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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

Characterization of chromosome instability (CIN) gene dosage sensitivity. (A) A bar plot shows CIN rate of nine strains each having heterozygous deletion of one icCIN gene (dark gray bar). These are thus two-way dosage-sensitive genes. Data are shown as mean ± SEM (n = 8). (B) A table displaying core complex involvement of six two-way dosage-sensitive CIN genes. (C) Bar plots showing the CIN rate of haploid strains with different copy number of genetically integrated NPL3. WT control has one native copy of NPL3. Data are shown as mean ± SEM, n = 8. P value was calculated from t-test. ****P < 0.0001. (D) Bar plots comparing the CIN rate of strains containing a blank centromeric plasmid, NPL3 wild-type centromeric plasmid, and NPL3 point mutant centromeric plasmid. Top panel indicates the position of the point mutation in the second RNA Recognition Motif (RRM2) of Npl3p. N (N terminus); C (C terminus). Mean ± SEM, n = 8. P value was calculated from t-test. **P < 0.01. (E) Bar plots showing the CIN rate of haploid strains with different copy number of genetically integrated MCD1. Data are shown as mean ± SEM, n = 8. P value was calculated from t-test. ****P < 0.0001. (F) Bar plots comparing CIN rate of strains containing two blank centromeric plasmids (Empty Plasmids Control), MCD1 centromeric plasmid plus one blank centromeric plasmid (MCD1), ESP1 centromeric plasmid plus one blank centromeric plasmid (ESP1), and MCD1 centromeric plasmid plus ESP1 centromeric plasmid (MCD1 + ESP1). Mean ± SEM, n = 8. P value was calculated from t-test. ***P < 0.001. (G) Bar plots comparing CIN rate of strains containing two blank centromeric plasmids (Empty Plasmids Control), MAD1 centromeric plasmid plus one blank centromeric plasmid (MAD1), MAD2 centromeric plasmid plus one blank centromeric plasmid (MAD2), and MAD1 centromeric plasmid plus MAD2 centromeric plasmid (MAD1 + MAD2). Mean ± SEM, n = 8. P value was calculated from t-test. ****P < 0.0001.
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fig5: Characterization of chromosome instability (CIN) gene dosage sensitivity. (A) A bar plot shows CIN rate of nine strains each having heterozygous deletion of one icCIN gene (dark gray bar). These are thus two-way dosage-sensitive genes. Data are shown as mean ± SEM (n = 8). (B) A table displaying core complex involvement of six two-way dosage-sensitive CIN genes. (C) Bar plots showing the CIN rate of haploid strains with different copy number of genetically integrated NPL3. WT control has one native copy of NPL3. Data are shown as mean ± SEM, n = 8. P value was calculated from t-test. ****P < 0.0001. (D) Bar plots comparing the CIN rate of strains containing a blank centromeric plasmid, NPL3 wild-type centromeric plasmid, and NPL3 point mutant centromeric plasmid. Top panel indicates the position of the point mutation in the second RNA Recognition Motif (RRM2) of Npl3p. N (N terminus); C (C terminus). Mean ± SEM, n = 8. P value was calculated from t-test. **P < 0.01. (E) Bar plots showing the CIN rate of haploid strains with different copy number of genetically integrated MCD1. Data are shown as mean ± SEM, n = 8. P value was calculated from t-test. ****P < 0.0001. (F) Bar plots comparing CIN rate of strains containing two blank centromeric plasmids (Empty Plasmids Control), MCD1 centromeric plasmid plus one blank centromeric plasmid (MCD1), ESP1 centromeric plasmid plus one blank centromeric plasmid (ESP1), and MCD1 centromeric plasmid plus ESP1 centromeric plasmid (MCD1 + ESP1). Mean ± SEM, n = 8. P value was calculated from t-test. ***P < 0.001. (G) Bar plots comparing CIN rate of strains containing two blank centromeric plasmids (Empty Plasmids Control), MAD1 centromeric plasmid plus one blank centromeric plasmid (MAD1), MAD2 centromeric plasmid plus one blank centromeric plasmid (MAD2), and MAD1 centromeric plasmid plus MAD2 centromeric plasmid (MAD1 + MAD2). Mean ± SEM, n = 8. P value was calculated from t-test. ****P < 0.0001.

Mentions: MAD1 and CLB2 represent overlapping hits from the dcCIN and icCIN screens (Figure S2D), suggesting the existence of two-way dosage sensitive CIN genes. To identify more of these genes, dcCIN analysis was performed for the 25 icCIN genes, which include 10 essential genes not previously included in the dcCIN screen but now deleted manually in a WT diploid qCTF strain. Altogether, 9 of 25 icCIN genes were found to be also dcCIN genes (Figure 5A). Interestingly, 6 of these genes encode components of core protein complexes (Figure 5B). For example, GLC7, encoding the catalytic subunit of protein phosphatase 1, is a stoichiometric subunit of the cleavage and polyadenylation factor complex, and Nuf2, is a component of the Ndc80 kinetochore complex. The fact that both increase and reduction in copy numbers of these genes enhanced CIN may support the idea that their dosage effect is due to an imbalanced protein stoichiometry rather than insufficiency or over-abundance in the activity of the encoded proteins (Papp et al. 2003).


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)

Characterization of chromosome instability (CIN) gene dosage sensitivity. (A) A bar plot shows CIN rate of nine strains each having heterozygous deletion of one icCIN gene (dark gray bar). These are thus two-way dosage-sensitive genes. Data are shown as mean ± SEM (n = 8). (B) A table displaying core complex involvement of six two-way dosage-sensitive CIN genes. (C) Bar plots showing the CIN rate of haploid strains with different copy number of genetically integrated NPL3. WT control has one native copy of NPL3. Data are shown as mean ± SEM, n = 8. P value was calculated from t-test. ****P < 0.0001. (D) Bar plots comparing the CIN rate of strains containing a blank centromeric plasmid, NPL3 wild-type centromeric plasmid, and NPL3 point mutant centromeric plasmid. Top panel indicates the position of the point mutation in the second RNA Recognition Motif (RRM2) of Npl3p. N (N terminus); C (C terminus). Mean ± SEM, n = 8. P value was calculated from t-test. **P < 0.01. (E) Bar plots showing the CIN rate of haploid strains with different copy number of genetically integrated MCD1. Data are shown as mean ± SEM, n = 8. P value was calculated from t-test. ****P < 0.0001. (F) Bar plots comparing CIN rate of strains containing two blank centromeric plasmids (Empty Plasmids Control), MCD1 centromeric plasmid plus one blank centromeric plasmid (MCD1), ESP1 centromeric plasmid plus one blank centromeric plasmid (ESP1), and MCD1 centromeric plasmid plus ESP1 centromeric plasmid (MCD1 + ESP1). Mean ± SEM, n = 8. P value was calculated from t-test. ***P < 0.001. (G) Bar plots comparing CIN rate of strains containing two blank centromeric plasmids (Empty Plasmids Control), MAD1 centromeric plasmid plus one blank centromeric plasmid (MAD1), MAD2 centromeric plasmid plus one blank centromeric plasmid (MAD2), and MAD1 centromeric plasmid plus MAD2 centromeric plasmid (MAD1 + MAD2). Mean ± SEM, n = 8. P value was calculated from t-test. ****P < 0.0001.
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fig5: Characterization of chromosome instability (CIN) gene dosage sensitivity. (A) A bar plot shows CIN rate of nine strains each having heterozygous deletion of one icCIN gene (dark gray bar). These are thus two-way dosage-sensitive genes. Data are shown as mean ± SEM (n = 8). (B) A table displaying core complex involvement of six two-way dosage-sensitive CIN genes. (C) Bar plots showing the CIN rate of haploid strains with different copy number of genetically integrated NPL3. WT control has one native copy of NPL3. Data are shown as mean ± SEM, n = 8. P value was calculated from t-test. ****P < 0.0001. (D) Bar plots comparing the CIN rate of strains containing a blank centromeric plasmid, NPL3 wild-type centromeric plasmid, and NPL3 point mutant centromeric plasmid. Top panel indicates the position of the point mutation in the second RNA Recognition Motif (RRM2) of Npl3p. N (N terminus); C (C terminus). Mean ± SEM, n = 8. P value was calculated from t-test. **P < 0.01. (E) Bar plots showing the CIN rate of haploid strains with different copy number of genetically integrated MCD1. Data are shown as mean ± SEM, n = 8. P value was calculated from t-test. ****P < 0.0001. (F) Bar plots comparing CIN rate of strains containing two blank centromeric plasmids (Empty Plasmids Control), MCD1 centromeric plasmid plus one blank centromeric plasmid (MCD1), ESP1 centromeric plasmid plus one blank centromeric plasmid (ESP1), and MCD1 centromeric plasmid plus ESP1 centromeric plasmid (MCD1 + ESP1). Mean ± SEM, n = 8. P value was calculated from t-test. ***P < 0.001. (G) Bar plots comparing CIN rate of strains containing two blank centromeric plasmids (Empty Plasmids Control), MAD1 centromeric plasmid plus one blank centromeric plasmid (MAD1), MAD2 centromeric plasmid plus one blank centromeric plasmid (MAD2), and MAD1 centromeric plasmid plus MAD2 centromeric plasmid (MAD1 + MAD2). Mean ± SEM, n = 8. P value was calculated from t-test. ****P < 0.0001.
Mentions: MAD1 and CLB2 represent overlapping hits from the dcCIN and icCIN screens (Figure S2D), suggesting the existence of two-way dosage sensitive CIN genes. To identify more of these genes, dcCIN analysis was performed for the 25 icCIN genes, which include 10 essential genes not previously included in the dcCIN screen but now deleted manually in a WT diploid qCTF strain. Altogether, 9 of 25 icCIN genes were found to be also dcCIN genes (Figure 5A). Interestingly, 6 of these genes encode components of core protein complexes (Figure 5B). For example, GLC7, encoding the catalytic subunit of protein phosphatase 1, is a stoichiometric subunit of the cleavage and polyadenylation factor complex, and Nuf2, is a component of the Ndc80 kinetochore complex. The fact that both increase and reduction in copy numbers of these genes enhanced CIN may support the idea that their dosage effect is due to an imbalanced protein stoichiometry rather than insufficiency or over-abundance in the activity of the encoded proteins (Papp et al. 2003).

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