by iG170Dhsp82 temperature sensitive allele, Sir2p level is found to be considerably diminished which is accompanied by complete loss of Sir2p function at restrictive temperature. Our result shows that at restrictive temperature, both the mating type silencing as well as telomere silencing function is completely lost, a phenotype similar to that observed in Dsir2 cells. This conclusively proves that Sir2p function is dependent on Hsp82. On the other hand, over- expression of Hsp82 shows negative regulatory effect on telomere silencing but has no effect on mating type silencing. Our result shows that Hsp82 over expression diminishes the level of Sir2p. We hereby speculate that under normal physiological condition, Hsp82 stabilizes Sir2p and thereby maintains repressed state of subtelomeric chromatin. On the other hand, under stressed condition, which results during over-expression of HSP82, the level of Sir2p is down regulated that de-represses the silent chromatin state. Thus, it seems that the central role of Hsp82 is to maintain a dynamic equilibrium of the level of silencing proteins which may be responsible for the reversible nature of telomere silencing. Materials and Methods Yeast strains Strains used in this study are listed in Table 1. The strain SLY20 used for TPE color assay is isogenic to W303a having ADE2 marked telomere VIIL. The mating tester strains used for these studies are YDS31 and YDS32. The temperature sensitive strain iG170Dhsp82 was kindly provided by Didier Picard [24]. This strain has the permissive temperature 25uC and restrictive temperature (35uC–37uC). Yeast knock out strains YSC1021- 551935 (HSP82::KANr), YSC1021-552834 (SBA1::KANr), YSC1021-551520 (HSC82::KANr) were purchased from Thermo Scientific, Open Biosystems, Huntsville, AL, USA. All primers used in this study were purchased from Sigma Aldrich. Using the genomic DNA of YSC1021-551520 as a template and the pair of primers OSB1 (59ACC AAG CGT TGG GTA ATG A39) and OSB2 (59 TGG TCA TTT GAC AGC TGA TG39) KANMX cassette with HSC82 flanking regions was amplified. The PCR product was then integrated into SLY20 and selected on G418 sulphate containing plates. The resultant hsc82 strain is referred as SLY4 in this paper. In a similar way, using genomic DNA of YSC1021-551935 (HSP82::KANr) as a template and the pair of primers OSB3 (59 TGA CAC ACT AGA CGC GTC GG39) and OSB4 (59TAC CAA CCA GGT CCT TCC GC39) KANMX cassette with HSP82 flanking regions was amplified. It was then integrated to SLY20 to generate hsp82 strain SLY5. To knock out SBA1 gene, the genomic DNA of YSC1021-552834 was used as a template to amplify KANMX cassette with sba1 flanking regions. Forward primer OSB5 (59 TGC TAC CCG CCT TCC GAG TG39) and the reverse primer OSB6 (59 CAC ATA CAG TTC CAT TAC TTG AC39) were used for this purpose. The PCR product was then integrated into SLY20 cells to generate sba1 strain SLY6. The sir2 strain SLY12 (sir2::KANr) was made by integration of KANMX cassette with SIR2 flanking regions in SLY20. All of the knockout genotypes were confirmed by PCR analysis. The HSP82 over expression plasmid pHCA/hsp82, which has HIS3 marker, was transformed into SLY20, SLY5 and SLY6 strains and the transformed colonies were selected by growing them on SC-his medium to derive SLY13, SLY10 and SLY31 strains respectively. The blank vector pHCA was transformed into SLY20 and the transformed colonies were selected on SC-his medium to derive the strain SLY46. The 2 m HSP82 over expression plasmid pTA/hsp82 which has TRP marker was transformed into SLY20 strain; the transformed colonies were Hsp82 Is Involved in Sir2p Abundance selected by growing them on SC-trp medium to derive SLY32 strain. Plasmids pHCA/HSP82 plasmid was a gift from Didier Picard [25]. It is a CEN/ARS plasmid, derived from pRS313 vector, which over expresses HSP82 under the control of GPD promoter. Using genomic DNA of W303a as a template we amplified the HSP82 gene using the forward primer OSB21 (59 GAC GGA TCC ATG GCT AGT GAA ACT TTT GAA TTT C 39) having BamH1 site and the reverse primer OSB22 (59 CGG GTC GAC CTA ATC TAC CTC TTC CAT TTC GG 39) having SalI sequence. The PCR amplified product was then cloned into BamH1, SalI double digested 2 m expression vector pTA, which expresses HSP82 under the control of GPD promoter. All the constructed plasmids were confirmed by sequencing. TPE color assay SLY20, SLY4, SLY5, SLY6, SLY13, SLY12, SLY31 and SLY46 cells were grown on appropriate medium and TPE was performed according to the protocol described [26]. Mating type frequency assessment Wild type strain (HSP82), Dhsp82, Dhsc82, YDS32 (MATa) and Dsir2 were grown to the mid exponential phase. About 400 cells from each culture were plated in YPD plate and incubated at 30uC. Also 400 cells of the wild type (HSP82), Dhsp82, Dhsc82, and Dsir2 were mixed with same number of cells of YDS32 (MATa) and incubated at 30uC shaker for 10 minutes. Then each of them was spreaded on SD (synthetic dextrose) plate and incubated at 30uC incubator for 30 hours. The number of cells grown on each YPD and SD plates were counted. The mating frequency was calculated as = (No. of cells grown on SD plate/No. of cells on YPD plate)* 100. The value obtained in case of Dsir2 strain was subtracted from each of the three strains. The experiment was repeated for 5 times and each bar shows the mean frequency 6 SD. Western blotting The temperature sensitive strain iG170Dhsp82 was grown at 25uC (permissive temperature) as well as 37uC (restrictive temperature) and protein was isolated from them by the procedure described below. Proteins were isolated from SLY4, SLY5, SLY6, SLY10, SLY13, SLY20 and SLY32 strains after growing them on proper medium at 30uC and equal amounts of proteins were loaded in SDS PAGE. For heat shock treatment, wild type cells (SLY20) were grown upto 0.3 O.D. at 595 nm. It was then divided into two batches, one batch of cells was subjected to heat shock at 39uC for 40 minutes, and other was grown at 30uC. Equal amount of cells were finally harvested and protein was isolated from them. We isolated the protein using trichloroacetic acid (TCA) as described in [27] with little modifications. Each of the above strains was grown on 20 ml of appropriate medium until the OD600 nm reaches 0.5. The cell was centrifuged and the pellet was washed first with distilled water and then with 500 ml 20% TCA. The pellet was finally resuspended in 200 ml 20% TCA and the cell lysis was done with glass beads keeping at cold temperature for 30 min. The TCA precipitated proteins were collected; washed with 5% TCA and dissolved in 60 ml 16 sample buffer (0.05 M Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate [SDS], 10% glycerol and 0.1% bromophenol blue), supplemented with 6.66 ml 1 M DTT, and 33 ml 1 M Tris HCl, [pH 9.0]. The protein samples were boiled for 3 minutes and appropriate volumes of samples were loaded on 10% SDS poly acrylamide gel. The gel PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23406