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Figure 1 Strategy for ligation-mediated primer extension: the duplex formed between the unique oligonucleotide and the 32P-labeled guide oligonucleotide is ligated to the 3' G-strand overhang. The guide oligonucleotide is extended with T4 polymerase and the primer extension products are separated on a 12% sequencing gel. An asterisk marks the 32P label; ∧ marks the site of ligation.
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 | Figure 2 Measurement of G-overhang length by ligation-mediated primer extension. (A) Extension products obtained using T.thermophila DNA and various guide oligonucleotides: lane 1, input 21 nt guide oligonucleotide; lane 10, 30 nt marker showing the expected size of the input guide oligonucleotide + 9 nt extension product (a 14 nt overhang); lanes 2–9, purified rDNA minichromosomes; lanes 11–18, total macronuclear DNA; lanes 2 and 11, guide oligonucleotide TG1; lanes 3 and 12, TG2; lanes 4 and 13, TG3; lanes 5 and 14, TG4; lanes 6 and 15, TG5; lanes 7 and 16, TG6; lanes 8 and 17, mix of TG1–TG6; lanes 9 and 18, mix of TG1–TG6, no DNA ligase. (B) Ligation occurs specifically to the telomeric G-strand overhang: alkaline agarose gel showing restriction fragments labeled by ligation of 32P-labeled unique oligonucleotide/TG6 duplex to total macronuclear DNA (lanes 1 and 2) or purified rDNA telomeres (lanes 3–6): lane 1, ligation followed by BamHI digestion; lane 2, ligation followed by PstI digestion; lane 3, BamHI digestion prior to ligation; lane 4, Exo1 then BamHI digestion and ligation; lane 5, T4 DNA polymerase then BamHI digestion and ligation; lane 6, samples from lane 3 were digested with PstI to release the 1.2 kb telomeric fragment. (C) Organization of Tetrahymena rDNA: hatched boxes represent telomeres; shaded boxes labeled 3'nts and 5'nts represent the 5' and 3' non-transcribed spacer regions; open boxes represent the rDNA transcription units. B and P mark BamHI and PstI sites. (D) Measurement of overhang length using Euplotes crassus DNA: lane 1, primer extension products; lane 2, minus ligase control; lane 3, marker oligonucleotides. The 21 nt marker corresponds to the input guide oligonucleotide, the 30 and 58 nt markers correspond to the expected product if the guide oligonucleotide is extended only to the end of the 14 nt G-strand overhang or all the way through the duplex region of the telomere. The arrow marks excess guide oligonucleotide to which a single untemplated nucleotide was added by the T4 polymerase. A diagram showing the organization of Euplotes telomeres and the products expected from ligation-mediated primer extension is shown below.
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Figure 3 Structure of Tetrahymena G-strand overhangs. (A) Identity of the terminal nucleotide. 3' end-labeled DNA was cleaved at DMS-modified G-residues: lane 1, control oligonucleotide 5' (T2G4)4; lane 2, control oligonucleotide 5'-G(T2G4)3T; lanes 3 and 4, rDNA from two different Tetrahymena clones; lane 5, Euplotes DNA. (B) Overhang length at rDNA and non-rDNA telomeres: lane 1, input guide oligonucleotide TG6; lane 2, marker oligonucleotide showing the expected size of the input oligonucleotide + 9 nt extension product; lanes 3–11, primer extension products obtained with T4 DNA polymerase; lane 12, products obtained with Sequenase; lanes 3 and 4, total DNA from exponentially growing and starved cells; lanes 5 and 6, non-rDNA from growing and starved cells; lanes 7 and 8, rDNA from growing and starved cells; lanes 9–12, rDNA from growing cells; lane 9, control reaction without ligase; lanes 10 and 11, Exo1 and T4 polymerase digestion prior to ligation. The overhang lengths corresponding to specific reaction products are indicated on the right.
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 | Figure 4 Cell cycle-related changes in G-overhang length. (A) Primer extension products obtained using cells synchronized by centrifugal elutriation: lanes 1–3, DNA isolated from three different batches of S phase cells; lanes 4–6, DNA isolated from a single batch of elutriated cells after 30 min growth (lane 4), 60 min growth (lane 5) and 90 min growth (lane 6); lane 7, DNA from unsynchronized exponentially growing cells; lane 8, DNA from starved cells; lanes 9–12, longer exposure of lanes 4–7. Positions of markers are shown on the left. The overhang lengths corresponding to specific reaction products are indicated on the right. (B) Distribution of G-overhangs throughout the cell cycle. G1, S and G2, the reaction products in lanes 4–6 of (A) were quantified by PhosphorImager analysis and the total amount of reaction product in each pair of bands (i.e. the 14 and 15 nt products, the 20 and 21 nt products, etc.) was normalized to the total amount of product in that lane: G1, cells in G1 and early S phase (50% BrdU staining); S phase, 95% BrdU staining; G2, cells in G2 and late S (40% BrdU staining); Unav, Stav, Sav, the reaction products were quantified from four different batches of unsynchronized growing cells, starved cells and cells synchronized in S phase.
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Figure 5 Are G overhangs present on both rDNA telomeres? (A) Organization of the 14 kb non-palindromic rDNA. The minichromosome carries a single rDNA transcription unit (unshaded box) with an extra BamHI site and a portion of the cloning vector (black line) used to engineer the molecule to be defective in palindrome formation. Hatched boxes represent telomeres, shaded boxes are the 3' and 5' non-transcribed spacer regions, B and P mark BamHI and PstI sites. (B) Primer extension products obtained from the left and right telomere of the non-palindromic rDNA: lane 1, input oligonucleotide; lanes 2–4, extension products from the left telomere; lanes 5–7, products from the right telomere; lanes 2, 3, 5 and 6, S phase cells; lanes 4 and 7, growing cells. (C) Strategy for cloning rDNA molecules with overhangs on both ends. Unique oligonucleotide/guide oligonucleotide duplexes were ligated to the 3' overhangs on purified rDNA and the guide oligonucleotide extended with T7 polymerase. Following ApaI digestion, molecules with two sticky ends were cloned into pBluescript. The sequences shown below were obtained from the cloned rDNA molecules using the M13 reverse primer (upper sequence) and the M13 universal primer (lower sequence). The heavy line marks the ApaI site at the junction with the pBluescript vector sequence; the lighter line marks the oligonucleotide adapter; the dotted line marks the start of G-strand overhang on the rDNA telomere; the capitalized A is complementary to the 3'-terminal T on the overhang.
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 | Figure 6 Leading strand telomeres have G-overhangs. (A) Strategy for detecting leading strand telomeres with G-overhangs. The ligation step was performed as previously described except that the unique oligonucleotide/guide oligonucleotide duplex was replaced by a single 5' end-labeled foldback oligonucleotide. This foldback oligonucleotide had the same 5'-ACCCC adapter sequence as the TG6 guide oligonucleotide. Lines marked with U represent BrdU-labeled DNA, the gray lines represent RNA primers, P marks PstI sites, the star marks the 32P label. (B) DNA immunoprecipitated with BrdU antibody. Samples of input and immunoprecipitated DNA were separated on a 1% agarose gel and blotted to nylon membrane. Upper panel: the membrane was directly exposed to a PhosphorImager screen to identify telomeric restriction fragments labeled by ligation to the 32P-labeled foldback oligonucleotide. Lanes 1–3, input DNA that had been labeled with BrdU for 10 h (lane 1), 2 h (lane 2) or 0 h (lane 3). Lanes 4–6, immunoprecipitates from samples labeled with BrdU for 10 h (lane 4), 2 h (lane 5) or 0 h (lane 6). The arrows mark the restriction fragments from the left (0.7 kb) and the right (1.2 kb) telomere. Lower panel: the membrane was hybridized to a probe for a 4.3 kb non-telomeric rDNA restriction fragment. Lanes 1–6 are the same as in the upper panel.
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