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. 2009 Nov;183(3):793–810. doi: 10.1534/genetics.109.108894

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Copyright © 2009 by the Genetics Society of America

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Figure 4.— — de novo telomere addition requires Ten1 in G2/M arrested cells. (A) Schematic diagram of the left arm of chromosome VII containing the HO endonuclease recognition site adjacent to 81 bp of telomeric repeats (Diede and Gottschling 1999). The ADE2 gene is located on the centromere proximal side of the inserted telomere repeat. The chromosome fragment from the HO site to the end of the chromosome does not contain any essential genes. A LYS2 marker is located in this region to retain this nonessential fragment. Note: this diagram is not to scale. (B) Telomere addition at 36°. Wild-type and ten1-105 strains containing the telomere healing cassette were grown in SD −Lys media at 23°, and then switched to YP-raffinose media containing nocodazole to arrest cells in G2/M. While maintaining the arrest, galactose was added to induce expression of the HO endonuclease, and the cultures were simultaneously shifted to 36°. Cells were collected at 0, 1, 3, and 5 hr following addition of galactose. Genomic DNA was isolated, digested with SpeI, and fragments were separated on a 1% agarose gel. The Southern blot was probed with a [³²P]-labeled fragment that hybridizes to the ADE2 gene (Diede and Gottschling 1999). Both the native ADE2 locus (INT) and the HO-adjacent ADE2 gene are recognized by this probe. The gel was exposed on a Phosphorimager screen and the total amount of signal in the HO-adjacent ADE2 locus determined for each lane. The numbers below each lane represent the sum of the precut and cut fragments, normalized to the amount present at T = 0. PRE, fragment prior to HO digestion; INT, internal ADE2 control; arrow, fragment after HO digestion. WT (wild-type, hc1943), ten1-105 (hc1946). (C and D) Ade⁺Lys⁻ colony formation as a means to assess the ability of ten1-105 strains to heal the DSB if released from the G2/M arrest and plated at 23°. In C, cells were incubated at 23° while HO was induced; in D, cells were shifted to 36° during the induction. Colonies on SD −Ade were replica-plated to SD −Ade −Lys (and SD −Ade) after 5 days at 23°. The lines on the graphs show the proportion of Ade⁺ colonies that are also Lys⁻ at each time point [(Ade⁺ Lys⁻)/(Ade⁺)]. TEN1, ten1-105. The bars on the graphs show the percentage of Ade⁺ Lys⁻ colonies relative to the number of Ade⁺ colonies at T = 0; cells unable to heal the DSB are not expected to form colonies. Lighted shaded bars, TEN1; shaded bars, ten1-105 solid bars, ten1-105 .

Inline graphic — de novo telomere addition requires Ten1 in G2/M arrested cells. (A) Schematic diagram of the left arm of chromosome VII containing the HO endonuclease recognition site adjacent to 81 bp of telomeric repeats (Diede and Gottschling 1999). The ADE2 gene is located on the centromere proximal side of the inserted telomere repeat. The chromosome fragment from the HO site to the end of the chromosome does not contain any essential genes. A LYS2 marker is located in this region to retain this nonessential fragment. Note: this diagram is not to scale. (B) Telomere addition at 36°. Wild-type and ten1-105 strains containing the telomere healing cassette were grown in SD −Lys media at 23°, and then switched to YP-raffinose media containing nocodazole to arrest cells in G2/M. While maintaining the arrest, galactose was added to induce expression of the HO endonuclease, and the cultures were simultaneously shifted to 36°. Cells were collected at 0, 1, 3, and 5 hr following addition of galactose. Genomic DNA was isolated, digested with SpeI, and fragments were separated on a 1% agarose gel. The Southern blot was probed with a [³²P]-labeled fragment that hybridizes to the ADE2 gene (Diede and Gottschling 1999). Both the native ADE2 locus (INT) and the HO-adjacent ADE2 gene are recognized by this probe. The gel was exposed on a Phosphorimager screen and the total amount of signal in the HO-adjacent ADE2 locus determined for each lane. The numbers below each lane represent the sum of the precut and cut fragments, normalized to the amount present at T = 0. PRE, fragment prior to HO digestion; INT, internal ADE2 control; arrow, fragment after HO digestion. WT (wild-type, hc1943), ten1-105 (hc1946). (C and D) Ade⁺Lys⁻ colony formation as a means to assess the ability of ten1-105 strains to heal the DSB if released from the G2/M arrest and plated at 23°. In C, cells were incubated at 23° while HO was induced; in D, cells were shifted to 36° during the induction. Colonies on SD −Ade were replica-plated to SD −Ade −Lys (and SD −Ade) after 5 days at 23°. The lines on the graphs show the proportion of Ade⁺ colonies that are also Lys⁻ at each time point [(Ade⁺ Lys⁻)/(Ade⁺)]. TEN1, ten1-105. The bars on the graphs show the percentage of Ade⁺ Lys⁻ colonies relative to the number of Ade⁺ colonies at T = 0; cells unable to heal the DSB are not expected to form colonies. Lighted shaded bars, TEN1; shaded bars, ten1-105 solid bars, ten1-105 .