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.

Isolation and transcriptional characterization of three genes which function at start, the controlling event of the Saccharomyces cerevisiae cell division cycle: CDC36, CDC37, and CDC39

1983 ◽  
Vol 3 (5) ◽  
pp. 881-891
Author(s):  
H J Breter ◽  
J Ferguson ◽  
T A Peterson ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Dna Sequences ◽  
Shuttle Vector ◽  
Control Process ◽  
Haploid Cell ◽  
Loop Analysis ◽  
Mrna Species ◽  
Coding Regions ◽  
Plasmid Library

The genes CDC36, CDC37, and CDC39, thought to function in the cell division control process in Saccharomyces cerevisiae, were isolated from a recombinant plasmid library prepared by partial digestion of S. cerevisiae genomic DNA with Sau3A and insertion into the S. cerevisiae-Escherichia coli shuttle vector YRp7. In each case, S. cerevisiae DNA sequences were identified which could complement mutant alleles of the gene in question and which could direct integration of a plasmid at the chromosomal location known to correspond to that gene. Complementing DNA segments were subcloned to remove extraneous coding regions. The coding regions corresponding to CDC36, CDC37, and CDC39 were then identified and localized by R-loop analysis. The estimated sizes of the three coding regions were 615, 1,400, and 2,700 base pairs, respectively. Transcriptional orientation of the coding regions was established by using M13 vectors to prepare strand-specific probes followed by hybridization to blots of electrophoresed S. cerevisiae mRNA. The intracellular steady-state abundance of the mRNA species corresponding to the genes was estimated by comparing hybridization signals on RNA blots to that of a previously determined standard, the cell cycle start gene CDC28. The quantities calculated for the three mRNA species were low, ranging from 1.5 +/- 1 copies per haploid cell for the CDC36 mRNA to 3.1 +/- 1.5 and 4.6 +/- 2 copies per haploid cell for the CDC37 and CDC39 mRNAs, respectively. The CDC28 mRNA had been previously estimated at 7.0 +/- 2 copies per cell.

Characterization of two telomeric DNA processing reactions in Saccharomyces cerevisiae

1988 ◽  
Vol 8 (11) ◽  
pp. 4642-4650
Author(s):  
A W Murray ◽  
T E Claus ◽  
J W Szostak
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Dna Polymerase ◽  
Dna Sequences ◽  
Inverted Repeat ◽  
Telomeric Sequence ◽  
Telomeric Dna ◽  
Telomeric Sequences ◽  
Telomere Elongation

We have investigated two reactions that occur on telomeric sequences introduced into Saccharomyces cerevisiae cells by transformation. The elongation reaction added repeats of the yeast telomeric sequence C1-3A to telomeric sequences at the end of linear DNA molecules. The reaction worked on the Tetrahymena telomeric sequence C4A2 and also on the simple repeat CA. The reaction was orientation specific: it occurred only when the GT-rich strand ran 5' to 3' towards the end of the molecule. Telomere elongation occurred by non-template-directed DNA synthesis rather than any type of recombination with chromosomal telomeres, because C1-3A repeats could be added to unrelated DNA sequences between the CA-rich repeats and the terminus of the transforming DNA. The elongation reaction was very efficient, and we believe that it was responsible for maintaining an average telomere length despite incomplete replication by template-directed DNA polymerase. The resolution reaction processed a head-to-head inverted repeat of telomeric sequences into two new telomeres at a frequency of 10(-2) per cell division.

scholarly journals Characterization of two telomeric DNA processing reactions in Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (11) ◽  
pp. 4642-4650 ◽  
Author(s):  
A W Murray ◽  
T E Claus ◽  
J W Szostak
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Dna Polymerase ◽  
Dna Sequences ◽  
Inverted Repeat ◽  
Telomeric Sequence ◽  
Telomeric Dna ◽  
Telomeric Sequences ◽  
Telomere Elongation

We have investigated two reactions that occur on telomeric sequences introduced into Saccharomyces cerevisiae cells by transformation. The elongation reaction added repeats of the yeast telomeric sequence C1-3A to telomeric sequences at the end of linear DNA molecules. The reaction worked on the Tetrahymena telomeric sequence C4A2 and also on the simple repeat CA. The reaction was orientation specific: it occurred only when the GT-rich strand ran 5' to 3' towards the end of the molecule. Telomere elongation occurred by non-template-directed DNA synthesis rather than any type of recombination with chromosomal telomeres, because C1-3A repeats could be added to unrelated DNA sequences between the CA-rich repeats and the terminus of the transforming DNA. The elongation reaction was very efficient, and we believe that it was responsible for maintaining an average telomere length despite incomplete replication by template-directed DNA polymerase. The resolution reaction processed a head-to-head inverted repeat of telomeric sequences into two new telomeres at a frequency of 10(-2) per cell division.

Chromosome instability mutants of Saccharomyces cerevisiae that are defective in microtubule-mediated processes

1990 ◽  
Vol 10 (1) ◽  
pp. 223-234
Author(s):  
M A Hoyt ◽  
T Stearns ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Instability ◽  
Null Alleles ◽  
Cold Sensitivity ◽  
Phenotypic Characterization ◽  
Chromosome Loss ◽  
Loss Of Function ◽  
Microtubule Stability ◽  
Chromosome Iii ◽  
Yeast Genes

By using a multiply marked supernumerary chromosome III as an indicator, we isolated mutants of Saccharomyces cerevisiae that display increased rates of chromosome loss. In addition to mutations in the tubulin-encoding TUB genes, we found mutations in the CIN1, CIN2, and CIN4 genes. These genes have been defined independently by mutations causing benomyl supersensitivity and are distinct from other known yeast genes that affect chromosome segregation. Detailed phenotypic characterization of cin mutants revealed several other phenotypes similar to those of tub mutants. Null alleles of these genes caused cold sensitivity for viability. At 11 degrees C, cin mutants arrest at the mitosis stage of their cell cycle because of loss of most microtubule structure. cin1, cin2, and cin4 mutations also cause defects in two other microtubule-mediated processes, nuclear migration and nuclear fusion (karyogamy). Overproduction of the CIN1 gene product was found to cause the same phenotype as loss of function, supersensitivity to benomyl. Our findings suggest that the CIN1, CIN2, and CIN4 proteins contribute to microtubule stability either by regulating the activity of a yeast microtubule component or as structural components of microtubules.

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 Tetrad analysis shows that gene conversion is the major mechanism involved in mutation at the human minisatellite MS1 integrated in Saccharomyces cerevisiae

2000 ◽  
Vol 75 (1) ◽  
pp. 1-12 ◽  
Author(s):  
INGRID BERG ◽  
HÅKAN CEDERBERG ◽  
ULF RANNUG
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Human Genome ◽  
Dna Sequences ◽  
Hot Spot ◽  
Tetrad Analysis ◽  
Strand Breaks ◽  
Major Mechanism ◽  
Marker Conversion ◽  
Repeat Units ◽  
Chromosome Iii

Minisatellites are arrays of tandemly repeated DNA sequences which occur at thousands of locations in the human genome. They are frequently hypervariable with respect to allele length as a result of high rates of complex and incompletely understood recombination-based germline mutation events that alter the repeat copy number. MS1 is one of the most variable minisatellites so far isolated from the human genome. We have integrated MS1, flanked by synthetic markers, in the vicinity of a hot spot for meiotic double-strand breaks upstream of the LEU2 locus in chromosome III of Saccharomyces cerevisiae. Here we present the first tetrad analysis of mutations at a human minisatellite locus. The data showed that mutant alleles occur as single mutants in one of the spores in a tetrad, also when the mutant structure was the result of a combination of intra- and inter-allelic rearrangements. The conversional transfer of repeat units from one allele to the other was associated with flanking marker conversion which always involved the same flank of the minisatellite. The results demonstrate that conversion is the predominant mechanism by which minisatellite alleles mutate to new lengths, and also support the assumption that cis-acting elements are involved in the regulation of the mutational process in humans.

scholarly journals BISEXUAL MATING BEHAVIOR IN A DIPLOID OF SACCHAROMYCES CEREVISIAE: EVIDENCE FOR GENETICALLY CONTROLLED NON-RANDOM CHROMOSOME LOSS DURING VEGETATIVE GROWTH

Genetics ◽  
1974 ◽  
Vol 78 (3) ◽  
pp. 843-858
Author(s):  
James E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Behavior ◽  
Mating Type ◽  
Vegetative Growth ◽  
Nuclear Gene ◽  
Chromosome Loss ◽  
Type Locus ◽  
Mating Efficiency ◽  
Single Mating ◽  
Chromosome Iii

ABSTRACT A diploid strain of Saccharomyces cerevisiae has been isolated which exhabits bisexual mating behavior. The strain mates with either a or α strains with a relative mating efficiency of 1 to 2%. The efficiency of mating is correlated with the frequency with which subclones of this strain revert to a single mating type. Crosses of the bisexual diploid with a/a or α/α diploids yield bisexual segregants with a frequency of approximately 3%. Analysis of the segregation of the mating type alleles and other markers on chromosome III indicates that the primary event which leads to the bisexual phenotype is the loss of one homolog of chromosome III during vegetative growth to produce a monosomic (2n-1) diploid. Evidence is presented that the loss of chromosome I11 and possibly of other chromosomes during vegetative growth is affected by a recessive nuclear gene-her (hermaphrodite)—which is not closely linked to the mating type locus.

scholarly journals ASSOCIATION OF DISOMIC CHROMOSOME LOSS WITH EMS-INDUCED CONVERSION IN YEAST

Genetics ◽  
1980 ◽  
Vol 96 (3) ◽  
pp. 613-625
Author(s):  
Douglas Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Causal Relationship ◽  
Mitotic Chromosome ◽  
Experimental Tests ◽  
Chromosome Loss ◽  
Constant Independent ◽  
Yeast Saccharomyces Cerevisiae ◽  
Proposed Model ◽  
Mitotic Gene Conversion ◽  
Chromosome Iii

ABSTRACT Experimental tests with the yeast Saccharomyces cerevisiae of a previously proposed model suggesting a causal relationship between disomic chromosome loss (n + 1 → n) and centromere-adjacent mitotic gene conversion were performed. Disomic haploid cells heteroallelic at two loci on the left arm of chromosome III were exposed to ethyl methanesulfonate (EMS) under nonlethal conditions; EMS-induced prototrophic gene convertants were selected and tested for coincident chromosome loss. The principal results are: (1) The frequency of chromosome loss among EMS-induced gene convertants selected to arise near the centromere is markedly enhanced over basal levels and remains constant, independent of EMS exposure. There is little such enhancement among EMS-induced convertants selected to arise far from the centromere. (2) Chromosome loss is almost completely associated with induced conversion of the centromere-proximal allele at the centromere-adjacent heteroallelic locus. This result is identical to (and confirms) results found previously for spontaneous loss-associated conversion. (3) The conversion polarity at the centromere-adjacent locus among unselected (nonloss-associated) induced or spontaneous mitotic convertants is identical to that among meiotic convertants and markedly favors the contromere-distal allele. These findings are wholly consistent with, and strengthen, the hypothesis that structural involvement of centromeric regions in nearby recombinational events may interfere with proper segregational function and lead to mitotic chromosome loss.

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