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Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity.

Zhu J, Heinecke D, Mulla WA, Bradford WD, Rubinstein B, Box A, Haug JS, Li R - G3 (Bethesda) (2015)

Bottom Line: Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells.Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately.Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110.

No MeSH data available.


Related in: MedlinePlus

Design and validation of the quantitative chromosome transmission fidelity (qCTF) assay. (A) Illustration of the organization of the mini-chromosome (MC) and how the presence of MC represses the expression of a-specific gene MFA1. The yellow bars flanking MATα and LEU2 loci represent pBR322 DNA. TEL, telomere; CEN, centromere. Chromosomes are not drawn proportionally to actual size. (B) Flow cytometry analysis of qCTF strain grown in nonselective media for MC. Black line outlines the small population of highly fluorescence cells that had lost MC. (C) Representative quantitative polymerase chain reaction karyotyping of colonies from GFP− (MC+) and GFP+ (MC−) cells. Roman number indicates each yeast chromosome. The green bar represents the left arm and the blue bar represents right arm. (D) Growth curves of cell with or without MC. Box-and-whisker plots summarize the minimum doubling time of cell with or without MC. The center line of the box indicates the median value, and boxes indicate the interquartile range (IQR). The bottom whisker contains data points that are within 1.5 IQR of the lower quartile and the top whisker contains data points that are within 1.5 IQR of the upper quartile. A P-value of 0.503 was calculated from t-test with n = 6. N.S., nonsignificant. (E) The linearized dependence of the loss rate of a given chromosome on the ratio of doubling times of cells with normal copy number of this chromosome (eup) and that of cells that have lost a copy of the chromosome (ane). An example was plotted with data in (D) using the equations (13) and (14) in File S1.
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fig1: Design and validation of the quantitative chromosome transmission fidelity (qCTF) assay. (A) Illustration of the organization of the mini-chromosome (MC) and how the presence of MC represses the expression of a-specific gene MFA1. The yellow bars flanking MATα and LEU2 loci represent pBR322 DNA. TEL, telomere; CEN, centromere. Chromosomes are not drawn proportionally to actual size. (B) Flow cytometry analysis of qCTF strain grown in nonselective media for MC. Black line outlines the small population of highly fluorescence cells that had lost MC. (C) Representative quantitative polymerase chain reaction karyotyping of colonies from GFP− (MC+) and GFP+ (MC−) cells. Roman number indicates each yeast chromosome. The green bar represents the left arm and the blue bar represents right arm. (D) Growth curves of cell with or without MC. Box-and-whisker plots summarize the minimum doubling time of cell with or without MC. The center line of the box indicates the median value, and boxes indicate the interquartile range (IQR). The bottom whisker contains data points that are within 1.5 IQR of the lower quartile and the top whisker contains data points that are within 1.5 IQR of the upper quartile. A P-value of 0.503 was calculated from t-test with n = 6. N.S., nonsignificant. (E) The linearized dependence of the loss rate of a given chromosome on the ratio of doubling times of cells with normal copy number of this chromosome (eup) and that of cells that have lost a copy of the chromosome (ane). An example was plotted with data in (D) using the equations (13) and (14) in File S1.

Mentions: The basic principle for our qCTF assay is to provide a direct readout of a chromosome loss event that can be detected in single cells. To this end, we engineered a system that leads to irreversible gain of GFP fluorescence signal soon after the loss of a tester chromosome, which can be detected and quantified in large populations of cells by the use of flow cytometry. Using the yeast mating type-determination system (Haber 2012) we first tagged the MATa-specific MFA1 gene with a 3×GFP fusion at the 3′ end of the ORF by homologous recombination, because Mfa1p is the greatest-expressed MATa specific protein (Ghaemmaghami et al. 2003). The MATα locus was then introduced into the tester chromosome (see below) in a haploid MATa strain. The α2 transcriptional repressor produced from the MATα locus strongly repressed MATa-specific genes, such as Mfa1p-3×GFP. Thus, when the tester chromosome is present, Mfa1p-3×GFP expression is strongly repressed; however, if this chromosome is lost, Mfa1p-3×GFP will be switched on and the cell will become highly fluorescent within one cell cycle after the chromosome loss event due to rapid proteasome degradation of the α2 repressor (Laney et al. 2006) (Figure 1A).


Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity.

Zhu J, Heinecke D, Mulla WA, Bradford WD, Rubinstein B, Box A, Haug JS, Li R - G3 (Bethesda) (2015)

Design and validation of the quantitative chromosome transmission fidelity (qCTF) assay. (A) Illustration of the organization of the mini-chromosome (MC) and how the presence of MC represses the expression of a-specific gene MFA1. The yellow bars flanking MATα and LEU2 loci represent pBR322 DNA. TEL, telomere; CEN, centromere. Chromosomes are not drawn proportionally to actual size. (B) Flow cytometry analysis of qCTF strain grown in nonselective media for MC. Black line outlines the small population of highly fluorescence cells that had lost MC. (C) Representative quantitative polymerase chain reaction karyotyping of colonies from GFP− (MC+) and GFP+ (MC−) cells. Roman number indicates each yeast chromosome. The green bar represents the left arm and the blue bar represents right arm. (D) Growth curves of cell with or without MC. Box-and-whisker plots summarize the minimum doubling time of cell with or without MC. The center line of the box indicates the median value, and boxes indicate the interquartile range (IQR). The bottom whisker contains data points that are within 1.5 IQR of the lower quartile and the top whisker contains data points that are within 1.5 IQR of the upper quartile. A P-value of 0.503 was calculated from t-test with n = 6. N.S., nonsignificant. (E) The linearized dependence of the loss rate of a given chromosome on the ratio of doubling times of cells with normal copy number of this chromosome (eup) and that of cells that have lost a copy of the chromosome (ane). An example was plotted with data in (D) using the equations (13) and (14) in File S1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4478535&req=5

fig1: Design and validation of the quantitative chromosome transmission fidelity (qCTF) assay. (A) Illustration of the organization of the mini-chromosome (MC) and how the presence of MC represses the expression of a-specific gene MFA1. The yellow bars flanking MATα and LEU2 loci represent pBR322 DNA. TEL, telomere; CEN, centromere. Chromosomes are not drawn proportionally to actual size. (B) Flow cytometry analysis of qCTF strain grown in nonselective media for MC. Black line outlines the small population of highly fluorescence cells that had lost MC. (C) Representative quantitative polymerase chain reaction karyotyping of colonies from GFP− (MC+) and GFP+ (MC−) cells. Roman number indicates each yeast chromosome. The green bar represents the left arm and the blue bar represents right arm. (D) Growth curves of cell with or without MC. Box-and-whisker plots summarize the minimum doubling time of cell with or without MC. The center line of the box indicates the median value, and boxes indicate the interquartile range (IQR). The bottom whisker contains data points that are within 1.5 IQR of the lower quartile and the top whisker contains data points that are within 1.5 IQR of the upper quartile. A P-value of 0.503 was calculated from t-test with n = 6. N.S., nonsignificant. (E) The linearized dependence of the loss rate of a given chromosome on the ratio of doubling times of cells with normal copy number of this chromosome (eup) and that of cells that have lost a copy of the chromosome (ane). An example was plotted with data in (D) using the equations (13) and (14) in File S1.
Mentions: The basic principle for our qCTF assay is to provide a direct readout of a chromosome loss event that can be detected in single cells. To this end, we engineered a system that leads to irreversible gain of GFP fluorescence signal soon after the loss of a tester chromosome, which can be detected and quantified in large populations of cells by the use of flow cytometry. Using the yeast mating type-determination system (Haber 2012) we first tagged the MATa-specific MFA1 gene with a 3×GFP fusion at the 3′ end of the ORF by homologous recombination, because Mfa1p is the greatest-expressed MATa specific protein (Ghaemmaghami et al. 2003). The MATα locus was then introduced into the tester chromosome (see below) in a haploid MATa strain. The α2 transcriptional repressor produced from the MATα locus strongly repressed MATa-specific genes, such as Mfa1p-3×GFP. Thus, when the tester chromosome is present, Mfa1p-3×GFP expression is strongly repressed; however, if this chromosome is lost, Mfa1p-3×GFP will be switched on and the cell will become highly fluorescent within one cell cycle after the chromosome loss event due to rapid proteasome degradation of the α2 repressor (Laney et al. 2006) (Figure 1A).

Bottom Line: Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells.Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately.Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110.

No MeSH data available.


Related in: MedlinePlus