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1.
Figure 7

Figure 7. From: Critical roles for a genetic code alteration in the evolution of the genus Candida.

Ambiguous cells have selective advantage under stress. Spores of S. cerevisiae control cells and cells expressing the G33 tRNACAGSer were grown for 7 days in agar plates of rich medium (YEPD), synthetic medium (SD) and minimal medium (MM) containing 100 μM CdCl2 or 1.5 mM H2O2. Spores from ambiguous cells grew much slower than non-ambiguous spores in rich medium, but recovered competitive capacity when growing under stress conditions, as shown by a similar colony size of control and ambiguous cells.

Raquel M Silva, et al. EMBO J. 2007 October 31;26(21):4555-4565.
2.
Figure 6

Figure 6. From: Critical roles for a genetic code alteration in the evolution of the genus Candida.

Genetic code alterations increase trehalose and glycogen accumulation. C and U33 represent S. cerevisiae control cells and cells expressing C. albicans U33 tRNACAGSer, respectively. The results are expressed as mean±s.d. of four independent experiments (*P<0.05 and **P<0.01 by Student's t-test). Trehalose (A) and glycogen (B) content were 6 fold higher in exponentially growing CUG ambiguous cells (OD=0.5), when compared with control cells. (C, D) Glycogen and trehalose accumulation were induced in ambiguous cells after a 30-min heat shock; glycogen content increased 8 fold under heat shock when compared with control cells, while trehalose accumulation increased in both ambiguous and control strains under heat stress.

Raquel M Silva, et al. EMBO J. 2007 October 31;26(21):4555-4565.
3.
Figure 4

Figure 4. From: Critical roles for a genetic code alteration in the evolution of the genus Candida.

Genetic code alterations reprogramme gene expression. (A) Transcriptome analysis indicates the percentage of genes with altered expression levels in ambiguous strains. Genes whose expression was both up- and downregulated by CUG ambiguity were grouped according to their functions. The genes that are included in the stress group from the pie-chart were further divided into the functional categories displayed on the adjacent column. (B) Proteome data show the percentage of proteins whose expression was altered in S. cerevisiae cells expressing C. albicans U33 tRNACAGSer, distributed by functional categories. The proteins that are included in the stress group from the pie-chart were further divided into the functional categories displayed on the adjacent column. Both analyses indicate that genetic code ambiguity extensively remodelled gene expression, altered the expression of genes and proteins belonging to the stress response, protein synthesis, folding and degradation pathways, and general metabolism.

Raquel M Silva, et al. EMBO J. 2007 October 31;26(21):4555-4565.
4.
Figure 3

Figure 3. From: Critical roles for a genetic code alteration in the evolution of the genus Candida.

Genetic code alterations induce ploidy variation. Flow cytometry analysis of haploid (A) and diploid (B) S. cerevisiae cell lines expressing the C. albicans G33 or U33 tRNACAGSer had a general increase of the nuclear DNA content, providing evidence of polyploidy and aneuploidy events in CUG ambiguous cells. (C) Ploidy shift was observed in 56% of the haploid G33 tRNACAGSer clones and 50% of diploid U33 tRNACAGSer clones tested by flow cytometry analysis. (D) Heterogeneity of the ambiguous cell population is shown by the variability in colony, cell and bud size and shape. The increase in cell volume is consistent with polyploidization of the ambiguous clones. DAPI staining highlights ambiguous cells with two nuclei or without nucleus, suggesting the presence of polyploid and aneuploid cells.

Raquel M Silva, et al. EMBO J. 2007 October 31;26(21):4555-4565.
5.
Figure 2

Figure 2. From: Critical roles for a genetic code alteration in the evolution of the genus Candida.

Genetic code alterations act as a barrier for sexual reproduction. The ability of S. cerevisiae cells expressing C. albicans G33 tRNACAGSerto reproduce sexually was determined by their sporulation and mating efficiencies. (A) The number of tetrads produced by diploid ambiguous cells decreased by 30% when compared with control cells, indicating that sporulation efficiency was lower in CUG ambiguous cells. (B) Spore viability was reduced in ambiguous cells, since most tetrads yielded only one (42%) or no viable spores (35%), while tetrads from control cells produced mainly two viable spores (92%). (C) Ambiguous cells produced fewer viable spores and spores grew slower, as shown by growth on solid selective medium. The U33 tRNA spores shown had mutations in the tRNACAGSer gene. (D) Haploid control and ambiguous cells with opposite mating types were mixed and serial dilutions of the mixtures were plated onto selective media. C × C, G × G and C × G indicate the crosses between the control cells, or ambiguous G33 tRNA cells or control and ambiguous G33 tRNA cells, respectively. The reduced amount of diploids produced by crossing ambiguous (G33 tRNA) cell lines showed that mating efficiency was also negatively affected by CUG ambiguity. In all cases, the U33 tRNACAG was lethal in haploid backgrounds. Alternatively, its gene acquired mutations that inactivated the U33 tRNA.

Raquel M Silva, et al. EMBO J. 2007 October 31;26(21):4555-4565.
6.
Figure 5

Figure 5. From: Critical roles for a genetic code alteration in the evolution of the genus Candida.

Genetic code alterations reprogramme the stress response. (A) Proteasome activity increased 3.6-fold in S. cerevisiae cells expressing C. albicans tRNACAGSer (U33), as shown by enhanced proteolysis of the chymotrypsin-like substrate SucLLVY-AMC. The results are expressed as mean±s.d. of 4–6 independent experiments (**P<0.01 by Student's t-test). Fluorescence intensity (FIU) is shown in arbitrary units. (B) Expression of proteasome subunits () was threefold induced by CUG ambiguity, as measured by proteome analysis. Control (C) and ambiguous (U33) cells were grown at 25°C (25), 37°C (37) or heat shocked (HS). Proteins were labelled in vivo with L-[35S]methionine and separated by 2D-PAGE as described in Materials and methods. The medium expression level of the selected proteins was calculated and normalized to the control to deduce general folds. (C) Ambiguity pre-adapted cells to tolerate adverse growth conditions. Expression of stress proteins () increased twofold in control cells at 37°C, but not in ambiguous cells that already had increased amounts (3 fold) of these stress-protective proteins. (D) Ambiguous cells retained the capacity to respond to additional stress. Expression of stress proteins () was induced in both strains under heat-shock (8- and 13-fold for the control and ambiguous cells, respectively).

Raquel M Silva, et al. EMBO J. 2007 October 31;26(21):4555-4565.
7.
Figure 1

Figure 1. From: Critical roles for a genetic code alteration in the evolution of the genus Candida.

Reconstruction model of the Candida genetic code alteration. (A) Redefinition of the identity of the CUG codon from leucine to serine in Candida started with a novel serine tRNA (tRNACAGSer) and evolved gradually over the last 272±25 million years. tRNACAGSer disappeared and the cognate leucine CUG decoder (tRNACAGLeu) was maintained in the S. cerevisiae lineage (standard genetic code), while the converse occurred in the C. albicans lineage (altered genetic code). (B) The tRNACAGSer contains guanosine at position 33 (G33), which is a conserved position occupied by uridine (U33; U-turn) in other tRNAs. (C) The upper panel shows a diagram of the reporter system used to quantify serine misincorporation at CUG codons in vivo in S. cerevisiae. A CUG cassette inserted in the CaPGK gene was flanked by two thrombin cleavage sites to facilitate the purification of the short reporter peptide encoded by the cassette. The recombinant protein was expressed and purified from S. cerevisiae cultures using nickel affinity chromatography, and was then cleaved with thrombin for 16 h at 26°C, in solution. The resulting peptides were analysed by mass spectrometry. The lower panel shows a 12% SDS–PAGE of the reporter protein. (D) Serine and leucine incorporation at the CUG position (see panel C) was determined by quantitative MRM methodologies using a hybrid quadrupole/linear ion-trap mass spectrometer. Synthetic peptides with sequences identical to those of the serine and leucine peptides shown in panel C were used as external controls and to build the calibration curves used for quantification.

Raquel M Silva, et al. EMBO J. 2007 October 31;26(21):4555-4565.

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