Protein Structure And Function

SC-Ura SC

SC-Ura SC

Fig. 3. The requirement of SIR2 for rDNA silencing. (A) Spot assay. Reporter strains containing mURA3 in the rDNA were spotted onto either SC-Ura or nonselective SC medium and allowed to grow for 3 days. Loss of silencing in the sir2 A strains is indicated by increased growth on the SC-Ura plates. (B) Colony color assay. Wild-type or sir2A strains containing MET15 in the rDNA were streaked onto rich medium containing lead nitrate. A lighter colony color and increased numbers of dark brown sectors indicate loss of silencing in the sir2 A strain.

Fig. 3. The requirement of SIR2 for rDNA silencing. (A) Spot assay. Reporter strains containing mURA3 in the rDNA were spotted onto either SC-Ura or nonselective SC medium and allowed to grow for 3 days. Loss of silencing in the sir2 A strains is indicated by increased growth on the SC-Ura plates. (B) Colony color assay. Wild-type or sir2A strains containing MET15 in the rDNA were streaked onto rich medium containing lead nitrate. A lighter colony color and increased numbers of dark brown sectors indicate loss of silencing in the sir2 A strain.

in a spectrophotometer. If testing multiple strains on the same plate, normalize each culture to an A6oo of 1.0 in a new 1.5-ml microcentrifuge tube. Starting with an A60o of 1.0, serially dilute the cells at a 1:5 ratio (40 ¡A into 160 ¡A) with water. The serial dilutions are conveniently carried out in a sterile 96-well microtiter dish, using an 8-channel multipipette. Using the multipipette, spot 5 /xl of each dilution onto a nonselective control plate such as YPD or synthetic complete (SC) medium, and onto SC-Ura medium for the analysis of URA3 silencing. The number of colonies growing on the selective medium compared with the nonselective SC medium is directly correlated with the expression level of URA3 in the rDNA. Colonies are grown at 30° for 2 days, and then photographed (Fig. 3A). The same procedure can be used to measure the silencing of telomeric or HM-specific reporter genes.

Colony Color Assays

In colony color assays, a white colony color indicates no silencing of the reporter gene, and a dark colony color indicates silencing. For telomeres and the HM loci, the ADE2 gene is used as the reporter. Cells mutated for ade2 form red colonies when grown on medium containing a limiting amount of adenine (64 pM). Therefore, when a telomeric ADE2 gene is expressed in an ade2 strain background, the resulting cells will grow white. Silencing of telomeric ADE2 results in a red color. Two or 3 days of growth at 30° followed by 4-5 days at 4° results in optimal color formation. In a wild-type strain, the normal pheno-type is a red-and-white variegated colony. A sir2 mutation turns on telomeric ADE2 and turns all the cells white. For the rDNA, a different colony color reporter gene is used, called MET15. Cells that are MET15+ have a white colony color when grown on rich medium containing lead nitrate (0.1%, w/v). When METIS is missing or silenced in the rDNA, the colony color turns to brown or tan, respectively. Colonies are grown for 5 days at 30° for optimal colony color development (Fig. 3B).

Cloning of SIR2 Homologs

We developed a set of degenerate PCR primers based on the conserved motifs in Sir2 proteins. These were originally used to clone HST3 and HST4 from S. cerevisiae, and subsequently to clone homologs from Schizosaccharomyces pombe and other organisms. The primer JB 710 [5'-GGNRTNCCNGAYTTY(A/C) G-3'], which recognizes the sequence encoding the peptide G(I/V)PDFR, is used in combination with either JB 708 [5'-RTC(A/G/T)ATRTTYTGNGTRTA-3'], which recognizes the sequence encoding the peptide YTQNID, or JB 709 [5'-RTC(A/G/T) ATRTTYTGNGT(A/G/T)AT-3'], which recognizes the sequence encoding the peptide ITQNID (see Fig. 1 for peptide sequences).

These oligonucleotides are were used in a touchdown PCR protocol using AmpliTaq polymerase (PerkinElmer, Norwalk, CT) under standard PCR conditions and the following cycling parameters:

(1 min at 94°, 30 sec at 54°, 30 sec at 72°), 2 cycles (1 min at 94°, 30 sec at 50°, 30 sec at 72°), 2 cycles (1 min at 94°, 30 sec at 46°, 30 sec at 72°), 2 cycles (1 min at 94°, 30 sec at 42°, 30 sec at 72°), 2 cycles (1 min at 94°, 30 sec at 38°, 30 sec at 72°), 2 cycles (1 min at 94°, 30 sec at 48°, 30 sec at 72°), 25 cycles 5 min at 72°

PCR products are then cloned by standard methods.

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