1. Genetics and Genomics
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Quiescence unveils a novel mutational force in fission yeast

  1. Serge Gangloff
  2. Guillaume Achaz
  3. Stefania Francesconi
  4. Adrien Villain
  5. Samia Miled
  6. Claire Denis
  7. Benoit Arcangioli  Is a corresponding author
  1. Institut Pasteur, France
  2. CNRS-Institut Pasteur, France
  3. UPMC, France
Research Article
Cite this article as: eLife 2017;6:e27469 doi: 10.7554/eLife.27469
8 figures, 6 tables, 1 data set and 3 additional files

Figures

Survival of ura- cells during two weeks of quiescence.

Wild-Type, ura4D18 cells and a 50% mixture of both cultures were put into quiescence and their survival was followed for two weeks. ura4D18 has no advantage compared to wild-type strain or mixed populations and the proportion of uracil auxotrophs is maintained constant for the two weeks.

https://doi.org/10.7554/eLife.27469.002
Figure 2 with 2 supplements
Mutations accumulate as a function of time in quiescence.

(A) slopes of FOAR accumulation as a function of time in quiescence from two independent experiments, determined by the least squares regression (R2 = 0.99 for all mutations, R2 = 0.96 for SNVs and R2 = 0.80 for indels). (B) mutation spectrum established on 105 SNVs found in the ura4+ and ura5+ genes at day 1 and 146 at days 8 + 15. (C) distribution among indels and SNVs of the mutations that result in FOAR for ura4+ and ura5+ genes over time. (D) distribution of the various sizes of indels over time in quiescence. The numbers directly above the histograms indicate the number of events, while those on the top of the figure are the sum of all the events for each class. The numbers at the bottom indicate the size range of the indels.

https://doi.org/10.7554/eLife.27469.003
Figure 2—figure supplement 1
SNVs distribution. 

(A) Mutation spectrum established on 105 SNVs found in the ura4+ and ura5+ genes at day 1 and 146 at days 8 + 15 (from Fig. Figure 2B). (B) Same as in A, but normalized to the total number of relevant scorable base pairs. (C) Counts of all possible STOP and nonsense mutations for each substitution in ura4 and ura5..

https://doi.org/10.7554/eLife.27469.004
Figure 2—figure supplement 2
Normalized percentage of each dinucleotide in the open reading frames of ura4+ and ura5+after one day in quiescence.

The percentage of every dinucleotide mutated at day one is compared to the frequency of occurrence of the same dinucleotide in the respective open reading frame.

https://doi.org/10.7554/eLife.27469.005
FOAR accumulation and corrected counts.

(A) slope of FOAR (as determined in Figure 2A). (B) slopes of FOAR SNVs and indels accumulation. (C) corrected counts determined on all experimental points using least squares regression (D) corrected slopes for SNVs and indels formation in the ura4+ and ura5+ genes.

https://doi.org/10.7554/eLife.27469.008
Survival curves (red: linear; blue: log) of prototrophic cells in G0 as a function of time.

The medium is being replaced every other week starting at day 15, while an aliquot is plated out to monitor the viability. The standard error of the experiments is indicated.

https://doi.org/10.7554/eLife.27469.013
Distribution of mutations among the 237 genomes sequenced.

The six strains exhibiting a phenotype were excluded from the analysis. The fit between the observed and theoretical Poisson distribution indicates that Chronos acts homogeneously on genomes over time. The goodness of fit is excellent (Pchi2 = 0.56).

https://doi.org/10.7554/eLife.27469.014
Corrected versus observed proportions of Indels.

In blue, we report the percentage of Indels expected from the FOA experiments, that is the ratio of the estimated slopes of accumulation of INDELS and SNVs (using method 1). In red, the observed fraction of INDELS after 3 months of quiescence. In green, the observed fraction of INDELs in the MA lines from (Farlow et al., 2015) P-values are computed using homogeneity chi2 tests.

https://doi.org/10.7554/eLife.27469.015
Mutation rate and spectrum in pcn1-K164R mutant strain.

(A) Survival curves in G0 of 3 independent prototrophic pcn1-K164R clones as a function of time. The standard error is indicated. (B) Frequency with standard error and slope of FOAR accumulation in G0 of 3 independent pcn1-K164R clones. The R square value is indicated. (C) Table and (D) graphical representation of the distribution among SNVs and indels of the ura4 and ura5 mutations accumulating over time in pcn1-K164R quiescent cells. The total number of the non-redundant events analyzed is indicated in the table.

https://doi.org/10.7554/eLife.27469.018
Schematic representation of a gene.

In all three methods, the gene model is the same. We observe only a subset of all possible SNVs (the back areas) among all SNVs that lead to a phenotype (the black and grey areas). All synonymous SNVs do not exhibit a phenotype. The methods aim at estimating the unknown proportion f of the non-synonymous SNVs that leads to a phenotype.

https://doi.org/10.7554/eLife.27469.019

Tables

Table 1
Proportion of septa observed among cycling and quiescent cells.
https://doi.org/10.7554/eLife.27469.006
DaysNumber of cellsNumber of SeptaPercentage
01 4001369.71%
13 50050.14%
410 50000.00%
616 00020.01%
814 00000.00%
1226 00000.00%
1526 25000.00%
Table 2
Type of mutation and AT bias found in ura4 and ura5 mutants at various time points of quiescence.
https://doi.org/10.7554/eLife.27469.007
day 1day 8day 15day 8+15
# of SNVs10510737144
Ts3326530
Tv728134116
Ts:Tv1:2.181:3.121:6.671:3.70
To A or to T63451560
To C or to G1730838
AT bias3.711.501.881.58
# of indels4013986220
# of deletions169971170
lost bases3348649071771
# of insertions24351550
gained bases26940199500
net loss654638081271
# of deletions / # of insertions0.72.84.73.4
average loss per event2191310
average gain per event1111710
  1. Ts: transitions; Tv: transversions

    AT bias: (GC→AT + GC→TA) to (AT→GC + AT→CG) mutations

Table 3
Sequencing analysis of all the FOAR colonies recovered after 6 days of quiescence.
https://doi.org/10.7554/eLife.27469.009
SNVsIndelsInsertionsDeletions
No divisionSingles819217
PairsNANANANA
One divisionSingles7936
Pairs91129
  1. NA: Not Applicable. All the FOAR isolated from the culture have been sequenced.

    For the first half of the cultures, the mutations found more than once in the clonal population were discarded.

  2. Similarly, identical mutations found in ‘no division’ and in ‘with one division’ were discarded.

Table 4
Estimation of the fraction of non-synonymous SNVs that lead to a phenotype.
https://doi.org/10.7554/eLife.27469.010
ura4ura5Total
(265 Aa)(216 Aa)(481 Aa)
Potential SNVsSynonymous530 (0.22)443 (0.23)973 (0.22)
Non-synonymous1739 (0.73)1405 (0.72)3144 (0.73)
STOPs116 (0.05)96 (0.05)212 (0.05)
Observed SNVsSynonymous3 (0.03)2 (0.03)5 (0.03)
Non-synonymous74 (0.69)58 (0.73)132 (0.71)
STOPs29 (0.27)19 (0.24)48 (0.26)
Non-coding1 (0.01)0 (0.00)1 (0.00)
Different mutations per Aa(41,13,3)(23,7,5,0,0,1)(64,20,8,0,0,1)
Independent identical mutations per non-syn SNV(64,9,1)(41,11,2,1,1,0,1, 0,1)(105,20,3,1,1,0,1, 0,1)
Method 1Essential Aa (ML)12454170
(Essential Aa)[CI 95%][93,202][43,81][140,228]
f0.470.250.35
[CI 95%][0.35,0.76][0.20,0.38][0.29,0.47]
Method 2Essential NS (ML)29688276
(Non-Syn)[CI 95%][195,394][73,118][228,362]
f0.170.060.09
[CI 95%][0.11,0.23][0.05,0.08][0.07,0.12]
Method 3# STOPs among all potentials STOPs0.250.200.23
(LM08)f0.170.210.19
Table 4—source data 1

Estimation of the fraction of SNVs and Indels that leads to a phenotype.

https://doi.org/10.7554/eLife.27469.011
Table 4—source data 2

Estimated mutation rates.

https://doi.org/10.7554/eLife.27469.012
Table 5
Type of mutation in whole genomes after 3 months in quiescence.
https://doi.org/10.7554/eLife.27469.016
Variants#
# of SNVs72
Ts26
Tv46
Ts:Tv1:21.77
to A or T33
to C or G26
AT bias1.27
# of indels77
# of deletions27
lost bases511
# of insertions50
gained of bases296
net loss215
# of deletions / # of insertions0.54
average loss per event19.6
average gain per event5.92
Table 6
Phenotypic alterations as a function of time spent in quiescence
https://doi.org/10.7554/eLife.27469.017
Time# of colonies w/phenotype18°C37°CKCl 1MCa(NO3)2 0.15MCaCl20.3MTBZ 15 μg/mlHU 4 mMSDS 0.01%
in G0
1 day0/384--------
1 month0/384--------
2 months4/376--114---
3 months6/33411113321
  1. (-) indicates the absence of a selected phenotype (all the cells form colonies) The numbers in the columns indicate the number of colonies exhibiting a sensitivity to a given treatment (some mutants display a sensitivity to multiple treatments).

    Serial dilutions were spotted on rich medium plates containing (or not) the drugs at the indicated concentrations. (TBZ): Thiabendazole; (HU): Hydroxyurea; (SDS): Sodium Dodecyl Sulfate

Data availability

The following data sets were generated
  1. 1
    Quiescence pombe sequencing
    1. Gangloff S
    (2017)
    Publicly available at the NCBI BioProject database (accession no: PRJNA413662).

Additional files

Supplementary file 1

Position and nature of the alterations yielding 5-FOA resistant colonies in ura4+ and ura5+ genes.

https://doi.org/10.7554/eLife.27469.020
Supplementary file 2

Mutations detected in the 243 colonies sequenced after 3 months in G0.

https://doi.org/10.7554/eLife.27469.021
Transparent reporting form
https://doi.org/10.7554/eLife.27469.022

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