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Functional mapping of yeast genomes by saturated transposition

  1. Agnès H Michel
  2. Riko Hatakeyama
  3. Philipp Kimmig
  4. Meret Arter
  5. Matthias Peter
  6. Joao Matos
  7. Claudio De Virgilio
  8. Benoît Kornmann  Is a corresponding author
  1. ETH Zurich, Switzerland
  2. University of Fribourg, Switzerland
Tools and Resources
Cite this article as: eLife 2017;6:e23570 doi: 10.7554/eLife.23570
6 figures, 4 tables, 1 data set and 5 additional files

Figures

Figure 1 with 3 supplements
Principle of the method.

(A) Outline of the experimental procedure. Left, the transposon (green) can insert either into non-essential DNA (blue) and give rise to a clone, or into essential DNA (orange), in which case no clone is formed. Right, procedure to identify transposon insertion sites by deep-sequencing. (B) Profile of the transposon density across the whole genome, when the transposon original location is either a centromeric plasmid (top) or the endogenous ADE2 locus on chromosome XV (bottom). The dashed lines indicate the chromosome centromeres. (C) Six examples of genomic regions and their corresponding transposon coverage in seven independent transposon libraries of indicated genotypes. Each vertical grey line represents one transposon insertion event. Genes annotated as essential are shown in orange, others in blue. Green arrowheads indicate the places where the absence of transposon coverage coincides with an essential gene. (D) Histogram of the number of transposons found in every annotated gene (CDS). The vertical dashed line is the median of the distribution. (E) Same as D, with genes categorized as non-essential (blue) and essential (orange) according to previous annotations.

https://doi.org/10.7554/eLife.23570.003
Figure 1—figure supplement 1
Size distribution of the colonies appearing on SD +Galactose -Ade.

(A) Histogram of colony area for a randomly chosen sample of 402 colonies. (B) Example of picture of colonies (top) and segmentation thereof (bottom) used to calculate histogram in (A). Scale bar, 1 mm.

https://doi.org/10.7554/eLife.23570.004
Figure 1—figure supplement 2
Genome-wide analysis of transposon insertion sites.

(A) Frequency plot of nucleotide composition around transposon insertion site. The strand is determined according to the orientation of the transposon insertion. Plot was calculated with a random sample of 50,000 transposon in the wild-type library 1. Note that GC-content of the yeast genome is 38%. (B) Preferential transposon insertion into internucleosomal DNA. Three genomic regions are shown. For each, the top panel shows the nucleosomal density as determined in (Lee et al., 2007), and the bottom panels show transposon insertion as in Figures 25. (C) Correlation analysis of nucleosome and transposon density. Transposon densities were calculated on the wild-type library 1 by averaging transposon number within a 40 bp moving average. Top, correlation coefficient calculated between the nucleosome and the transposon data offset by the indicated number of bp. The correlation is most negative when offset = 0. The periodicity is ~160 bp. Middle, autocorrelation of transposon data. The periodicity is identical as above. Bottom, autocorrelation of the nucleosome density data. The periodicity is identical as above. (D) Analysis of transposon number relative to distance from centromere. For each chromosome, the number of transposons mapping within a certain distance from their respective centromere was computed and plotted (grey lines). The number of transposons increases linearly with the distance, except in the vicinity of the centromere, where transposon enrichment can be observed. The intercept of the linear regression, computed on the linear part of the plot and multiplied by 16 chromosomes, gives a rough estimate of the numbers of transposons enriched at pericentromeric regions (~20% of the total transposon number).

https://doi.org/10.7554/eLife.23570.005
Figure 1—figure supplement 3
Transposon density in essential and non-essential genes.

As in Figure 1D–E, except that, for each gene, the transposon density (i.e. number of transposons divided by length of the gene) is shown.

https://doi.org/10.7554/eLife.23570.006
Figure 2 with 5 supplements
Examples of genes showing partial loss of transposon coverage.

The grey level is proportional to the number of sequencing reads. Known functional domains are indicated. (A) Essential genes for which C-terminal truncations yield a viable phenotype. (B) Essential genes for which N-terminal truncations yield a viable phenotype. (C) Essential genes for which various truncations yield a viable phenotype.

https://doi.org/10.7554/eLife.23570.008
Figure 2—figure supplement 1
Detection of essential protein domains.

Top, algorithm to detect essential protein domains. This algorithm is implemented in Source code 2. For each gene, a score is computed as follows: the longest interval between transposon n and transposon n + 5, multiplied by the total number of transposons mapping to that gene, divided by the gene length to the power of 1.5. A score of 0 is assigned to genes targeted by less than 20 transposons, in which the longest interval is smaller than 300 bp, and/or in which the longest interval represents more than 90% or less than 10% of the CDS length. Bottom, yeast genes sorted according to their domain likelihood score. Vertical black bars above the graph indicate previously annotated essential genes.

https://doi.org/10.7554/eLife.23570.009
Figure 2—figure supplement 2
Transposon maps in the 100 highest scoring genes.

Grey scale indicates the number of sequencing reads as in Figure 2.

https://doi.org/10.7554/eLife.23570.010
Figure 2—figure supplement 3
Transposon maps in the genes scoring 101 to 200.
https://doi.org/10.7554/eLife.23570.011
Figure 2—figure supplement 4
Transposon maps in the genes scoring 201 to 300.
https://doi.org/10.7554/eLife.23570.012
Figure 2—figure supplement 5
Transposon maps in the genes scoring 301 to 400.
https://doi.org/10.7554/eLife.23570.013
TAF3 and PRP45 can be truncated without visible effects on cell growth.

(A) A truncation of TAF3 was generated in a heterozygous diploid strain (left) by introduction of an HA tag and a G418-resistance cassette (HA kanr). The strain was tetrad dissected (middle). Tetrads 2 and 3 were further analyzed by PCR to confirm the Mendelian segregation of the truncated allele (right). (B) A complete TAF3 deletion was generated in a heterozygous diploid strain (left) by introduction of a G418-resistance cassette (kanr). Meiosis yields only two viable, G418-sensitive spores per tetrad, confirming that TAF3 complete deletion is lethal. (C–D) As in (A–B) but applied to PRP45. Asterisks in the right panel designate PCR reactions that were inefficient at amplifying the large truncated allele. The genotype of these spores can nevertheless be inferred from the Mendelian segregation of the G418 resistance. (E) Top, cryo-EM structure of the S. cerevisiae spliceosome (PDB accession 5GMK, Wan et al., 2016). Bottom, the same structure stripped of every protein except Prp45. The essential portion of Prp45 as defined in (C) is in green and the non-essential part is in red and yellow. U2, U6, U5 and substrate RNAs are depicted in pale blue, pink, dark blue and orange, respectively. The red circle indicates the catalytic active site of the spliceosome. (F) Alignment of the Human, S. cerevisiae, and S. pombe Prp45 orthologs. The green, red and yellow boxes are colored as in (E). The yellow box features the most conserved region of the protein.

https://doi.org/10.7554/eLife.23570.014
Figure 4 with 1 supplement
Genetic interaction analyses.

Libraries in panels B, C, E and G are displayed in the same order as in Figure 1C. (A) Comparison of the number of transposons inserted in each of the 6603 yeast CDSs in the wild-type (x-axis) and mmm1Δ VPS13(D716H) (y-axis) libraries. (B) Transposon coverage of genes encoding ERMES components is increased in libraries from strains bearing the VPS13(D716H) allele. (C) Examples of genes showing synthetic sick/lethal interaction with mmm1Δ VPS13(D716H). (D) Comparison of the number of transposons inserted in each of the 6603 yeast CDSs in the wild-type (x-axis) and dpl1Δ (y-axis) libraries. (E) Transposon coverage of the HIP1 locus in the dpl1Δ his3Δ library and in all the other libraries (HIS3). (F) Comparison of the number of transposons inserted in each of the 6603 yeast CDSs in the dpl1Δ (x-axis) and dpl1Δ psd2Δ (y-axis) libraries. (G) Transposon coverage of the PSD1 locus in the dpl1Δ psd2Δ and in all other libraries.

https://doi.org/10.7554/eLife.23570.015
Figure 4—figure supplement 1
Volcano plots comparing libraries or combinations of libraries as indicated.

The calculated fold-change in transposon density between the two sets of libraries is plotted in log2 scale on the x-axis. The -log10(p-value) (computed using the Student’s t-test) is plotted on the y-axis. (1)The VPS13(D716H) and the mmm1Δ VPS13(D716H) strains were generated in a MET17 background while all other libraries where generated in a met17Δ background. As a result MET17 and the overlapping ORF YLR302C appear as transposon free in the reference set. MET6 is more targeted by transposons in met17Δ libraries, likely because Met17 produces homocysteine, which needs to be converted to methionine by Met6, or might otherwise accumulate to toxic levels. (2)The VPS13(D716H) and mmm1Δ VPS13(D716H) strains were generated in a Matα background, while the others were generated in a Mata background. (3)YGR190C overlaps with HIP1. (4)GPP1 shows synthetic lethality with a recessive Mendelian variant present in the psd2Δ dpl1Δ strain. However, this variant is neither linked to DPL1 nor to PSD2 (data not shown). (5)ERMES component genes scores very high with respect to fold change, because two of the five libraries in the reference set bear the VPS13(D716H) allele. The p-value, by contrast, is not significant.

https://doi.org/10.7554/eLife.23570.016
Synthetic rescue of lethal phenotypes.

(A) Transposon coverage of CDC10 in the seven libraries. The coverage is increased in the dpl1Δ psd2Δ library. (B) Tetrad dissection of a PSD2/psd2Δ DPL1/dpl1Δ CDC10/cdc10Δ triple heterozygote at 30°C (left) and 25°C (right). The cdc10Δ spores of ascertained genotype are circled with a color-coded solid line. cdc10Δ spores for which the genotype can be inferred from the other spores of the tetrad are circled with a color-coded dashed line. (C) Quantification of growing and non-growing cdc10Δ spores of the indicated genotype obtained from 48 tetrads (three independent diploids). (D) Transposon coverage of DNA2 in the seven libraries. The coverage is increased in the YEN1on library. (E) Tetrad dissection of a DNA2/dna2Δ YEN1/YEN1 single heterozygote and of a DNA2/dna2Δ YEN1/YEN1on double heterozygote at 30°C (left) and 25°C (right). All viable dna2Δ spores additionally carry the YEN1on allele (red circle). (F) FACS profile of propidium-iodide-stained DNA content in DNA2 and dna2Δ YEN1on strains exponentially growing at 30°C (left) and 25°C (right). For DNA2 panels, each profile is an overlay of two independent strains. For dna2Δ YEN1on panels, each profile is an overlay of four independent strains.

https://doi.org/10.7554/eLife.23570.017
Figure 6 with 1 supplement
Detection of rapamycin resistant strains.

(A) Comparison of the number of sequencing reads mapping to each of the 6603 yeast CDSs in rapamycin-untreated (x-axis) and -treated (y-axis) libraries. Note the difference in scale between both axis due to the high representation of rapamycin-resistant clones. (B) Distribution of transposons and number of associated sequencing reads on the PIB2 gene. Transposons with high number of sequencing reads in the rapamycin-treated library are clustered at the 5’-end of the CDS. (C) Wild-type (WT) and pib2Δ strains were transformed with either an empty plasmid (∅) or plasmids encoding full-length (FL) or indicated fragments of Pib2 (see E, numbers refer to the amino acid position in the full-length Pib2 protein). 5-fold serial dilutions of these strains were spotted on YPD or YPD containing 10 ng/ml rapamycin. Centromeric plasmids were used in all strains, except in those denoted with 2 µ, which carried multi-copy plasmids. (D) Strains of the indicated genotypes, transformed with either an empty plasmid (∅) or plasmids encoding full length (pPIB2) or truncated (pPIB2165-635) versions of Pib2, were grown exponentially in minimal medium with proline as nitrogen source. 3 mM glutamine was added to the culture and the cells were harvested 2 min later. Protein extracts were resolved on an SDS-page and probed with antibodies either specific for Sch9-pThr737 (P-Sch9), or for total Sch9 to assess TORC1 activity. (E) Schematic overview of Pib2 architecture and of the fragments used for genetic studies. (F) Summary of yeast-two-hybrid interactions between Pib2 fragments and the TORC1 subunit Kog1 (Figure 6—figure supplement 1). Fragments indicated by a black box interacted with Kog1, fragments indicated by a white box did not. (G) pib2Δ cells expressing the indicated Pib2 fragments from plasmids (see E) were assayed for their sensitivity to rapamycin (2.5 or 5 ng/ml) as in C. (H) WT or pib2Δ cells expressing the indicated Pib2 fragments from plasmids were assayed as in G, except that cells were spotted on synthetic medium to apply a selective pressure for plasmid maintenance.

https://doi.org/10.7554/eLife.23570.018
Figure 6—figure supplement 1
A) Transposon coverage of the PIB2 gene.

Top row is the rapamycin-treated library and rows below are presented as in Figures 25. The gray scale has been adjusted to account for the large number of sequencing reads mapping in the 5’ region of the gene. (B) Yeast-two-hybrid assay assessing the interaction of the indicated Pib2 fragments encoded on the pCAB plasmid, with full-length Kog1 encoded on the pPR3N plasmid. (C) gtr1Δ gtr2Δ cells expressing indicated, plasmid-encoded Pib2 fragments (see Figure 6E) were assayed for their sensitivity to rapamycin. Note that expressing Pib2Δ533-620 in gtr1Δ gtr2Δ cells appears to inhibit growth even in the absence of rapamycin. (D) gtr1Δ gtr2Δ cells, transformed with centromeric plasmids expressing Pib2 fragments (see Figure 6E) and alleles of Gtr1 and Gtr2 as indicated, were assayed for their sensitivity to rapamycin.

https://doi.org/10.7554/eLife.23570.019

Tables

Table 1

Characteristics of the libraries

https://doi.org/10.7554/eLife.23570.007
LibraryNumber of coloniesReads mappedTransposons mappedMedian read per transposonNumber of MiSeq runsOverlap between MiSeq runs
Wild-type 1~1.6×10631794831284162222*54%, 88%*
Wild-type 2~2.4×10615303285258568121NA
VPS13(D716H)~4.7×1062495845641411413241%, 42%
Mmm1Δ
VPS13(D716H)
~1.9×10617799948303323121NA
dpl1Δ~2.3×1061507715640112681NA
dpl1Δ psd2Δ~2.9×1061164956136317991NA
YEN1on~2.8×106951787749512561NA
Wild-type 2 + rapamycin~2.4×106966495616932291NA
  1. * The harvested library was grown in two flasks, one at 30°C and the other at 37°C. DNA was extracted separately from the two cultures and sequenced in two separate MiSeq runs

  2. † The library was harvested as ten subpools, which were grown in ten separate flasks. DNA was extracted separately. In one case, DNA from all ten subpools was pooled and processed to sequencing in one MiSeq run. In the other case, DNAs were kept separate and processed until the PCR step (1 × 100 µl PCR by subpool). PCR products were pooled and sequenced as another MiSeq run.

Table 2

Yeast strains used in this study.

https://doi.org/10.7554/eLife.23570.020
NameParentGenotypeReference
 CWY1BY4723MATa his3Δ0 ura3Δ0 ade2:Ds-1Weil and Kunze (2000)
 ByK157BY4743MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 VPS13(D716H)Lang et al. (2015b)
 ByK352BY4741MATa his3Δ1 leu2Δ0 met17Δ0 ura3Δ0 ade2Δ::HIS3*This study
 ByK484By4742MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 ade2Δ::HIS3*This study
 ByK485ByK352 and ByK484MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met17Δ0/MET17 ura3Δ0/ura3Δ0 ade2Δ::HIS3*/ade2Δ::HIS3*This study
 ByK446ByK157MATα his3Δ1 leu2Δ0 ura3Δ0 ade2Δ::HIS3* VPS13(D716H)This study
 ByK528ByK446MATα his3Δ1 leu2Δ0 ura3Δ0 ade2Δ::HIS3* VPS13(D716H) mmm1Δ::KanMX6This study
 ByK530ByK352MATa his3Δ1 leu2Δ0 met17Δ0 ura3Δ0 ade2Δ::NAT* dpl1Δ::KanMX6This study
 ByK533ByK352 MATa his3Δ1 leu2Δ0 met17Δ0 ura3Δ0 ade2Δ::HIS3* psd2Δ::KanMX6 dpl1Δ::NATThis study
 ByK576ByK485MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met17Δ0/MET17 ura3Δ0/ura3Δ0 ade2Δ::HIS3*/ade2Δ::HIS3* prp45Δ::KanMX6/PRP45This study
 ByK579ByK485MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met17Δ0/MET17 ura3Δ0/ura3Δ0 ade2Δ::HIS3*/ade2Δ::HIS3* PRP451-462-HA(KanMX6)/ PRP45This study
 ByK583ByK485MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met17Δ0/MET17 ura3Δ0/ura3Δ0 ade2Δ::HIS3*/ade2Δ::HIS3* taf3Δ::KanMX6/TAF3This study
 ByK588ByK485MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met17Δ0/MET17 ura3Δ0/ura3Δ0 ade2Δ::HIS3*/ade2Δ::HIS3* TAF31-270-HA(KanMX6)/ TAF3This study
 ByK725ByK533 and ByK484MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met17Δ0/MET17 ura3Δ0/ura3Δ0 ade2Δ::HIS3*/ade2Δ::HIS3* psd2Δ::KanMX6/PSD2 dpl1Δ::NAT /DPL1This study
 ByK726ByK533 and ByK484MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met17Δ0/MET17 ura3Δ0/ura3Δ0 ade2Δ::HIS3*/ade2Δ::HIS3* psd2Δ::KanMX6/PSD2 dpl1Δ::NAT /DPL1This study
 ByK739ByK725MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met17Δ0/MET17 ura3Δ0/ura3Δ0 ade2Δ::HIS3*/ade2Δ::HIS3* psd2Δ::KanMX6/PSD2 dpl1Δ::NAT /DPL1 cdc10Δ::URA3/CDC10This study
 ByK740ByK726MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met17Δ0/MET17 ura3Δ0/ura3Δ0 ade2Δ::HIS3*/ade2Δ::HIS3* psd2Δ::KanMX6/PSD2 dpl1Δ::NAT /DPL1 cdc10Δ::URA3/CDC10This study
 ByK741ByK726MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met17Δ0/MET17 ura3Δ0/ura3Δ0 ade2Δ::HIS3*/ade2Δ::HIS3* psd2Δ::KanMX6/PSD2 dpl1Δ::NAT /DPL1 cdc10Δ::URA3/CDC10This study
 YJM3916ByK352MATa his3Δ1 leu2Δ0 met17Δ0 ura3Δ0 ade2Δ::HIS3* YEN1onThis study
 YL516BY4741/BY4742MATa his3Δ1 leu2Δ0 ura3Δ0Binda et al. (2009)
 MB32YL516MATa his3Δ1 leu2Δ0 ura3Δ0 gtr1Δ::kanMXBinda et al. (2009)
 RKH106YL516MATa his3Δ1 leu2Δ0 ura3Δ0 pib2Δ::kanMXThis study
 RKH241MB32MATa his3Δ1 leu2Δ0 ura3Δ0 gtr1Δ::kanMX gtr2Δ::hphMX4This study
 NMY51
his3∆200 trp1-901 leu2-3,112 ade2 LYS::(lexAop)4-HIS3 ura3::(lexAop)8- lacZ ade2::(lexAop)8-ADE2 GAL4Dualsystems Biotech AG
  1. *ADE2 deleted −56 before ATG +62 after STOP with PCR primers #6 and #7 on pFA6a-His3MX6.

Table 3

Oligonucleotides used in this study.

https://doi.org/10.7554/eLife.23570.021
#Original nameSequencePurpose
1P5_MiniDsAATGATACGGCGACCACCGAGATCTACtccgtcccgcaagttaaataamplify library
2MiniDs_P7CAAGCAGAAGACGGCATACGAGATacgaaaacgaacgggataaaamplify library
3688_minidsSEQ1210tttaccgaccgttaccgaccgttttcatccctasequence library
4ADE2FwdGGTTCGAGCTCCCTTTTGATGCGGAATTGACclone ADE2 MiniDS
5ADE2RevGACCTGAGCTCTTACTGGATATGTATGTATGclone ADE2 MiniDS
6Ade2PriFwdGTATAAATTGGTGCGTAAAATCGTTGGATCTCTCTTCTAAcggatccccgggttaattaadelete ADE2
7Ade2PriRevTATGTATGAAGTCCACATTTGATGTAATCATAACAAAGCCgaattcgagctcgtttaaacdelete ADE2
8Dpl1_Janke_S1AGCAAGTAGGCTAGCTTCTGTAAAGGGATTTTTCCATCTAATACAcgtacgctgcaggtcgacdelete DPL1
9Dpl1_Janke_S2GCACTCTCGTTCTTTAAATTATGTATGAGATTTGATTCTATATAGatcgatgaattcgagctcgdelete DPL1
10Psd2_pringle_FGATGCTGTATCAATTGGTAAAGAATCCTCGATTTTCAGGAGCATCCAACGcgtacgctgcaggtcgacdelete PSD2
11Psd2_pringle_RCTTGTTTGTACACGCTATAGTCTATAATAAAGTCTGAGGGAGATTGTTCATGatcgatgaattcgagctcgdelete PSD2
12TAF3_R1TGGATGAGATAATGACGAAAGAAAATGCAGAAATGTCGTTgaattcgagctcgtttaaacTAF3 partial deletion
13TAF3_aa90_F2AGGTATTGTTAAGCCTACGAACGTTCTGGATGTCTATGATcggatccccgggttaattaaTAF3 partial deletion
14Taf3_FwdGGCAAGATGTGATCAGGACGcheck TAF3 partial deletion
15Taf3_RevTCTTGAAGAAGCGAAAGTACACTcheck TAF3 partial deletion
16TAF3_R1TGGATGAGATAATGACGAAAGAAAATGCAGAAATGTCGTTgaattcgagctcgtttaaacTAF3 complete deletion
17TAF3_aa1_
F1
GAAAACAGCGATATCTTTGGGTCAATAGAGTTCCTCTGCTtgaggcgcgccacttctaaaTAF3 complete deletion
18PRP45_R1ACTCAAGCACAAGAATGCTTTGTTTTCCTAGTGCTCATCCTGGGCgaattcgagctcgtttaaacPRP45 partial deletion
19PRP45_aa154_F2AACGACGAAGTCGTGCCTGTTCTCCATATGGATGGCAGCAATGATcggatccccgggttaattaaPRP45 partial deletion
20PRP45_FwdAGGTTGTAGCACCCACAGAAcheck PRP45 partial deletion
21PRP45_RevCAATCATCACACCTCAGCGAcheck PRP45 partial deletion
22PRP45_R1ACTCAAGCACAAGAATGCTTTGTTTTCCTAGTGCTCATCCTGGGCgaattcgagctcgtttaaacPRP45 complete deletion
23PRP45_aa1_F1GCTCTGAGCCGAGAGGACGTATCAGCAACCTCAACCAAATtgaggcgcgccacttctaaaPRP45 complete deletion
24CDC10-Ura3_fwdAAGGCCAAGCCCCACGGTTACTACAAGCACTCTATAAATATATTAtgacggtgaaaacctctgacCDC10 complete deletion
25URA3-CDC10_revTTCTTAATAACATAAGATATATAATCACCACCATTCTTATGAGATtcctgatgcggtattttctccCDC10 complete deletion
26OJM370ATGGGTGTCTCACAAATATGGGAmplify YEN1
27OJM371TTCAATAGTGCTACTGCTATCACAmplify YEN1
28OJM372TTCAATAGTGCTACTGCTATCACTGTCACAGGCTCAAACCGGTCGACTG TTCGTACGCTGCAGGTCGACDelitto perfetto on YEN1
29OJM373ATGGGTGTCTCACAAATATGGGAATTTTTGAAGCCATATCTGCAAGATTCCCGCGCGTTGGCCGATTCATDelitto perfetto on YEN1
30o3958gacggtatcgataagcttgatatcgGCGCTGGCATCTTTAATCTCPIB2 cloning
31o3959actagtggatcccccgggctgcaggTGCTTGGATCCTTCTTGGTCPIB2 cloning
32o3224TAATA CGACT CACTA TAGGGvarious PIB2 truncations
33o3225ATTAA CCCTC ACTAA AGGGA Avarious PIB2 truncations
34o4034atctagttcagggttcgacattctggtctccactacPIB2165-635 truncation
35o4010gtagtggagaccagaatgtcgaaccctgaactagatPIB2165-635 truncation
36o4012tagtggagaccagaatgttaccgcagcctgctPIB2304-635 truncation
37o4035tcaaattagaactagcattcattctggtctccactacaactgtgPIB2221-635 truncation
38o4011cacagttgtagtggagaccagaatgaatgctagttctaatttgaPIB2221-635 truncation
39o4062atagttggtattaagttgattctcattctggtctccactacaactgPIB2426-635 truncation
40o3996cagttgtagtggagaccagaatgagaatcaacttaataccaactatPIB2426-635 truncation
41o4063cgtgtttgcgttatggttgtcgctgttcggaatagaPIB2Δ426-532 truncation
42o3997tctattccgaacagcgacaaccataacgcaaacacgPIB2Δ426-532 truncation
43o4064cacagagccgataacactcgtggttgaaaggttctcPIB2Δ533-620 truncation
44o3998gagaacctttcaaccacgagtgttatcggctctgtgPIB2Δ533-620 truncation
45o4065gtctcgcaaaaaatgttcatcagcccaaaacatcattaccttctPIB21-620 truncation
46o3999agaaggtaatgatgttttgggctgatgaacattttttgcgagacPIB21-620 truncation
47o1440GCTAGAGCGGCCATTACGGCCCCGGAGATTTATGGACCTCKOG1 cloning into pPR3N
48o1442CGATCTCGGGCCGAGGCGGCCTCAAAAATAATCAATTCTCTCGTCKOG1 cloning into pPR3N
49o3787GCTAGAGCGGCCATTACGGCC GAATTGTACAAATCTAGAACTAGTcloning PIB2 fragments into pCabWT*
50o3788CGATCTCGGGCCGAGGCGGCCAA GAAACTACTCCAATTCCAGTTTGCcloning PIB2 fragments into pCabWT*
51o3872CGATCTCGGGCCGAGGCGGCCAAGCCCAAAACATCATTACCTTCTTCTcloning PIB2 fragments into pCabWT*
52o3871CGATCTCGGGCCGAGGCGGCCAAATCTTCGCCCTCCTCAACGTcloning PIB2 fragments into pCabWT*
53o3870CGATCTCGGGCCGAGGCGGCCAAGTTGATTCTGTCGCTGTTCGcloning PIB2 fragments into pCabWT*
54o3933GCTAGAGCGGCCATTACGGCCAGGAAGAAATTACGCAATTACTACcloning PIB2 fragments into pCabWT*
55o3934GCTAGAGCGGCCATTACGGCC AGTGTTATCGGCTCTGTGCCcloning PIB2 fragments into pCabWT*
56o3868CGATCTCGGGCCGAGGCGGCCAAATTAGTGCTCGAAGCAGGCTcloning PIB2 fragments into pCabWT*
57o3867CGATCTCGGGCCGAGGCGGCCAAGTCATCCGTGAATGGCAACGcloning PIB2 fragments into pCabWT*
58o3866CGATCTCGGGCCGAGGCGGCCAAGCCTGCCCCTGTTGAGCTCTcloning PIB2 fragments into pCabWT*
59o3865CGATCTCGGGCCGAGGCGGCCAAGTCAGCACCGCTTTCCTCATcloning PIB2 fragments into pCabWT*
  1. Oligonucleotides #1 and #2, ordered as PAGE-purified and lyophilized, are resuspended at 100 μM in water. Oligonucleotide #3, ordered as HPLC-purified and lyophilized, is resuspended at 100 μM in water and distributed into single-use aliquots.

Table 4

Plasmids used in this study.

https://doi.org/10.7554/eLife.23570.022
NameParentDescriptionReference
 pBK257pWL80R_4xCEN/URA3, carries MiniDs in ADE2 and hyperactive Ac transposase under GAL1 promoterThis study
 pWL80R_4x
CEN/URA3, carries hyperactive Ac transposase under GAL1 promoterLazarow et al. (2012)
 pCORE-UH
Delitto pefetto URA3 cassetteStorici and Resnick (2003)
 pJM7
pENTRY-YEN1ONThis study
 pRS413
CEN/HIS3, empty vectorSikorski and Hieter, 1989
 pRS415
CEN/LEU2, empty vectorSikorski and Hieter, 1989
 pRS416
CEN/URA3, empty vectorSikorski and Hieter, 1989
 p1822pRS413CEN/HIS3, GTR1This study
 p1451pRS415CEN/LEU2, GTR2This study
 p1821pRS413CEN/HIS3, GTR1Q65LThis study
 p1452pRS415CEN/LEU2, GTR2S23LThis study
 p3084pRS416CEN/URA3, PIB2This study
 p3099p3084CEN/URA3, PIB2165-635This study
 p3097p3084CEN/URA3, PIB2304-635This study
 p3101p3084CEN/URA3, PIB2221-635This study
 p3253pRS4262 μ/URA3, PIB2This study
 p3255pRS4262 μ/URA3, PIB2165-635This study
 p3163p3084CEN/URA3, PIB2426-635This study
 p3153p3084CEN/URA3, PIB2Δ426-532This study
 p3154p3084CEN/URA3, PIB2Δ533-620This study
 p3156p3084CEN/URA3, PIB21-620This study
 pPR3N
2 μ/TRP1, NubG-HADualsystems Biotech AG
 pCabWT
CEN/LEU2, Aβ-Cub-LexA-VP16Dualsystems Biotech AG
 p3081pPR3N2 μ/TRP1, NubG-HA-KOG1This study
 p2966pCabWTCEN/LEU2, Aβ-PIB2-Cub-LexA-VP16This study
 p3002pCabWTCEN/LEU2, Aβ-PIB21-620-Cub-LexA-VP16This study
 p3007pCabWTCEN/LEU2, Aβ-PIB21-550-Cub-LexA-VP16This study
 p3001pCabWTCEN/LEU2, Aβ-PIB21-428-Cub-LexA-VP16This study
 p3051pCabWTCEN/LEU2, Aβ-PIB2440-550-Cub-LexA-VP16This study
 p3054pCabWTCEN/LEU2, Aβ-PIB2556-620-Cub-LexA-VP16This study
 p3052pCabWTCEN/LEU2, Aβ-PIB2621-635-Cub-LexA-VP16This study
 p3000pCabWTCEN/LEU2, Aβ-PIB21-312-Cub-LexA-VP16This study
 p2987pCabWTCEN/LEU2, Aβ-PIB2304-635-Cub-LexA-VP16This study
 p2999pCabWTCEN/LEU2, Aβ-PIB21-162-Cub-LexA-VP16This study
 p2986pCabWTCEN/LEU2, Aβ-PIB2165-635-Cub-LexA-VP16This study
 p2998pCabWTCEN/LEU2, Aβ-PIB21-101-Cub-LexA-VP16This study
 p2991pCabWTCEN/LEU2, Aβ-PIB2102-635-Cub-LexA-VP16This study
 p2997pCabWTCEN/LEU2, Aβ-PIB21-49-Cub-LexA-VP16This study
 p2990pCabWTCEN/LEU2, Aβ-PIB250-635-Cub-LexA-VP16This study

Data availability

The following data sets were generated
  1. 1

Additional files

Supplementary file 1

Processed dataset containing (1) the position and number of reads for all transposons in each library (in the WIG format),

Processed dataset - Dpl1del.wig

Processed dataset - Mmm1Del_Vps13D716H.wig

Processed dataset - Psd2Del_Dpl1del.wig

Processed dataset - V13D716H.wig

Processed dataset - WildType1.wig

Processed dataset - WildType2.wig

Processed dataset - WT_plus_rapamycin.wig

Processed dataset - Yen1on.wig

summaries of the number of transposon and number of reads per gene, for all genes in each library (in the TXT format).

Processed dataset - Dpl1del_pergene.txt

Processed dataset - Mmm1Del_Vps13D716H_pergene.txt

Processed dataset - Psd2Del_Dpl1del_pergene.txt

Processed dataset - V13D716H_pergene.txt

Processed dataset - WildType1_pergene.txt

Processed dataset - WildType2_pergene.txt

Processed dataset - WT_plus_rapamycin_pergene.txt

Processed dataset - Yen1on_pergene.txt

https://doi.org/10.7554/eLife.23570.023
Supplementary file 2

Table of genes appearing as essential in our analysis (i.e., their density of transposon is below 1/400 bp), but were not previously annotated as essential.

The likely explanation for the low transposon density is written in column B for each gene.

https://doi.org/10.7554/eLife.23570.024
Supplementary file 3

Data computed to draw the volcano plots (Figure 4—figure supplement 1)

https://doi.org/10.7554/eLife.23570.025
Source code 1

MatLab Script 1.

https://doi.org/10.7554/eLife.23570.026
Source code 2

MatLab Script 2.

https://doi.org/10.7554/eLife.23570.027

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