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1.
FIGURE 6.

FIGURE 6. From: Regulation of Telomere Length by Fatty Acid Elongase 3 in Yeast.

Measurement of IP levels in elo3Δ yeast strains. The levels of IPs in WT and elo3Δ (A), ipk1Δ, and elo3Δipk1Δ (B), or ipk2Δ and elo3Δ ipk2Δ (C) yeast strains were measured using HPLC subsequent to [3H]inositol labeling of cells as described under “Experimental Procedures,” and normalized to the total inositol incorporation.

Suriyan Ponnusamy, et al. J Biol Chem. 2008 October 10;283(41):27514-27524.
2.
FIGURE 5.

FIGURE 5. From: Regulation of Telomere Length by Fatty Acid Elongase 3 in Yeast.

Deletion of IPK2 or KCS1, which are involved in inositol phosphate generation, protects telomere attrition in elo3Δ cells. A, the metabolic pathways of IP generation. B, the telomere length in WT, elo3Δ, ipk1Δ, elo3Δipk1Δ, ipk2Δ, elo3Δipk2Δ, kcs1Δ, and elo3Δkcs1Δ cells (BY4742 strain) was measured by Southern blotting (lanes 18). The vertical dashed lines indicate the gap between lanes 1–2 and 3–8 on the blot. Molecular weight markers are indicated on the left. These data represent experiments performed in at least three independent trials.

Suriyan Ponnusamy, et al. J Biol Chem. 2008 October 10;283(41):27514-27524.
3.
FIGURE 3.

FIGURE 3. From: Regulation of Telomere Length by Fatty Acid Elongase 3 in Yeast.

Effects of the expression of WT- and mutant-ELO3 on FA synthesis, and regulation of telomere length in elo3Δ cells. A, the roles of WT and mutant Elo3p in the generation of VLCFA were determined as described above. B, the reduction in telomere restriction fragment length of elo3Δ, elo3Δ/wt-ELO3, and elo3Δ/mutant-ELO3 were analyzed and represented in a graphical format. C, telomere length was measured by Southern blotting as described under “Experimental Procedures” using genomic DNA, isolated from WT, elo3Δ, elo3Δ/wt-ELO3, and elo3Δ/mutant-ELO3 (lanes 14). Molecular weight markers are indicated on the left. Experiments were performed in at least three independent trials.

Suriyan Ponnusamy, et al. J Biol Chem. 2008 October 10;283(41):27514-27524.
4.
FIGURE 2.

FIGURE 2. From: Regulation of Telomere Length by Fatty Acid Elongase 3 in Yeast.

Reconstitution of WT ELO3 expression prevents telomere attrition in elo3Δ cells. A, the levels of FAs were measured as described under “Experimental Procedures.” B, the plasmid containing the full-length WT-ELO3 under a galactose (Gal)-sensitive promoter was transformed into elo3Δ cells. Then, telomere length was measured after cells were grown in the presence (“+”) or absence (“–”) of galactose for 24, 48, and 72 h (lanes 2, 4, 6, and 3, 5, 7, respectively). Lane 1 contains samples obtained from WT cells. Molecular weight markers are indicated on the left. Experiments were performed in at least two independent trials.

Suriyan Ponnusamy, et al. J Biol Chem. 2008 October 10;283(41):27514-27524.
5.
FIGURE 1.

FIGURE 1. From: Regulation of Telomere Length by Fatty Acid Elongase 3 in Yeast.

Roles of ELO1, ELO2, and ELO3 in the generation of FAs, and regulation of telomere length. A, the generation of VLCFAs by ELO1–3 in yeast. B, FAs were measured in WT versus ELO1, ELO2, and ELO3 mutants (BY4742 strain) as described under “Experimental Procedures.” FA levels were normalized to the levels of Pi. C, telomere length was measured by Southern blotting using genomic DNA, isolated from ELO1, -2, and -3 mutant strains (lanes 24, respectively) as described under “Experimental Procedures.” Lanes 1 and 5 contain DNA samples isolated from the WT strain. Lane M contains molecular weight (Mr) markers. The vertical dashed lines indicate a gap between lanes M and 1 on the blot. FAs and telomere lengths were measured in duplicates at least in two independent trials. Error bars represent standard deviations.

Suriyan Ponnusamy, et al. J Biol Chem. 2008 October 10;283(41):27514-27524.
6.
FIGURE 4.

FIGURE 4. From: Regulation of Telomere Length by Fatty Acid Elongase 3 in Yeast.

Roles of ceramide metabolism in telomere shortening in response to the loss of Elo3. A, involvement of VLCFA synthesis in the regulation of the generation of phytoceramide (PHC) via the acylation phytosphingosine (PHS) is shown. Phytoceramide can be a precursor for the generation of complex lipids, such as IPC, mannosylinositol phosphorylceramide (MIPC), and mannosyldiinositol phosphorylceramide (M[IP]2C). B, effects of the loss of ELO3 on the generation/accumulation of phytoceramides were using LC/MS/MS as described under “Experimental Procedures.” Error bars represent S.D. ± mean. C, length of WT, elo3Δ, lag1Δ, elo3Δ lag1Δ, lac1Δ, elo3Δ lac1Δ, isc1Δ, and elo3Δ isc1Δ cells (Jk9–3D strain) measured by Southern blotting (lanes 18, respectively). The genomic DNAs from WT and lag1Δlac1Δ (W303-1A strain) were used in lanes 9 and 10 for telomere length measurements. Molecular weight markers indicated on the left. Experiments were performed in at least three independent trials.

Suriyan Ponnusamy, et al. J Biol Chem. 2008 October 10;283(41):27514-27524.
7.
FIGURE 7.

FIGURE 7. From: Regulation of Telomere Length by Fatty Acid Elongase 3 in Yeast.

Roles of FA elongation and inositol diphosphate signaling in chronologic aging and replicative life span in yeast. A, WT, elo3Δ, elo3Δipk2Δ, and elo3Δkcs1Δ (lanes 14) cells were spotted on YPD plates in different dilutions following growth in stationary phase in water for several days, and then grown at 30 °C for 24 h. B, the replicative life span characteristics of cells were analyzed as described under “Experimental Procedures.” The number of daughter cells produced by a single mother cell was counted using microdissection. In each life span, determination of 43 cells/strain were used, which were performed in three independent experiments. The statistical analysis of the data were performed using Mann-Whitney (rank test), and p < 0.05 was considered significant. The differences in life span between WT and elo3Δ, and kcs1Δ were significant (p = 0.0043, and p < 10–6, respectively), whereas the difference between WT and elo3Δkcs1Δ was not (p = 0.37). The mean (maximum) life spans for these strains were 28.1 (48), 23 (44), 13.3 (23), and 26.4 (43) generations, respectively.

Suriyan Ponnusamy, et al. J Biol Chem. 2008 October 10;283(41):27514-27524.
8.
FIGURE 8.

FIGURE 8. From: Regulation of Telomere Length by Fatty Acid Elongase 3 in Yeast.

Elo3 shares a common pathway with Ku70/80 for the regulation of telomere length. A, the telomere length was measured in WT, elo3Δ, yku80Δ, elo3Δyku80Δ, tel1Δ, elo3Δtel1Δ, rad50Δ, elo3Δrad50Δ, est1Δ, elo3Δest1Δ, yku70Δ, and elo3Δyku70Δ cells (lanes 112, respectively). The vertical dashed lines indicate a gap between lanes 1–10 and 11–12 on the blot. Molecular weight markers are indicated on the left. B, the average reductions in telomere length in mutants mentioned above were detected in at least three independent experiments, and the data are shown in a graphical format. C, effects of loss of ELO3 in telomere binding/protective function of endogenous Ku-GFP in WT versus elo3Δ cells (left panel, lane 2) were measured using ChIP analysis as described under “Experimental Procedures.” Lane 1 contains input DNA used as loading controls. The expression of Yku80p-GFP protein in WT and elo3Δ (C, right panel, lanes 3 and 4, respectively) cells was measured by immunoprecipitation using anti-GFP antibody-coupled microbeads, followed by Western blotting using a soluble antibody against GFP protein. D, the role of ELO3 deletion in the regulation of the NHEJ function of Ku was examined as described under “Experimental Procedures.” The linearized and circular pYES2 plasmids, harboring the URA gene, were transformed into WT, elo3Δ, and yku80Δ cells. Then, the percent of WT, elo3Δ, and yku80Δ colonies grown in URA-negative media were counted in at least two independent trials as duplicates. Error bars represent S.D. ± mean.

Suriyan Ponnusamy, et al. J Biol Chem. 2008 October 10;283(41):27514-27524.

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