Summary of <i>spdri</i>'s <i>cis</i>-regulatory region [7].

<p>All constructs are depicted with a black horizontal line, which represents genomic DNA, and a red box, which is <i>spdri</i>'s first exon. Constructs 4.7IL-GFP, 4.6IL-GFP, 4.0IL-GFP, 3.0IL-GFP and 1.8IL-GFP were obtained by fusing a GFP coding cassette downstream of the first exon of <i>spdri</i> (GFP cassette is indicated with a green box in the diagrams); the function of the OEE module was studied by cloning it directly into the EpGFP vector. To test the function of the E0L3L fragment, a version of 4.7IL-GFP where this element was deleted was produced <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004025#pone.0004025-Mahmud1" target="_blank">[10]</a>. Abbreviations are as follows. OE: oral ectoderm; AE: aboral ectoderm; G: gut. (A) A table showing (on the left) the PMCs modules. Here, the name of each module is given according to the function assigned (on the left of each module). Numbers on the right of each module are used to indicate the position of the extremities of the module with respect to the transcriptional start site. The size in nucleotides of each module is given in parenthesis. On the right part of the table, a brief description of the regulators operating on each module is provided. (B) A picture of a live 24 h embryo with GFP fluorescent PMCs (green cells) is shown on the left. The vegetal pole is at the bottom. The expression pattern of the indicated constructs is reported as observed in 24 h live embryos. At this stage, GFP is seen in PMCs (blue bars), at ectopic locations (purple bars), or both. (C) Regulatory modules responsible for oral ectoderm expression are shown similarly to (A). The four functions assigned to the OEE are indicated. OE+: oral specific activation; AE/G−: repression of expression in aboral ectoderm and gut; PMC−: repression of expression in PMCs; Gen+: activation in non oral territories. The position of AE/G− and OE+ is explicitly indicated. (D) A picture of a live 48 h embryo with GFP fluorescence in the oral ectoderm is show on the left. Oral ectoderm is on the right and the embryo is shown from the side. Invaginated gut is visible in its length. The vegetal pole is at the bottom. The expression pattern of the indicated constructs is reported as observed in 48 h live embryos; the consequence of removing the OEE module from 4.0IL construct is illustrated. Expression in the different territories is indicated according to the legend provided.</p

Changing the genomic context's GC level interferes with control of gene expression.

<p>(A) After injection, embryos were scored for GFP fluorescence at the times indicated in parentheses. Scoring results are given in a histogram form. Percentages of expression in the different territories are indicated according to the legend. Note that in the same embryos expression can happen in more than one territory. “Control” is used to indicate results from embryos injected with non-fractionated carrier DNA, or with carrier DNA from the central fraction of the gradient; in both cases the same result was obtained and the construct expressed appropriately. “GC-poor” and “GC-rich” indicate embryos injected with GC-poor, or GC-rich carrier DNA respectively. Abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004025#pone-0004025-g001" target="_blank">fig. 1</a>. (B–G) Representative pictures of injected embryos at 24 h (B–D) or 48 h (E–G; embryos are shown from the vegetal pole; in this view the gut appears in cross section and the PMCs form a chain around it). GFP fluorescence is present only in PMCs or oral ectoderm (B and E; control embryos), PMCs and ectopic locations (C) or in just at ectopic location (D); oral ectoderm and ectopic locations (gut in F and PMCs in G). (H) 4.7IL-GFP transcriptional output is measured at 48 h in the different injection conditions indicated. Transcriptional output measured upon injection with WGD is taken as reference and equated to 1. (I) 4.7IL-GFP is represented and aligned with its <i>cis</i>-regulatory modules. Red and green boxes over the length of 4.7IL-GFP indicate regions of the <i>cis</i>-regulatory DNA whose function is interfered with or left unaffected, upon alteration of the genome context. Abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004025#pone-0004025-g001" target="_blank">Fig. 1</a>. Note that upon injection with “GC-poor” carrier DNA the interference on the OEE function is mostly limited to its 5′ portion, where the “AE/G−” element is located.</p

Integrated construct molecules intersperse within the concatemer.

<p>(A) A schematic view of construct 4.7IL-GFP showing the upstream portion, the first exon (red) and the GFP coding sequence (green). The portion between the upstream regulatory modules and the first exon is shown with a dashed line (not in scale). Primer couples used for quantitative PCR are shown below: in green primers used to amplify the GFP coding sequence: these two primers will only give an amplification signal if a construct-construct concatenate forms; in light blue those utilized to amplify at the junction between two adjacent construct molecules; in pale yellow those utilized to amplify at the junction between construct and carrier (HGC4 in the figure) molecules. The distance between primers is maintained around 120 nt. Carrier molecule is not shown. (B) Construct-construct (or construct-carrier) concatenation is measured Vs construct incorporation. The amount of construct molecules is measured by the level of the amplification of GFP. Construct incorporation level is equated to 1 in each experiment and the level of concatenation is measured by comparing the Ct value obtained for the amplification of construct-construct (or construct-carrier) amplification to that of construct incorporation. This allows to asses how many times concatenation occurs per each construct molecule incorporated in the genome. About 10% of the incorporated construct molecules concatenate when the WGD, a specific sequence (HGC4) or the DNA from shallow gradient fractions is used as carrier. In each experiment 100–150 embryos are utilized. Experiments were repeated using at least two different batches of embryos. (C) In order to prepare carrier DNA of chosen GC level, <i>S. purpuratus</i> genomic DNA was extracted and fractionated by CsCl shallow gradient ultracentrifugation. DNA from fractions at average GC (37.9%; green bars), GC-poor (34–35%; red bars) or GC-rich (about 40%; yellow bars) portions was utilized as carrier, in experiments where the 4.7IL-GFP construct was injected in zygotes. Upon concatemer formation, incorporation of the construct in the genomic DNA is obtained in a defined compositional context. (D) Incorporation of 4.7IL construct molecules is equal or higher when “GC-poor” or “GC-rich” carrier DNA is used compared to WGD. All the results presented here were verified in at least three separate experiments where independent batches of embryos were utilized.</p

Analysis of fermentation vigor (top) and sulfite tolerance (bottom) after two days (left) and seven days (right) of 129 <i>Saccharomyces</i> strains isolated from spontaneous fermentations.

<p>A large number of isolates show values higher than the control strain L404.</p

Mt-DNA RFLP patterns obtained at the end of fermentation from Nero d'Avola (lane 3) and Frappato (lane 4), inoculated with B2–48 (A), B2–25 (B), ICV-D254 (C) and QD-145 (D).

<p>Control RFLP patterns obtained with DNA from the pure cultures of each isolate are shown in lane 2 of each panel. Lane 1, molecular marker (1 kb ladder, BDH).</p

CO₂ production (as derived from the measured weight loss) of 129 <i>Saccharomyces</i> strains, upon 300 g/l sugar supplementation.

<p>The weight loss of L404 is indicated by an arrow (12.48 g). The number of strains per each frequency class is indicated.</p

Mt-DNA RFLP patterns of 14 isolates (indicated) used for the 100 liter fermentations of 2004.

<p>A. Patterns obtained with the <i>RsaI</i> enzyme. B. patterns obtained with the <i>HinfI</i> enzyme. M, molecular marker (1 kb ladder, BHD).</p

Research area (A) and location of the wineries (B) where must sampling was carried out (collection sites are indicated by capital letters).

<p>Research area (A) and location of the wineries (B) where must sampling was carried out (collection sites are indicated by capital letters).</p

Wine tasting results for the 14 experimental wines.

<p>The name of each starter is indicated.</p

Results of the sensory analyses performed on the Nero d'Avola (A) and Frappato (B) wines.

<p>See text for further explanation.</p