(A) Telomere lengthening in hpr1 and tho2 strains. Yeast DNA was isolated from the YPH499 yeast strain with indicated mutations, digested with XhoI, separated on a 1% agarose gel and analyzed by Southern blotting. The blots were hybridized with probes prepared using the 600-bp sequence from the Y′ element 3′ end. The position of the Y′ telomere is indicated. (B) Temperature-sensitive growth of THO complex mutants. Aliquots of ten-fold serial dilutions of yeast cells were spotted on YEPD plates and incubated at 30 or 37°C for three days. (C) Telomerase is required for the telomere lengthening effects in hpr1 and tho2 cells. Yeast cells carrying the indicated mutations were freshly sporulated and grown on YEPD plates at 30°C after 1 or 2 restreaks. Total yeast DNA was then isolated and analyzed by Southern blotting. (D) Recombination is not required for telomere lengthening in tho2 cells. Total yeast DNA from the indicated strains was isolated and analyzed by Southern blotting.

(A) Yeast DNA from the indicated mutations was isolated and analyzed by Southern blotting assays using the Y′ element probe. (B) The decreased Tel1p level does not contribute to telomere lengthening in tho2 and hpr1 cells. Total mRNA from the indicated strains was isolated and analyzed for GCN1 and TEL1 RNA levels using real time RT-PCR (left panel). Results were presented as relative levels normalized to the wild-type expression level. The bars were standard deviations determined from three independent experiments. Yeast DNA from indicated strains was analyzed for telomere length (right panel). (C) Overexpressing Rif1p suppresses the telomere lengthening in tho2 and hpr1 cells. Wild-type, tho2, or hpr1 yeast cells carrying plasmids pRS426 or pRS426-RIF1 (o/e RIF1) were cultured at 30°C. Telomere lengths of these cells were then analyzed using Southern blotting assays (top panel). Immunoblotting analysis of the Rif1p level was also performed (bottom panel).

(A) Overexpressing SUB2 suppressed the growth defect of tho2 or hpr1 cells. Strains of the indicated genotypes derived from THO2/tho2, or HPR1/hpr1 diploids carrying SUB2 or sub2-5 overexpressing plasmids were grown on YC plates at 30° or 37°C. (B) SUB2 or sub2-5 overexpression did not restore the Rif1p level in tho2 or hpr1 cells. Immunoblotting assays were carried out as described. (C) SUB2 overexpression did not restore the RIF1 level in tho2 or hpr1 cells. Total mRNA from the indicated strains was isolated and analyzed for the RIF1 RNA level using real time RT-PCR. Results were presented as relative levels normalized to the wild-type expression level. The bars were standard deviations calculated using data from three independent experiments. (D) SUB2 or sub2-5 overexpression did not restore the telomere length in tho2 or hpr1 cells. Telomere length analyses were performed as previously described.

(A) The Rif1p protein level is decreased in tho2 and hpr1 cells. Total yeast proteins from wild-type, tho2 or hpr1 yeast cells were precipitated with TCA, separated on an 8% SDS-polyacrylamide gel, and analyzed by immunoblotting using antibodies against Rap1p, Cdc13p, α-tubulin, α-actin, Myc (for myc9-tagged Rif1p and Rif2p), or TAP (for TAP-tagged Sir4p) (top panel). The percentages of protein expression relative to the wild-type cells are quantified (bottom panel). The bars were standard deviations determined using data from three different colonies of the indicated yeast strain. (B) The myc9-epitope tagging does not affect the effect of Rif1p on telomere length and telomere silencing. Telomere length of wild-type RIF1 or RIF1-myc9 cells were determined by Southern blotting using the Y′ element probe (left panel). Telomere silencing effects were determined in RIF1 or RIF1-myc9 cells. Yeast cells in 10-fold serial dilutions were spotted on YC, YC lacking uracil, or plates containing 5-FOA (right panel). (C) The Rif1p protein level is not affected in mft1 and thp2 cells. The Rif1p level is analyzed in mft1 and thp2 cells using immunoblotting analysis. (D) tho2 and hpr1 reduced the RIF1 RNA levels. The RNA transcripts of GCN1, RIF1, RIF2, and TUB1 were analyzed using Northern blotting assays (left panel). The expression levels of the indicated RNA transcripts were quantified and displayed as the percentages relative to the expression level in wild-type cells (right panel). The error bars were standard deviations calculated using data from three different colonies of the indicated yeast strain.

(A) tho2 enhances the telomere position effect. Wild-type or tho2 cells with URA3 at 1.1 kbp (UCC507), 2.5 kbp (UCC509), or 5.5 kbp (UCC511) from the telomere of chromosome VII–L were analyzed. Cells in 10-fold serial dilutions were spotted on YC, YC lacking uracil, or plates containing 5-FOA, and incubated until colonies formed. (B) THO2 and HPR1 function in the same pathway as RIF1 to regulate the telomere position effect. Samples of serial dilutions of UCC509 cells with the indicated mutations were spotted on plates as indicated.