Limits...
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 for increased in copy number (icCIN) genes with the quantitative chromosome transmission fidelity (qCTF) assay. (A) A schematic representation of the strain generation and screen procedure for a genome-wide screen for icCIN genes using the yeast MoBY open-reading frame (ORF) library. Experimental details are given in the section Materials and Methods. (B) Box-and-whisker plots comparing the CIN rate of the wild-type qCTF strain without (W/O) or with the MoBY empty control plasmid and qCTF strains with the MoBY ORF plasmids, n = 8 for WT qCTF strain; n = 58 for qCTF strain with control plasmid; n = 4932 for all MoBY ORFs in the primary screen. P value was calculated from the Mann-Whitney U test. ****P < 0.0001; N.S., nonsignificant. (C). Bar plots showing the rate of chromosome instability (CIN) of the 25 icCIN genes (orange bars) and two genes (blue bars) that suppressed basal CIN when increased in copy number. Data are shown as mean ± SEM (n= 8). (D) Protein interaction network among icCIN genes. Genes that cause different fold changes in CIN rate are differentially color coded as indicated. (E) Gene Ontology Slim functional classification of icCIN genes. Only groups with at least two genes are shown with some highly similar groups being omitted. (F) Bar plots comparing the frequency of essential gene and genes involved in core protein complex from icCIN genes or all MoBY plasmids screened (Background). P value was calculated from Fisher’s exact test. **P < 0.01; N.S., nonsignificant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4478535&req=5

fig4: Genome-wide screen for increased in copy number (icCIN) genes with the quantitative chromosome transmission fidelity (qCTF) assay. (A) A schematic representation of the strain generation and screen procedure for a genome-wide screen for icCIN genes using the yeast MoBY open-reading frame (ORF) library. Experimental details are given in the section Materials and Methods. (B) Box-and-whisker plots comparing the CIN rate of the wild-type qCTF strain without (W/O) or with the MoBY empty control plasmid and qCTF strains with the MoBY ORF plasmids, n = 8 for WT qCTF strain; n = 58 for qCTF strain with control plasmid; n = 4932 for all MoBY ORFs in the primary screen. P value was calculated from the Mann-Whitney U test. ****P < 0.0001; N.S., nonsignificant. (C). Bar plots showing the rate of chromosome instability (CIN) of the 25 icCIN genes (orange bars) and two genes (blue bars) that suppressed basal CIN when increased in copy number. Data are shown as mean ± SEM (n= 8). (D) Protein interaction network among icCIN genes. Genes that cause different fold changes in CIN rate are differentially color coded as indicated. (E) Gene Ontology Slim functional classification of icCIN genes. Only groups with at least two genes are shown with some highly similar groups being omitted. (F) Bar plots comparing the frequency of essential gene and genes involved in core protein complex from icCIN genes or all MoBY plasmids screened (Background). P value was calculated from Fisher’s exact test. **P < 0.01; N.S., nonsignificant.

Mentions: Gene or segmental chromosome amplification often lead to increased expression of the affected genes and is a frequently observed genetic change in evolving genomes (Santarius et al. 2010). It has been shown that increased expression of certain mitotic regulators disrupt chromosome transmission fidelity (Ricke et al. 2011; Ryan et al. 2012; Sotillo et al. 2007), but a comprehensive analysis of genes that elevates CIN at increased copy number has not be reported. To this end, we used qCTF assay in combination with the MoBY-ORF library containing 4956 yeast ORFs with their natural promoter and terminator (Ho et al. 2009) to perform a genome-wide profiling of genes for which a moderate increase in copy number affects CIN (referred as icCIN genes). The presence of a centromeric sequence on MoBY-ORF plasmids ensures that the copy number of the gene associated with each ORF is only increased by 1- to 3-fold. The individual centromeric MoBY-ORF plasmids were transformed in a high throughput manner into the qCTF yeast strain with an efficiency of 88%, resulting in 4392 strains (Figure 4A). The transformants were then subjected to high-throughput qCTF assay by flow cytometry as with the dcCIN screen, with the qCTF strain carrying an empty MoBY plasmid as the control. It is interesting to note that the CIN rate in this control strain is 1.4 fold higher (P < 0.0001) than the parental strain without the centromeric MoBY vector (Figure 4B), and additional centromeric plasmids leads to further increase in CIN (Figure S4). This finding suggests that extra centromeres in an otherwise-euploid genome elevate CIN.


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 for increased in copy number (icCIN) genes with the quantitative chromosome transmission fidelity (qCTF) assay. (A) A schematic representation of the strain generation and screen procedure for a genome-wide screen for icCIN genes using the yeast MoBY open-reading frame (ORF) library. Experimental details are given in the section Materials and Methods. (B) Box-and-whisker plots comparing the CIN rate of the wild-type qCTF strain without (W/O) or with the MoBY empty control plasmid and qCTF strains with the MoBY ORF plasmids, n = 8 for WT qCTF strain; n = 58 for qCTF strain with control plasmid; n = 4932 for all MoBY ORFs in the primary screen. P value was calculated from the Mann-Whitney U test. ****P < 0.0001; N.S., nonsignificant. (C). Bar plots showing the rate of chromosome instability (CIN) of the 25 icCIN genes (orange bars) and two genes (blue bars) that suppressed basal CIN when increased in copy number. Data are shown as mean ± SEM (n= 8). (D) Protein interaction network among icCIN genes. Genes that cause different fold changes in CIN rate are differentially color coded as indicated. (E) Gene Ontology Slim functional classification of icCIN genes. Only groups with at least two genes are shown with some highly similar groups being omitted. (F) Bar plots comparing the frequency of essential gene and genes involved in core protein complex from icCIN genes or all MoBY plasmids screened (Background). P value was calculated from Fisher’s exact test. **P < 0.01; N.S., nonsignificant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Genome-wide screen for increased in copy number (icCIN) genes with the quantitative chromosome transmission fidelity (qCTF) assay. (A) A schematic representation of the strain generation and screen procedure for a genome-wide screen for icCIN genes using the yeast MoBY open-reading frame (ORF) library. Experimental details are given in the section Materials and Methods. (B) Box-and-whisker plots comparing the CIN rate of the wild-type qCTF strain without (W/O) or with the MoBY empty control plasmid and qCTF strains with the MoBY ORF plasmids, n = 8 for WT qCTF strain; n = 58 for qCTF strain with control plasmid; n = 4932 for all MoBY ORFs in the primary screen. P value was calculated from the Mann-Whitney U test. ****P < 0.0001; N.S., nonsignificant. (C). Bar plots showing the rate of chromosome instability (CIN) of the 25 icCIN genes (orange bars) and two genes (blue bars) that suppressed basal CIN when increased in copy number. Data are shown as mean ± SEM (n= 8). (D) Protein interaction network among icCIN genes. Genes that cause different fold changes in CIN rate are differentially color coded as indicated. (E) Gene Ontology Slim functional classification of icCIN genes. Only groups with at least two genes are shown with some highly similar groups being omitted. (F) Bar plots comparing the frequency of essential gene and genes involved in core protein complex from icCIN genes or all MoBY plasmids screened (Background). P value was calculated from Fisher’s exact test. **P < 0.01; N.S., nonsignificant.
Mentions: Gene or segmental chromosome amplification often lead to increased expression of the affected genes and is a frequently observed genetic change in evolving genomes (Santarius et al. 2010). It has been shown that increased expression of certain mitotic regulators disrupt chromosome transmission fidelity (Ricke et al. 2011; Ryan et al. 2012; Sotillo et al. 2007), but a comprehensive analysis of genes that elevates CIN at increased copy number has not be reported. To this end, we used qCTF assay in combination with the MoBY-ORF library containing 4956 yeast ORFs with their natural promoter and terminator (Ho et al. 2009) to perform a genome-wide profiling of genes for which a moderate increase in copy number affects CIN (referred as icCIN genes). The presence of a centromeric sequence on MoBY-ORF plasmids ensures that the copy number of the gene associated with each ORF is only increased by 1- to 3-fold. The individual centromeric MoBY-ORF plasmids were transformed in a high throughput manner into the qCTF yeast strain with an efficiency of 88%, resulting in 4392 strains (Figure 4A). The transformants were then subjected to high-throughput qCTF assay by flow cytometry as with the dcCIN screen, with the qCTF strain carrying an empty MoBY plasmid as the control. It is interesting to note that the CIN rate in this control strain is 1.4 fold higher (P < 0.0001) than the parental strain without the centromeric MoBY vector (Figure 4B), and additional centromeric plasmids leads to further increase in CIN (Figure S4). This finding suggests that extra centromeres in an otherwise-euploid genome elevate CIN.

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