Complementation experiment of yeast single knockout strains with mtnBD fusion gene from <i>Tetrahymena</i>.

<p>Cells were grown to late exponential phase in −Leu +Met liquid media, transferred to −Leu−Met for an overnight to deplete internal Methionine pool, and serial dilutions for each strain were prepared in a 96-well plate with 1×10⁸ cells/ml, 1×10⁷ cells/ml, 1×10⁶ cells/ml, 1×10⁵, 1×10⁴ cells/ml, and 1×10³ cells/ml. 3 µl of each diluted culture was spotted with a 96-well pin replicator onto (A) −Met−MTA (negative control plate). (B) +Met plates, (positive control). (C) −Met +MTA (5 mM) (experimental plate). The strains assayed are: 1. <i>mtnB</i>Δ + pGREG505/<i>SYN-MTNBD</i>; 2. <i>mtnB</i>Δ + pGREG505; 3. <i>mtnC</i>Δ + pGREG505/<i>SYN-MTNBD</i>; 4. <i>mtnC</i>Δ + pGREG505; 5. <i>mtnD</i>Δ + pGREG505/<i>SYN-MTNBD</i>; 6. <i>mtnD</i>Δ + pGREG505; columns 7–12 are replicates of columns 1 through 6. After four days, none of the six yeast strains grew on the negative control plate. All six strains grew on the positive control plate. Only the strains transformed with pGREG/<i>SYN-MTNBD</i> (columns 1, 3, 5, 7, 9, 11) grew in the experimental plate.</p

The methionine salvage pathway.

<p>The enzyme names are from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Sekowska2" target="_blank">[4]</a>, and compound names are from KEGG <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Kanehisa1" target="_blank">[18]</a>. The reactions in black are known in bacteria <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Sekowska2" target="_blank">[4]</a>. The yeast pathway is indicated by blue gene names under the corresponding enzymes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Pirkov1" target="_blank">[9]</a>. Dashed lines indicate variants of the pathway (see text). In <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Ashida1" target="_blank">[5]</a> it was noted that the genes coding for mtnB and mtnC appear to be fused in <i>Arabidopsis thaliana</i>, and the genes for mtnB and mtnD appear to be fused in <i>Tetrahymena thermophila</i>, which indicates that the pathway in these organisms proceeds through the green and red reaction lines, respectively. We identified another fusion gene, between mtnK and mtnA, in <i>Tetrahymena</i> (red line).</p

Screenshot of a tblastn search of the mtnAK enzyme from <i>Tetrahymena</i> (XP_001031773) against the EST sequences from <i>Tetrahymena</i> in GenBank.

<p>The EST sequences TT1BI24TH (acc: FF565362; evalue = 1×10⁻¹²⁵) and TT1BI24TV (acc: FF565363; evalue = 2×10⁻¹²⁵) correspond to the 5′- and 3′-end of a single cDNA clone, indicating that the fusion protein is expressed in <i>Tetrahymena</i>.</p

Homologs of <i>B. subtillis</i> and yeast methionine salvage pathway enzymes in <i>Tetrahymena</i>.

<p>*Enzyme names and EC numbers are from KEGG (Kyoto Encyclopedia of Genes and Genomes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Kanehisa1" target="_blank">[18]</a>.</p

Flowchart of simulation.

<p>Cycles continue for 100,000 iterations.</p

Properties of individual ciliates during simulation.

<p>Properties of individual ciliates during simulation.</p

Definition, default values and ranges for parameters used in the simulations.

<p>Definition, default values and ranges for parameters used in the simulations.</p

Simulation data for default values given in Table 1.

<p>The top, middle, and bottom graph show copy number, <b>X</b>, elimination coefficient, <b>E</b>, and chromosome number, <b>N</b>, respectively versus number of iterations. Each value represents the average of the population, and each colored line represents a different trial, all with the same parameters. Note that due to the stochastic nature of our simulation we see significant variation between trails using the same parameters, yet the final result is consistent.</p