Expression levels of <i>DAL2</i>, <i>DAL4</i> and <i>DAL1</i> genes in the DAL2.I₁ and CI₁ strains.

<p>Real time PCR to show the expression of <i>DAL1</i> (<b>A</b>), <i>DAL2</i> (<b>B</b>) and <i>DAL4</i> (<b>C</b>). Blue, green and red, boxes represents the FY3 control and inverted strains, respectively. Error bars are from three technical replicas for each of the three independent biological samples. Relative normalized fold expression was calculated by using ΔΔCt method and <i>ACT1</i> as a reference gene.</p

Strategies followed to engineer strains possessing <i>N. castellii</i> like DAL cluster.

<p>In strategy one the <i>DAL2</i> was inverted first followed by the larger inversion of the three genes <i>DAL1</i>, <i>DAL4</i> and <i>DAL2</i>. In strategy two the block of the three genes, <i>DAL1</i>, <i>DAL4</i> and <i>DAL2</i>, was inverted first followed by the single inversion of the <i>DAL2</i> gene. The control strains had the <i>lox</i>P and <i>lox</i>2272 insertions in the intergenic regions, but they do not present the inversion. The red, yellow and green blocks indicate the single inverted, double inverted and non-inverted genes respectively. The <i>lox</i>P and <i>lox</i>2272 scars are represented as blue and violet triangles respectively.</p

Schematic representation of the yeast DAL gene cluster involved in the allantoin degradation pathway.

<p>The panel <b>A</b> shows the six DAL genes located on chromosome IX at the same position and orientation in all <i>S. cerevisiae</i> “sensu stricto” species. In <i>N. castellii</i> the cluster presents two nested inversions involving <i>DAL1, DAL2</i> and <i>DAL4</i> (marked with a double ended arrow). The colour orange indicates collinear genes, the pale green arrows shows genes which underwent one inversion event and the dark green colour indicate the <i>DAL2</i> gene which inverted twice in <i>N. castellii</i>. (Figure adapted from Wolf, 2006). The panel <b>B</b> shows how allantoin is converted to allontoate by allantoinase and degraded to produce glyoxalate and urea. In the final stage of the pathway, the glyoxalte is converted to malate and the urea to ammonia.</p

Structure of the DAL cluster.

<p>A schematic representation of the size of the DAL cluster and the intergenic regions of <i>S. cerevisiae</i> and <i>N. castellii</i>. The genes located on the watson and crick strand are coloured red and blue respectively.</p

Northern analysis of <i>DAL4</i> sense and antisense transcripts.

<p>RNA was extracted from cells grown under non-induced conditions (YPD medium, panel <b>A</b>) and induced conditions (F1 medium containing Proline+Allantoin as N-source, panel <b>B</b>). Oligonucleotides specific for the <i>DAL4</i> sense and anti-sense strand were used. As expected no expression of <i>DAL4</i> sense transcript was observed in YPD medium while, the antisense <i>DAL4</i> signal was very strong (<b>A</b>). Under induced conditions the expression of <i>DAL4</i> sense was greatly reduced in the inverted strain as compared to the control while the expression of antisense transcript remained same in both strain backgrounds (<b>B</b>). <i>ACT1</i> was used as the reference gene for expression comparisons.</p

Fitness assays of the engineered <i>Sc/Su</i> hybrids carrying different type of <i>TRP2/TRP3</i> chimeric complexes.

<p><i>Sc/Su</i> hybrids were genetically modified to carry either the two different chimeric complexes, Trp2p^Su/Trp3p^Sc and Trp2p^Sc/Trp3p^Su, or the two parental hemizygous controls, Trp2p^Su/Trp3p^Su and Trp2p^Sc/Trp3p^Sc (panel A). The growth curves of <i>S. cerevisiae</i>, <i>S. uvarum</i>, the hybrid <i>Sc/Su</i> and the engineered hybrids shows that Trp2p^Su/Trp3p^Sc grows better than the other combinations in SD media lacking tryptophan (panel B). The fitness competition assay between <i>Sc/Su</i> hybrids with different combination of the <i>TRP2/TRP3</i> complex and the GFP reference strain shows again that Trp2p^Su/Trp3p^Sc grows faster (panel C). The competitive fitness coefficient Sg represents the difference between the ln of the ratio of hybrid and reference strain between final and initial time points, normalized for the number of generations. An equal fitness between hybrid and reference strains would be indicated by a value of zero (see Method section).</p

Growth assays of <i>Sc/Su</i> hybrids carrying different types of MBF chimeric complexes.

<p><i>Sc/Su</i> hybrids were genetically modified either to carry the two different chimeric complexes, Mbp1^Su/Swi6^Sc and Mbp1^Sc/Swi6^Su, or the two uni-parental controls, Mbp1^Su/Swi6^Su and Mbp1^Sc/Swi6^Sc (Panel A). The growth spot assay of the engineered hybrids in rich YPD and YP-glycerol media are shown in Panel B. The strain carrying the <i>S. uvarum</i> homologous Mbp1^Su and Swi6^Su is the only one that performs respiratory growth and grows normally in the presence of glycerol a sole carbon source.</p

TAP-strategy for recovery and identification of hybrid protein complexes.

<p><i>S. cerevisiae</i> strains with the TAP cassette inserted into the C-terminal of one member of the complex (TAP-tag A) were crossed with <i>S. mikatae</i> and <i>S. uvarum</i> species. The complexes that freely formed in the hybrids were then isolated and the interacting members identified via MS analysis. A', B' and C' represent the orthologs of the <i>S. cerevisiae</i> A, B, C proteins, respectively.</p

Supplementary Figures and tables Legends from Rapid functional and evolutionary changes follow gene duplication in yeast

Duplication of genes or genomes provides the raw material for evolutionary innovation. After duplication a gene may be lost, recombine with another gene, have its function modified, or be retained in an unaltered state. The fate of duplication is usually studied by comparing extant genomes and reconstructing the most likely ancestral states. Valuable as this approach is, it may miss the most rapid evolutionary events. Here, we engineered strains of <i>Saccharomyces cerevisiae</i> carrying tandem and non-tandem duplications of the singleton gene <i>IFA38</i> to monitor (i) the fate of the duplicates in different conditions, including timescale and asymmetry of gene loss and (ii) the changes in fitness and transcriptome of the strains immediately after duplication and after experimental evolution. We found that the duplication brings widespread transcriptional changes but a fitness advantage is only present in fermentable media. In respiratory conditions, the yeast strains consistently lose the non-tandem <i>IFA38</i> gene copy in a surprisingly short time, within only few generations. This gene loss appears to be asymmetric and dependent on genome location since the original <i>IFA38</i> copy and the tandem duplicate are retained. Overall, this work shows for the first time that gene loss can be extremely rapid and context dependent

Table S2 from Rapid functional and evolutionary changes follow gene duplication in yeast

Duplication of genes or genomes provides the raw material for evolutionary innovation. After duplication a gene may be lost, recombine with another gene, have its function modified, or be retained in an unaltered state. The fate of duplication is usually studied by comparing extant genomes and reconstructing the most likely ancestral states. Valuable as this approach is, it may miss the most rapid evolutionary events. Here, we engineered strains of <i>Saccharomyces cerevisiae</i> carrying tandem and non-tandem duplications of the singleton gene <i>IFA38</i> to monitor (i) the fate of the duplicates in different conditions, including timescale and asymmetry of gene loss and (ii) the changes in fitness and transcriptome of the strains immediately after duplication and after experimental evolution. We found that the duplication brings widespread transcriptional changes but a fitness advantage is only present in fermentable media. In respiratory conditions, the yeast strains consistently lose the non-tandem <i>IFA38</i> gene copy in a surprisingly short time, within only few generations. This gene loss appears to be asymmetric and dependent on genome location since the original <i>IFA38</i> copy and the tandem duplicate are retained. Overall, this work shows for the first time that gene loss can be extremely rapid and context dependent