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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

Genome-wide screen of nonessential yeast open-reading frame deletions for decreased in copy number (dcCIN) genes with the quantitative chromosome transmission fidelity (qCTF) assay. (A) A schematic representation of the strain generation and screen procedure for identifying dcCIN genes. In brief, qCTF strains hemizygous for each ORF deletion were obtained by mating as described in detail in the section Materials and Methods. OD and mini-chromosome negative (MC−) cell frequency (flow cytometry) were monitored at the beginning and end points for each culture grown in no-selective media for MC. Analysis details are given in the section Materials and Methods. (B) Gene Ontology Slim functional classification of dcCIN genes, showing only major groups with at least 10 genes with some highly similar groups being omitted. (C) A bar plot shows the rate of chromosome instability (CIN) of the top 25 dcCIN genes (orange bars). Data are shown as Mean ± SEM (n = 8). (D) Protein interaction network among dcCIN genes. Genes that cause different fold changes in CIN rate are differentially color coded as indicated.
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fig3: Genome-wide screen of nonessential yeast open-reading frame deletions for decreased in copy number (dcCIN) genes with the quantitative chromosome transmission fidelity (qCTF) assay. (A) A schematic representation of the strain generation and screen procedure for identifying dcCIN genes. In brief, qCTF strains hemizygous for each ORF deletion were obtained by mating as described in detail in the section Materials and Methods. OD and mini-chromosome negative (MC−) cell frequency (flow cytometry) were monitored at the beginning and end points for each culture grown in no-selective media for MC. Analysis details are given in the section Materials and Methods. (B) Gene Ontology Slim functional classification of dcCIN genes, showing only major groups with at least 10 genes with some highly similar groups being omitted. (C) A bar plot shows the rate of chromosome instability (CIN) of the top 25 dcCIN genes (orange bars). Data are shown as Mean ± SEM (n = 8). (D) Protein interaction network among dcCIN genes. Genes that cause different fold changes in CIN rate are differentially color coded as indicated.

Mentions: The MATa locus of the WT qCTF yeast strain was deleted so that it would behave as an MATα haploid strain and can be mated to the nonessential MATa ORF deletion strain library, generating a library of diploid strains carrying heterozygous deletion of 4919 ORFs, of 4977 nonessential ORFs and containing the qCTF system in the genetic background (Figure 3A). We first used qCTF to validate 254 previously identified nonessential dcCIN genes. The assay was performed in 96-well sample format, where ∼0.3 million cells per replicate and eight replicates were analyzed for each strain at 0- and 24-hr time points after switching the cultures to nonselective media (see the section Materials and Methods). OD at each time point also was measured for each strain to estimate cell doubling number. We found 44% (111/254) of previously identified dcCIN genes were verified by qCTF to produce significant increase in CIN (fold change >1.5, P < 0.01) compared with a WT diploid control, and 87% of these caused elevation of CIN by less than twofold (Table S4). These results also validated the ability of qCTF to quantify small changes in CIN. We therefore went on to screen the remaining heterozygous deletion strains by qCTF. Because the primary screen of 4914 genes by flow cytometry was performed without replicates, strains with the top 105 highest and the top 101 lowest CIN rates were picked as candidate hits. These candidate hits were then rescreened with eight biological replicates, and 80 of them were validated to cause significant change in CIN (fold change >1.5, P < 0.01) compared with the WT control (Table S4).


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)

Genome-wide screen of nonessential yeast open-reading frame deletions for decreased in copy number (dcCIN) genes with the quantitative chromosome transmission fidelity (qCTF) assay. (A) A schematic representation of the strain generation and screen procedure for identifying dcCIN genes. In brief, qCTF strains hemizygous for each ORF deletion were obtained by mating as described in detail in the section Materials and Methods. OD and mini-chromosome negative (MC−) cell frequency (flow cytometry) were monitored at the beginning and end points for each culture grown in no-selective media for MC. Analysis details are given in the section Materials and Methods. (B) Gene Ontology Slim functional classification of dcCIN genes, showing only major groups with at least 10 genes with some highly similar groups being omitted. (C) A bar plot shows the rate of chromosome instability (CIN) of the top 25 dcCIN genes (orange bars). Data are shown as Mean ± SEM (n = 8). (D) Protein interaction network among dcCIN genes. Genes that cause different fold changes in CIN rate are differentially color coded as indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Genome-wide screen of nonessential yeast open-reading frame deletions for decreased in copy number (dcCIN) genes with the quantitative chromosome transmission fidelity (qCTF) assay. (A) A schematic representation of the strain generation and screen procedure for identifying dcCIN genes. In brief, qCTF strains hemizygous for each ORF deletion were obtained by mating as described in detail in the section Materials and Methods. OD and mini-chromosome negative (MC−) cell frequency (flow cytometry) were monitored at the beginning and end points for each culture grown in no-selective media for MC. Analysis details are given in the section Materials and Methods. (B) Gene Ontology Slim functional classification of dcCIN genes, showing only major groups with at least 10 genes with some highly similar groups being omitted. (C) A bar plot shows the rate of chromosome instability (CIN) of the top 25 dcCIN genes (orange bars). Data are shown as Mean ± SEM (n = 8). (D) Protein interaction network among dcCIN genes. Genes that cause different fold changes in CIN rate are differentially color coded as indicated.
Mentions: The MATa locus of the WT qCTF yeast strain was deleted so that it would behave as an MATα haploid strain and can be mated to the nonessential MATa ORF deletion strain library, generating a library of diploid strains carrying heterozygous deletion of 4919 ORFs, of 4977 nonessential ORFs and containing the qCTF system in the genetic background (Figure 3A). We first used qCTF to validate 254 previously identified nonessential dcCIN genes. The assay was performed in 96-well sample format, where ∼0.3 million cells per replicate and eight replicates were analyzed for each strain at 0- and 24-hr time points after switching the cultures to nonselective media (see the section Materials and Methods). OD at each time point also was measured for each strain to estimate cell doubling number. We found 44% (111/254) of previously identified dcCIN genes were verified by qCTF to produce significant increase in CIN (fold change >1.5, P < 0.01) compared with a WT diploid control, and 87% of these caused elevation of CIN by less than twofold (Table S4). These results also validated the ability of qCTF to quantify small changes in CIN. We therefore went on to screen the remaining heterozygous deletion strains by qCTF. Because the primary screen of 4914 genes by flow cytometry was performed without replicates, strains with the top 105 highest and the top 101 lowest CIN rates were picked as candidate hits. These candidate hits were then rescreened with eight biological replicates, and 80 of them were validated to cause significant change in CIN (fold change >1.5, P < 0.01) compared with the WT control (Table S4).

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