus Nidulans ◽  
Plasmid Dna ◽  
Chromosome Instability ◽  
Chromosome Stability ◽  
Chromosomal Integration ◽  
Centromeric Dna ◽  
Preferential Loss ◽  
Chromosome Iii

We constructed Aspergillus nidulans transformation plasmids containing the A. nidulans argB+ gene and either containing or lacking centromeric DNA from Saccharomyces cerevisiae chromosome XI (CEN11). The plasmids transformed an argB Aspergillus strain to arginine independence at indistinguishable frequencies. Stable haploid transformants were obtained with both plasmids, and strains were identified in which the plasmids had integrated into chromosome III by homologous recombination at the argB locus. Plasmid DNA was recovered from a transformant containing CEN11, and the sequence of the essential portion of CEN11 was determined to be unaltered. The transformants were further characterized by using them to construct heterozygous diploids and then testing the diploids for preferential loss of the plasmid-containing chromosomes. The CEN11 sequence had little or no effect on chromosome stability. Thus, CEN11 does not prevent chromosomal integration of plasmid DNA and probably lacks centromere activity in Aspergillus spp.

scholarly journals Size threshold for Saccharomyces cerevisiae chromosomes: generation of telocentric chromosomes from an unstable minichromosome.

1986 ◽  
Vol 6 (3) ◽  
pp. 925-932 ◽  
Author(s):  
V A Zakian ◽  
H M Blanton ◽  
L Wetzel ◽  
G M Dani
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telocentric Chromosome ◽  
Stable Transformants ◽  
Large Deletions ◽  
Telocentric Chromosomes ◽  
Orthogonal Field ◽  
The Right ◽  
Linear Chromosomes ◽  
Size Threshold

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.

scholarly journals Mitotic transmission of artificial chromosomes in cdc mutants of the yeast, Saccharomyces cerevisiae.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 763-774 ◽  
Author(s):  
R E Palmer ◽  
E Hogan ◽  
D Koshland
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Relative Rate ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Linear Chromosome ◽  
Chromosome Fragment ◽  
Artificial Chromosomes ◽  
Loss Event ◽  
Efficient Transmission

Abstract In the yeast, Saccharomyces cerevisiae, cell division cycle (CDC) genes have been identified whose products are required for the execution of different steps in the cell cycle. In this study, the fidelity of transmission of a 14-kb circular minichromosome and a 155-kb linear chromosome fragment was examined in cell divisions where specific CDC products were temporarily inactivated with either inhibitors, or temperature sensitive mutations in the appropriate CDC gene. All of the cdc mutants previously shown to induce loss of endogenous linear chromosomes also induced loss of a circular minichromosome and a large linear chromosome fragment in our study (either 1:0 or 2:0 loss events). Therefore, the efficient transmission of these artificial chromosomes depends upon the same trans factors that are required for the efficient transmission of endogenous chromosomes. In a subset of cdc mutants (cdc6, cdc7 and cdc16), the rate of minichromosome loss was significantly greater than the rate of loss of the linear chromosome fragment, suggesting that a structural feature of the minichromosome (nucleotide content, length or topology) makes the minichromosome hypersensitive to the level of function of these CDC gene products. In another subset of cdc mutants (cdc7 and cdc17), the relative rate of 1:0 events to 2:0 events differed for the minichromosome and chromosome fragment, suggesting that the type of chromosome loss event observed in these mutants was dependent upon chromosome structure. Finally, we show that 2:0 events for the minichromosome can occur by both a RAD52 dependent and RAD52 independent mechanism. These results are discussed in the context of the molecular functions of the CDC products.

A 125-base-pair CEN6 DNA fragment is sufficient for complete meiotic and mitotic centromere functions in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (8) ◽  
pp. 3342-3349
Author(s):  
G Cottarel ◽  
J H Shero ◽  
P Hieter ◽  
J H Hegemann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Base Pair ◽  
Base Pairs ◽  
Linear Chromosome ◽  
Chromosome Fragment ◽  
Dna Elements ◽  
Dna Fragment ◽  
Conserved Region

Saccharomyces cerevisiae centromeres contain a conserved region ranging from 111 to 119 base pairs (bp) in length, which is characterized by the three conserved DNA elements CDEI, CDEII, and CDEIII. We isolated a 125-bp CEN6 DNA fragment (named ML CEN6) containing only these conserved elements and assayed it completely separated from its chromosomal context on circular minichromosomes and on a large linear chromosome fragment. The results show that this 125-bp CEN6 DNA fragment is by itself sufficient for complete mitotic and meiotic centromere functions.

Sign in / Sign up

Export Citation Format

Share Document