copy, 2 copy, 4 copy and 6 copy); experiment 2 contained the second dataset for the Zip1 1 copy and the Zip1 6 copy strain; experiment 3 contained the second dataset for the Zip1 2 copy and the Zip1 4 copy strain. Imaging was carried out using a Deltavision RT imaging system (Applied Precision) adapted to an Olympus (IX71) microscope. Zip1, Zip1-GFP and Zip3-MYC lengths were measured using the Softworx Measure Distance Tool. Graphpad Prism software was used for scatterplot generation and statistical analysis. All slides used for quantitative fluorescence analysis were assayed for intensity of the native fluorophore (Zip1-YFP) and were stained only with DAPI to visualize DNA. Samples prepared for fluorescence quantitation experiments were imaged with a fixed exposure time so that intensity measurements could be compared between time points and strains. For experiments not requiring fluorescence quantitation, exposure times were opti- mized on a slide-by-slide basis to obtain linear range. All fluorescence quantitation experiments used the following imaging conditions. 7 Z-stack sections of 0.2 mm were collected in the FITC channel using a 1 second exposure per section. This exposure time was selected based on the minimal time to achieve a quantifiable fluorescent image, above background levels, in the dimmest SCs (early synapsis in a strain carrying only one copy of ZIP1-YFP). As reference, an additional DAPI channel image was acquired at the middle section, with a 0.5 second exposure. Projections were constructed from raw Z-stack data by building a summed-intensity projection of the 5 best-resolved 0.2 mm sections of the Z-stack (usually the 5 middle sections). The intensity in the FITC channel along well-spread SCs was analyzed by using the Softworx Data Inspector tool. Three adjacent 0.257260.2572 mm square region-of-interest boxes were placed over the region of the SC to be analyzed in the summed-intensity projection. The total intensity of the region of interest (ROI), in units of arbitrary fluorescence intensity, was recorded. The ROI for all intrinsic fluorescence measurements were three-dimensional volumes of 3 0.066 mm , calculated as follows: 1 pixel = 0.0643 mm; 4 pix- els = 0.2527 mm (length of side of 464 pixel box); Area of box = (0.2572 mm)2 = 0.06615 mm 2 ; Volume of a projected box (5 sections of 0.2 mm) = (0.06615 mm2)(5 sections)(0.2 mm/sec- tion) = 0.066 mm3). Statistical Analysis Monte Carlo simulations were done to assess the statistical significance of the distribution of Zip1-GFP foci, relative to Zip3- MYC foci, on 69 well-spread Zip1 linear stretches exhibiting Zip1- GFP foci whose largest dimension spanned 0.58 mm or less of the Zip1 stretch (average size = 0.31 mm). Two distinct analyses were performed. First, we analyzed ‘‘adjacency’’, defined as the presence of a Zip1-GFP focus directly adjacent to (touching), partially overlapping, or completely encompassed by a Zip3 focus. In this case, the SC domain in which a Zip1-GFP focus would be considered adjacent to, partially overlapping with or encompassed by a Zip3 focus corresponds to the length of the Zip3-MYC focus plus half of the length of a Zip1-GFP focus on each side of the Zip3 focus. For the second analysis, we analyzed ‘‘complete overlap’’, defined as Zip1-GFP completely encompassed by a Zip3-MYC focus. In this case, the SC domain in which a Zip1- GFP focus would be considered completely overlapping corre- sponds to the length of the Zip3-MYC focus minus half the length of a Zip1-GFP from the boundaries of that Zip3 focus. Both analyses followed a similar procedure. First, the size of Zip1-GFP is randomly sampled from the experimental data (117 distinct foci). For each of 69 Zip1 stretches of any given size, Zip1-GFP foci of that fixed size are randomly distributed, considering the SC Dynamics at Steady State observed coordinates of Zip3-MYC foci and limiting the allowed number of Zip1-GFP foci to the observed number of Zip1-GFP foci on a particular stretch (i.e. if a given Zip1 stretch displayed three Zip1-GFP foci, each random distribution procedure will place three Zip1-GFP foci onto that stretch). For a generated random distribution, the total number of Zip1-GFP foci falling within the SC domain of ‘‘adjacency’’ or ‘‘complete overlap’’ on the 69 Zip1 stretches is calculated, out of a possible 117. This random assignment of Zip1-GFP foci of the same fixed size on the 69 Zip1 stretches, constraining the allowed number of Zip1-GFP foci on a given Zip1 stretch to the observed number of Zip1-GFP foci on that stretch, is performed 1000 times. Next, a new Zip1- GFP size is randomly selected from the observed Zip1-GFP focus sizes, and the aforementioned steps are repeated. In total, 1000 random samplings of Zip1-GFP sizes are performed, bringing the total number of individual iterations to 1,000,000. The observed number of adjacent Zip1-GFP foci (78/117) or completely encompassed (42/117) is compared to the number of iterations in which they were equal or greater, thus the reported p-value represents the fraction of random iterations in which the number of adjacent/encompassed Zip1-GFP was equal or superior to the observed frequency. Western Blot Protein pellets were isolated by TCA precipitation using 10 mL of sporulating cell culture [15]. The final protein pellet was resuspended at a concentration of 10 mg/ml in 26 Laemmli sample buffer supplemented with 30 mM DTT. Protein samples were heated for 10 minutes at 65u, centrifuged at top speed and 150 mg was loaded onto an 8% polyacrylamide/SDS gel. Proteins were transferred to Whatman Protran nitrocellulose membrane. Rabbit anti-Zip1 antibody was used at 1:2500 dilution and Alkaline Phosphatase-conjugated AffiniPure Donkey anti-Rabbit (Jackson ImmunoReasearch) was used at 1:2500 dilution. Southern Blot Yeast genomic DNA was digested with BglII (NEB) overnight at 37uC and separated on a 0.8% agarose gel. Subsequent transfer to nitrocellulose membrane (Roche) was carried out by the alkali method described in Sambrook and Russell [47]. A 1.8 Kb PCR fragment containing the LEU2 gene was used as a probe against genomic DNA, using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche). Membrane-bound probe was visualized by incubating with an alkaline phosphatase-conjugated anti-DIG antibody followed by BCIP/NBT addition in alkaline phosphatase buffer. Genomic DNA without an integrated ZIP1- YFP plasmid gives a 2.8 Kb band whereas one, two, or three ZIP1- YFP integration events are predicted to result in a 12.7 Kb, 22.6 Kb, or 32.5 Kb band, respectively. Supporting Information Figure S1 An inducible Zip1 system. Table in (A) gives sporulation efficiency and spore viability of wild type (1919) and homozygous PGAL1[ZIP1] NDT80+ GAL4.ER (K62) strains (in which ZIP1 expression is driven by an inducible promoter at its endogenous locus), with or without extended incubation with b- estradiol. Images in (B) show three examples of surface-spread chromosomes from homozygous PGAL1[ZIP1] meiotic nuclei sporulated in the absence of b-estradiol, immunostained for the meiotic axis protein, Red1 (red) and for Zip1 (green). In these uninduced nuclei, Zip1 signal is rare, and axial associations (arrows) are visible owing to the absence of Zip1 at the interface between aligned homologous axes. The nucleus at far right is one PLOS Genetics | www.plosgenetics.org 15 October 2012 | Volume 8 | Issue 10 | e1002993