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1.
Figure 5

Figure 5. Nucleosome profile of P. pastoris.. From: GC-Rich DNA Elements Enable Replication Origin Activity in the Methylotrophic Yeast Pichia pastoris.

Nucleosome density is plotted for sites centered on all TSSs as a control to test the overall quality of the mapping data (left), non-overlapping GC-ARS sites with a single match to the GC-ACS (middle), or the A/T-rich motif shown in Figure 3C (right). TSS sites are ranked based on expression in the SDEG condition [55]. GC-ARS and AT-ARS sites are ranked by the strength of the best match to the G/C- and the A/T-rich motif respectively.

Ivan Liachko, et al. PLoS Genet. 2014 March;10(3):e1004169.
2.
Figure 2

Figure 2. The GC-ACS is required for GC-ARS function.. From: GC-Rich DNA Elements Enable Replication Origin Activity in the Methylotrophic Yeast Pichia pastoris.

Wild type (WT) and mutant (MUT) alleles of the twelve ARSs indicated were cloned into a URA3 ARS-less vector and used to transform ura3 yeast on selective medium plates lacking uracil. Plates were grown at 30°C for five days before pictures were taken. Colony formation indicates plasmid maintenance and ARS activity. The GC-ACS was positioned <15 bp away from the 5′ endpoint in all ARS sequences. The sequences of the fragments tested are listed in Table S4.

Ivan Liachko, et al. PLoS Genet. 2014 March;10(3):e1004169.
3.
Figure 3

Figure 3. Deep mutational scanning of P. pastoris ARSs.. From: GC-Rich DNA Elements Enable Replication Origin Activity in the Methylotrophic Yeast Pichia pastoris.

(A) Schematic of the mutARS-seq deep mutational scanning experiment. Auxotrophic ura3 yeast were transformed with a library of mutant ARS variants and competed in selective medium. The abundance of different ARS variants was determined by deep sequencing at intervals during competitive growth. (B) Results of mutARS-seq of ARS-C379. The relevant sequence of ARS-C379 is shown with the best match to the GC-ACS motif highlighted in red (and a 3′ constrained dinucleotide highlighted in blue). The log-transformed enrichment ratio is shown for each nucleotide at each position along the sequence. (C) Results of mutARS-seq of ARS-A2772. Same as in (B), except that the motif logo shown was constructed from the enrichment ratio scores post-analysis, whereas the motif shown in (B) was constructed from ARS alignments.

Ivan Liachko, et al. PLoS Genet. 2014 March;10(3):e1004169.
4.
Figure 6

Figure 6. Sequence features of GC-ARSs.. From: GC-Rich DNA Elements Enable Replication Origin Activity in the Methylotrophic Yeast Pichia pastoris.

(A) Average nucleotide frequencies around 107 GC-ARS sites (top) and twenty-eight non-ARS intergenic occurrences of the GC-ACS (bottom), centered on the best match of the GC-ACS. The nucleotide frequencies are calculated at all flanking regions around the motif independent of whether the flanking region is present in ARS contigs or cores. (B) The distribution of distances between the GC-ACS motif (in the orientation shown) and the TSS for adjacent genes transcribing away from the ARS with available TSS annotations. Distances to the 5′ side of the motif are shown in blue; distances to the 3′ side of the motif are shown in red. (C) The distribution of sequence lengths between the GC-ACS and the end of the inferred functional core region for each GC-ARS. The 5′ distance is indicated in blue; the 3′ distance is indicated in red. Numbers indicate the upper limit of the bin.

Ivan Liachko, et al. PLoS Genet. 2014 March;10(3):e1004169.
5.
Figure 1

Figure 1. Mapping of replication origins in P. pastoris.. From: GC-Rich DNA Elements Enable Replication Origin Activity in the Methylotrophic Yeast Pichia pastoris.

(A) Schematic of ARS-seq and miniARS-seq screens. Fragmented genomic DNA was ligated into non-replicating URA3 vectors and screened for ARS activity followed by deep sequencing of the resultant plasmid inserts (ARS-seq, top). ARS-seq plasmid inserts were amplified and sheared using DNase I. Short fragments of ARSs were ligated into the URA3 vectors and screened for ARS activity followed by deep sequencing of the plasmid inserts (miniARS-seq, bottom). (B) The GC-ACS motif identified by the MEME algorithm. (C) The distribution of MAST motif scores of the best match to the GC-ACS in every PpARS. (D) 2D gel analysis at loci A2772 (putative AT-ARS at chromosome 1: 2,772 kb) and C379 (putative GC-ARS at chromosome 3: 379 kb). The red arrows highlight arcs corresponding to replication bubble intermediates.

Ivan Liachko, et al. PLoS Genet. 2014 March;10(3):e1004169.
6.
Figure 4

Figure 4. Replication timing of the P. pastoris genome.. From: GC-Rich DNA Elements Enable Replication Origin Activity in the Methylotrophic Yeast Pichia pastoris.

(A) Genomic DNA from G1 and S phase cells was sheared and sequenced. Normalized S/G1 DNA copy ratios (in 1 kbp windows) were smoothed and plotted against chromosomal coordinates. Peaks correspond to positions of replication initiation. The profile of chromosome 4 is shown (all chromosomes are shown in Figure S6) with ARS locations indicated by open (AT-ARSs) and shaded (GC-ARSs) circles. Un-smoothed ratio data for one of the replicates is shown are grey. Coordinates of replication timing peaks are indicated by dashed vertical lines. (B) The distributions of smoothed S/G1 ratio data. The distribution of all ratios (“Genome”) is shown adjacent to the distribution of values at bins containing midpoints of GC-ACSs (“GC”) or AT-ARSs (“AT”). Values for ARSs that have no other ARSs within 40 kb in both directions are shown on the right (“isolated”). (C) The complete genomic ratio distribution is shown relative to distributions after removal of data within 60 kb ranges centered on AT-ARSs (“AT”), GC-ARSs (“GC”), or all ARSs (“all ARS”). (D) For each ARS, the distance to the nearest replication peak was calculated. The ARS-peak distances are shown as distributions separately for GC-ARSs (blue) and AT-ARSs (orange). Peak distances from simulated random sets of loci are shown in grey.

Ivan Liachko, et al. PLoS Genet. 2014 March;10(3):e1004169.

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