The ARS assay. (A) Sheared genomic DNA was cloned into a plasmid that contained a centromere but lacked a yeast origin of replication. The markers on the plasmid, indicated by boxes, in clockwise order and starting at the 1:00 position are LacZ (blue) multiple cloning site (contains SmaI, KpnI, and SacI sites), Amp^R (pink), KanMX^R (green), L. waltii CEN7 (black). Plasmids with genomic inserts were transformed into L. waltii and plated on G418. Colonies growing on G418 were presumed to have ARS elements in their inserts. These colonies were scraped and plasmids were extracted. Primers flanking the LacZ cloning site were used to identify the genomic insert (the ARS). (B) ARSs sequenced by Sanger sequencing were confirmed by genomic 2D gel analysis. The presence of a bubble arc (arrow) indicates that the sequence acts as a chromosomal origin. (C) All candidate ARSs were identified using Illumina sequencing. The top panel shows the raw sequencing data binned in 500-bp bins, shifting every 100 bp. The bottom panel shows the data after normalization against the genomic input library, removing all bins in the lower 97.5% of the data, summing adjacent remaining bins, and converting sequence read counts to Z-scores. Those remaining peaks with a summed Z-score of 12 or greater (above the shaded box) were scored as ARSs. The data for chromosome II are plotted with the centromere illustrated by a yellow ellipse. Plots for all chromosomes are shown in Supplemental Figure S1.

The ssDNA and density transfer assays. (A) Outline of ssDNA-based mapping of early-firing origins. L. waltii cells were treated with HU (200 mM) to enrich for ssDNA around origins of replication, or with low nitrogen medium to maintain cells in G1. ssDNA regions were labeled by random primed labeling without template denaturation and hybridized to a microarray. (B) The ratio of ssDNA in S/G1 is plotted for chromosome II. Early-firing origins are revealed as peaks in the plot. (Inset) Broadening in ssDNA peaks as S phase progresses. Plots for all chromosomes are shown in Supplemental Figure S2. (C) Outline of density transfer experiment to monitor replication dynamics. L. waltii cells were pregrown in a heavy isotope medium and then transferred to a light isotope medium containing HU (100 mM). After 2 h, HU was removed and cells were collected over the course of the S phase. DNA isolated from these samples were fragmented and subjected to ultracentrifugation to separate the heavy-heavy (HH), unreplicated DNA from the heavy-light (HL), replicated DNA. The HH and HL DNAs for each sample were labeled and competitively hybridized on a microarray. (D) Replication of chromosome II as revealed by the density transfer. The different colored lines correspond to samples taken at different times in the S phase: black (arrest), blue (15% HL), purple (25% HL), red (45% HL). The centromere is shown by a yellow circle on the x-axis. Color-coded diamonds above the plots indicate locations and samples in which origin activity (peaks of HL DNA) was detected. Plots for all chromosomes are shown in Supplemental Figure S3. (E) 2D gel analysis across a representative HL DNA peak confirms that the peak contains an origin.

All L. waltii replication data for chromosome V. Profiles of % HL and HL DNA peak locations (color-coded as in Fig. 2) are shown above the ssDNA profile. (Gray vertical lines) ARS locations. (Blue vertical lines) Sites that are redundant in the genome and cannot be mapped by Illumina sequencing data. (Filled squares) L. waltii ARSs that show syntenic conservation with ARSs in one or more other species (S. cerevisiae, L. kluyveri, and K. lactis). Orange, green, or brown squares indicate conservation with one, two, or all three of these species, respectively. (No instances of conservation with all three species were seen on this chromosome.) The yellow circle at the bottom represents the centromere. Plots for all chromosomes are shown in Supplemental Figure S4.

ACS alignment for S. cerevisiae, L. kluyveri, L. waltii, and K. lactis. The consensus sequence for L. waltii ARSs as compared with those in S. cerevisiae, L. kluyveri, and K. lactis are plotted showing the T-rich strand. (Blue box) 13-bp A element; (orange box) extended 17-bp A element. The purple, green, and red boxes show the B1 element. The S. cerevisiae ACS was taken from Eaton et al. (2010), the L. kluyveri ACS was taken from Liachko et al. (2011), and the K. lactis ACS was taken from Liachko et al. (2010). The tree phylogeny is based on Jeffroy et al. (2006).

A-T asymmetry and the nucleosome profile surrounding the L. waltii ACS. (A) The ratios of A/T and C/G bases around the L. waltii ACS are shown. All sequences were plotted such that the ACS begins at position 0 and are oriented such that the T-rich ACS strand is plotted. (Left plot) ACSs present in ARSs; (right plot) ACS matches found in intergenic, non-ARS locations. (B) The nucleosome profile surrounding the L. waltii ACS is shown. All nucleosome data are orientated as in A. The colored lines show the nucleosome profile: red, all ARSs; blue, HU-pos ARSs; orange, HU-neg ARSs; black, intergenic non-ARS ACS matches; gray, genome-wide average.