sizes in kilobases); a, total RNA from AMTa cells; a, total RNA from MATa cells. observed that the TBF1 gene hybridizes to chromosome XVI. (data not shown) ...
Vol. 13, No. 2
MOLECULAR AND CELLULAR BIOLOGY, Feb. 1993, P. 1306-1314
0270-7306/93/021306-09$02.00/0 Copyright C 1993, American Society for Microbiology
An Essential Yeast Gene Encoding a TTAGGG Repeat-Binding Protein CLAUDIO BRIGATI,1'2 STEPHEN KURTZ,1t DINA BALDERES,1 GIORGIO VIDALI,2 AND DAVID SHORE'*
Department of Microbiology, College of Physicians and Surgeons of Columbia University, 701 West 168th Street, New York, New York 10032,1 and Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa, Italy2 Received 14 September 1992/Returned for modification 19 October 1992/Accepted 6 November 1992
A yeast gene encoding a DNA-binding protein that recognizes the telomeric repeat sequence TTAGGG found in multicellular eukaryotes was identified by screening a Agtll expression library with a radiolabeled TTAGGG multimer. This gene, which we refer to as TBFI (TTAGGG repeat-binding factor 1), encodes a polypeptide with a predicted molecular mass of 63 kDa. The TBF1 protein, produced in vitro by transcription and translation of the cloned gene, binds to (TTAGGG). probes and to a yeast telomeric junction sequence that contains two copies of the sequence TTAGGG separated by 5 bp. TBF1 appears to be identical to a previously described yeast TTAGGG-repeat binding activity called TBFa. TBF1 produced in vitro yields protein-DNA complexes with (TTAGGG)" probes that have mobilities on native polyacrylamide gels identical to those produced by partially purified TBFa from yeast cells. Furthermore, when extracts are prepared from a strain containing a TBFJ gene with an antigen tag, we find that the antigen copurifies with the predominant (TTAGGG)n-binding activity in the extracts. The DNA sequence of TBF1 was determined. The predicted protein sequence suggests that TBF1 may contain a nucleotide-binding domain, but no significant similarities to any other known proteins were identified, nor was an obvious DNA-binding motif apparent. Diploid cells heterozygous for a tbfl::URA3 insertion mutation are viable but upon sporulation give rise to tetrads with only two viable spores, both of which are Ura, indicating that the TBFI gene is essential for growth. Possible functions of TBF1 (TFBa) are discussed in light of these new results.
Among all eukaryotes examined to date, the telomere is a highly conserved structure designed to protect chromosomes from degradation and fusion (for reviews, see references 6 and 32). Telomeres are composed of multiple repeats of short sequence elements (typically 5 to 8 bp in length, with a GT-rich strand oriented 5' to 3' toward the end of the chromosome) and range in length from a few repeat units to >10 kb. The repeated sequence (TTAGGG)n is found at telomeres in all vertebrates, certain slime molds, and trypanosomes; (TTGGGG)n and (TlTITGGGG)n are found in the ciliated protozoan Tetrahymena and Oxytricha species, respectively; and (TG1_3)n is found in the yeast Saccharomyces cerevisiae. In organisms whose telomeres have been examined in detail, the GT strand extends 12 to 16 nucleotides (two repeats) beyond the complementary C-rich strand. Proteins that associate with or contribute to telomere structure and function have been identified in several organisms. In Oxyticha nova, a DNA-binding protein specific for the terminus of the chromosome has been characterized and shown to consist of two subunits, a and (14, 15), that form a heterodimer. Recent work has shown that the ao subunit is the predominant DNA-binding subunit, although the subunit also contacts DNA and contributes to telomere structure. The a/13 heterodimer recognizes a guanosine-rich single-stranded 3' overhang at the very end of the telomere, however, and does not bind to internal T4G4 repeats. In yeast cells several proteins that bind telomeric sequences
* Corresponding author. t Present address: Microbial Molecular Biology, Bristol-Myers Squibb Co., Princeton, NJ 08543-4000.
have been identified. Multiple binding sites for RAP1, a transcriptional regulatory protein, are found within the TG1_3 telomeric repeat sequences (5, 8, 19). Biochemical (11, 31) and cytological (17) studies have shown that RAP1 is bound to these sites in vivo. Furthermore, mutations in RAP1 affect telomere length, clearly establishing a role for RAP1 in the regulation of telomere structure (11, 22, 29). Two proteins (TBFa and TBF1) were identified in yeast extracts on the basis of their ability to bind to a yeast telomeric sequence and to telomeric repeat sequences found in Tetrahymena species (T2G4) and vertebrates (TTAGGG). TBFot was purified and shown by footprinting to bind to telomeric sequences that occur proximal to the TG13 repeat in yeast cells in which two copies of the sequence TTAGGG occur (18). Proteins related in binding specificity to TBFa have been described to occur in the slime mold Physarum polycephalum (12) and more recently in HeLa cell extracts (33). In both Physarum and human cells, the telomere repeat sequence is T1AGGG. Because the sequence TTAGGG is present at the telomeres of many divergent organisms, it is likely that it constitutes a binding site for a highly conserved protein(s) with important roles in chromosomal structure and function. The occurrence of a ]ITAGGG-binding activity in S. cerevisiae (18) and the presence of ITAGGG sequences at telomere junctions (20) raise the possibility that there is a related factor with a functional role at telomeres in S. cerevisiae. As a first step toward testing this hypothesis, we report the cloning and initial characterization of a yeast gene encoding a TTAGGG repeat-binding protein, referred to here as TBF1 (TTAGGG repeat-binding factor 1). We show that TBF1 is an essential gene and that it probably encodes the TBFa 1306
VOL. 13, 1993
activity previously described (18). Possible in vivo functions of the TBF1 (TBFa) protein are discussed. MATERUILS AND METHODS Agtll library screening. Xgtll expression libraries of human cDNA (Clontech) and yeast genomic DNA (generous gift of R. Young and M. Snyder) were screened with radiolabeled DNA according to the method of Singh et al. (27, 28). Briefly, Escherichia coli Y1090 was infected with library phage and plated on LB agarose plates (35 cm) to generate -3 x 104 plaques per plate. After 2 h of incubation at 42°C, nitrocellulose filters (previously immersed in 10 mM isopropylthio-13-D-galactoside [IPTG] and dried) were overlaid onto the plates. The plates were incubated for an additional 5 h at 37°C, after which the filters were removed and allowed to dry. Duplicate IPTG-impregnated filters were overlaid and incubated for 2 h. After removal, all filters were submerged in binding buffer (50 mM NaCl, 10 mM Tris [pH 7.5], 1 mM EDTA, 1 mM dithiothreitol, 2.5% dry milk) and gently shaken for 30 min at room temperature. The solution was then decanted and replaced by new binding buffer containing 0.25% dry milk. A telomere probe containing 50 tandem copies of the sequence TTAGGG was generated by EcoRI and HindlIl digestion of plasmid pT6 (30) and radiolabeled to a specific activity of > 108 cpm/,ug. The probe was added directly to the filters along with sheared, doublestranded salmon sperm DNA at 10 ,ug/ml and incubated at 4°C for 2 h. The filters were then briefly washed three times in binding buffer, dried, and exposed to XAR-5 film for 16 h at -80°C. Plaque purification and preparation of phage DNA and bacterial lysogen extracts were carried out by standard
techniques. Cloning and DNA sequencing. The Agtll phage 15.4 contains a 3.6-kb EcoRI insert that was subcloned into M13
mpl8. DNA sequencing revealed an 800-bp open reading frame (ORF) extending from the end joined to lacZ in phage 15.4. The 800-bp ORF was isolated after digestion with EcoRI and HpaI and was subcloned into pUC19. This fragment was used to probe a yeast genomic library in YEp24 (a gift from M. Carlson) by E. coli colony hybridization. Positive clones were analyzed by restriction mapping and Southern blotting. Appropriate fragments were subcloned into M13 mpl8 and mpl9 vectors for DNA sequence analysis using the Sanger dideoxynucleotide chain termination method (3). Yeast strains and manipulations. Genetic analyses were done in two strains of widely divergent genetic backgrounds: W303 (a derivative of strain LL20; AL4Ta/at ade2-1 canl-100 his3-11 15 leu2-3 112 trpl-l ura3-1) and LN224 (a derivative of S288c; AM Ta/a leu2 ura3-52). Diploid strains were transformed (4) to uracil prototrophy with a purified fragment in which the URA3 gene was inserted into the XbaI site (nucleotide 1301) of the TBF1 coding region. Appropriate transformants, verified by Southern blotting, were induced to undergo meiosis, and spores were dissected by microma-
nipulation onto YEPD agar.
To facilitate subsequent biochemical and genetic analyses, strain W303 was modified by deleting the protease encoded by the PEP4 locus and by deleting the entire TBF1 locus. The PEP4 locus was deleted by transformation into a diploid strain with plasmid pTS17, which contains an insertion of a HpaI fragment of LEU2 in the PEP4 coding region (1). This strain was then transformed with EcoRI-digested DNA from plasmid CDS99, which contains an insertion of the HIS3 gene in the TBF1 coding region (described below). Histidine
YEAST TTAGGG REPEAT-BINDING FACTOR GENE
1307
prototrophs were analyzed by Southern blotting to confirm the disruption of one of the TBF1 alleles. Sporulation and dissection of this strain yields only two viable spores per tetrad, none of which are His'. This diploid was transformed with pCDS63, a centromere-containing plasmid that also carries the URA3, sup4-o, and TBF1 genes. Transformants were induced to undergo meiosis, and spores were dissected onto YEPD agar. A haploid colony prototrophic for leucine, histidine, and uracil was recovered and used in a plasmid shuffle assay (26) to determine the ability of plasmids carrying mutations in TBF1 to complement the null mutation. Extracts for gel mobility shift assays were also prepared from the protease-deficient strain BJ2168 (A 4Ta leu2 trpl ura3-52 prbl-1122 pep4-3 prcl-407 gal2) carrying plasmids with an epitope-tagged TBFI gene (strain CYS40). Plasmids. To construct a null mutation of TBF1, we first subcloned a 2.5-kb SphI-HpaI fragment containing the entire gene into pUC19 (digested with SphI and SmaI) to create pCDS47. A fragment containing the TBF1 gene was recovered from pCDS47 after digestion with SphI and SacI and inserted into a Bluescript vector, using identical restriction sites in the superpolylinker to create pCDS50. The TBF1 fragment was then subcloned into pIC20R (digested with SacI and EcoRV) to create pCDS66. This plasmid was digested with HindIll and XbaI to remove 1,274 bp of TBF1 coding region and end filled with Klenow enzyme in the presence of deoxynucleoside triphosphates (dNTPs). After ligation of BamHI linkers and digestion with BamHI, a fragment containing the HIS3 gene was inserted into this site to create pCDS99. A fragment containing the tbfl::HI53 insertion was recovered after digestion with EcoRI and used for transformation into yeast cells. Plasmids that complement the tbfl::HIS3 mutation were constructed by cloning a 2.5-kb BamHI-SacI fragment containing the TBF1 gene from pCDS50 into BamHI-SacIdigested pRS314 (a CEN-TRPI vector) to create pCDS58. The same TBFJ-containing fragment was ligated into the pRS316 (CEN-URA3) vector after insertion of the sup4-o gene at the EcoRI site in this plasmid, creating pCDS63. Both plasmids (CDS58 and CDS63) complement the tbfl ::HIS3 mutation. An 11-amino-acid antigen tag, derived from the influenza virus hemagglutinin antigen (HA1) (reference 13 and references therein), was inserted into the TBF1 coding region as follows. Plasmid CDS66 was digested with HindIII, treated with calf alkaline phosphatase, and ligated with annealed oligonucleotides encoding the HAl peptide synthesized with HindIll ends (5'-AGCTTATACCCATACGATGTTCCAGA TTACGCT-3' and 5'-AGCTAGCGTAATCTGGAACATCG TATGGGTATA-3'). Transformants were recovered with single and double tandem insertions of the epitope. A fragment containing the epitope-tagged TBF1 gene was obtained after digestion with ClaI and Sacl and inserted into pRS306 to create an integrating plasmid (CDS109) and into pRS314 to create an episomal (CEN) plasmid (CDS111). Both the integrating and CEN plasmid versions of the epitope-tagged TBF1 gene containing either single or double epitope insertions complement the tbf ::HIS3 null mutation. The epitope-tagged TBF1 protein was detected on Western immunoblots with monoclonal antibody 12CA5 (ascitic fluid; BABCO) diluted 1:5,000 and horseradish peroxidase-conjugated second antibody. To produce the TBF1 protein in vitro, the TBF1 coding region was placed under the control of the bacteriophage T7 promoter as follows. Oligonucleotide primers were designed for polymerase chain reaction amplification and subsequent
1308
BRIGATI ET AL.
cloning of the first 275 bp of the coding region. An NcoI site which provided the ATG was synthesized at the 5' end. After polymerase chain reaction amplification and digestion with NcoI and BglII, the fragment was ligated, together with a fragment containing the remaining portion of the TBFI coding region (a BglII-SacI fragment recovered from pCDS66), into a T7 expression vector (T7,BSal, digested with NcoI and Sacl). The resulting plasmid, pCDS125, was linearized with SacI and transcribed in vitro with T7 RNA polymerase. RNA was translated in vitro by using a rabbit reticulocyte lysate supplemented with 35S-labeled methionine (Promega transcription/translation system). The telomere-derived probe for gel mobility shift assays was constructed from telomere clone 120 (10) after digestion with FokI, end filling with Klenow enzyme in the presence of dNTPs, and digestion with EcoRI. A 180-bp fragment was recovered and subcloned into pUC18 (EcoRI-SmaI digested) to create pCDS58. The plasmid was sequenced to verify the cloning. Probes from this plasmid were radiolabeled after digestion with EcoRI and HindIII and purified on acrylamide gels. Plasmid pTT6 (a gift of L. Van der Ploeg) contains 50 tandem copies of the sequence TTAGGG cloned into the BamHI site of pBluescript. Derivatives containing fewer copies of the TTAGGG sequence were generated by BAL 31 digestion, fragment isolation, and subcloning into pUC18. Preparation of cell extracts. Protease-deficient strains carrying plasmids with the epitope-tagged TBFI gene were grown at 30°C and harvested at a density of 2 x 107 cells per ml. Eight liters of cells was centrifuged, washed once in ice-cold water, and resuspended in 10 ml of breakage buffer (0.2 M Tris [pH 8.0], 10% glycerol, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol) supplemented with 2 ,ug of pepstatin per ml and 1 mM phenylmethylsulfonyl fluoride. Glass beads were added up to one-half volume of the cell suspension. Cells were broken on a Vortex mixer with 12 1-min bursts alternating with incubations on ice. Debris was removed by centrifugation in an SS34 rotor at 8,000 x g for 15 min. The supernatant was further cleared by ultracentrifugation in a Ti5O rotor at 100,000 x g for 45 min and then fractionated by ammonium sulfate precipitation. Proteins precipitating between 25 and 50% ammonium sulfate saturation were resuspended in 3 ml of A50 (20 mM Tris [pH 8.0], 0.5 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol, 50 mM ammonium sulfate) containing protease inhibitors and dialyzed against A50 overnight. The sample was loaded onto a heparin-agarose column and developed with a linear salt gradient of 50 to 600 mM ammonium sulfate in A50. The elution of TBF1 was monitored by gel mobility shift assay and by Western blotting with HAl monoclonal antibody 12CA5. Electrophoretic mobility shift assays. Assays were performed as described previously (24, 25) except that the buffer contained 15 mM MgCl2, 50 mM KCl, 10 mM N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.5), and 12.5% (vol/vol) glycerol (5). Radiolabeled DNA probes either contained 3, 6, 12, 17, or 50 tandem repeats of the sequence ITAGGG or were derived from telomere clone 120 (10). RESULTS Cloning of a yeast gene encoding a TTAGGG repeat-binding protein. In an attempt to identify both human and yeast genes encoding (1TAGGG)n-binding proteins, we screened Xgtll expression libraries for plaques that would specifically bind a radiolabeled (TTAGGG)50 probe (28). We failed to
MOL. CELL. BIOL. A
B 1
2
3
4
5
6
7
8
9
c 3 4ZzZ_i1 5 6 ._ 23456
2
1
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__-., L--
FIG. 1. Cloning of a yeast gene whose product binds TTAGGG repeats. (A) Purification of Xgtll phage 15.4 encoding an activity that binds TTAGGG. Approximately 200 PFU was plated, adhered to nitrocellulose, and probed with a radiolabeled TTAGGG multimer as described in Materials and Methods. (B) Electrophoretic mobility shift assay with crude extracts of bacterial strains lysogenic for phage 15.4 (lanes 2 to 8) and an unrelated control phage encoding Epstein-Barr virus nuclear antigen (lane 9). The probe is radiolabeled (TTAGGG)50, and its mobility is shown in lane 1 in the absence of extract. The reaction in lane 2 contained only poly(dIdC) and vector DNA as carrier. Specific competitor DNA [unlabeled (TTAGGG)50-containing plasmid DNA] was added at 10-, 4-, 2-, and 1-fold molar excesses (lanes 3 to 6). In lane 7, a 20-fold molar excess of double-stranded oligonucleotide containing a high-affinity RAPlbinding site (TEF2 [24]) was added to the binding reaction. In lane 8, the reaction contained a 20-fold molar excess of a (TTACGG)3 oligonucleotide. (C) Electrophoretic mobility shift assay with in vitro-synthesized protein. The 800-bp ORF from clone 15.4 was subcloned into an expression vector and produced by transcription and translation in vitro. Competitor DNAs [unlabeled (TTAGGG)50 plasmid DNA] were used at 0-, 1-, 2-, 4-, and 10-fold molar excesses (lanes 2 to 6). Lane 1 contained probe alone in the absence of extract.
isolate a reactive phage from a human cDNA library but did identify a single plaque from a yeast genomic library that reacted strongly with the (1TAGGG)50 probe. The positive phage (called 15.4) was purified by two subsequent rounds of screening (Fig. 1A). Binding of 15.4 plaques to the (TTAGGG)50 probe appeared to be specific, since a number of unrelated probes failed to react with this phage (data not shown). To confirm that the 15.4 phage encoded a (TTAGGG)nbinding protein, we performed a solution binding assay using the electrophoretic mobility shift assay. Protein extracts prepared from E. coli strains lysogenic for phage 15.4 or an unrelated control phage (encoding a LacZ-Epstein-Barr virus nuclear antigen fusion) were mixed with end-labeled (TTAGGG)50 and electrophoresed in 4% native polyacryl-
VOL. 13, 1993
amide gels. As shown in Fig. 1B, extracts containing the 15.4 lysogen produce two prominent complexes in this assay, one a discrete band of low mobility relative to the free probe and the other a broad smear of even lower mobility. Both complexes are competed for by increasing molar amounts of (TTAGGG)50 competitor DNA but not by either a RAPlbinding site competitor or a multimer of a single-point mutant of the TTAGGG hexamer, TTACGG. The extract from the control phage lysogen fails to react with the (TTAGGG)50 probe. We have detected binding of the 15.4 lysogen extract to (TTAGGG)12 and (TTAGGG)6 probes. Binding to a shorter repeat, (TTAGGG)3, is weaker but still detectable (data not shown). The 15.4 Xgtll clone contains a 3.6-kb insert of yeast genomic DNA present as an EcoRI fragment. Sequence analysis revealed an 800-bp ORF fused in frame to the lacZ gene in the Xgt1l vector. This ORF, which fortuitously contained a methionine codon near its 5' end, was subcloned downstream from the bacteriophage SP6 promoter. Transcription and translation of this clone in vitro yields a product which produces a series of complexes with the TTAGGG multimer probe which are competed for with increasing molar amounts of TTAGGG-containing competitor DNA (Fig. 1C). This result demonstrates that the 800-bp ORF contains sufficient information for sequence-specific binding, ruling out a possible role for the lacZ sequences in this process. The lower mobility of complexes produced from the lysogen extract presumably reflects the large lacZ sequence fused to the DNA-binding sequences within the 800-bp ORF. Isolation, map location, and DNA sequence of TBF1. To examine the expression of the 800-bp ORF sequence in yeast cells, we probed Northern (RNA) blots of total and poly(A)selected yeast RNA. The 800-bp ORF detects an mRNA of approximately 2,100 nucleotides in both AM Ta and MATa cells (Fig. 2). Because the mRNA detected with this probe was substantially larger than the 800-bp ORF from the initial Xgtll clone, we screened a library of yeast genomic sequences in the multicopy vector YEp24 by DNA hybridization with the 800-bp fragment to isolate a full-length copy of the gene. Positive clones were characterized by restriction endonuclease mapping and DNA sequence analysis, the
results of which define a gene which we refer to as TBF1. The nucleotide and predicted amino acid sequences of TBFI are presented in Fig. 3. The TBFI coding region consists of 562 amino acids with a predicted molecular mass of 62,996 Da, consistent with the anticipated size of the gene based on the Northern blot analysis. The predicted protein sequence of TBF1 is not similar to any sequences in current data bases (SWISSPROT and GenPept [13a]). We did note the presence of a potential nucleotide-binding site at residues 295 to 302, as defined by a match to the consensus sequence GxxGxGKS (7; for a review, see reference 16). However, we were unable to identify any other conserved sequences associated with ATP-binding subunits or GTPases in the TBF1 sequence. The region important for DNA binding, which was broadly defined by the initial 15.4 Xgtll clone, is located at the carboxy terminus of the protein. This region has no obvious similarities to known DNA-binding motifs, e.g., helix-turnhelix, Zn finger, or b-ZIP. In the course of constructing the TBFJ null mutations in diploid W303, we noticed that sporulation of strains containing both the pep4::LEU2 and tbfl::HIS3 alleles produced predominantly ditype tetrads, suggesting that the two loci are genetically linked. Consistent with this observation, we
YEAST TTAGGG REPEAT-BINDING FACTOR GENE
M a
1309
cc
3.6 2.3 1.9
1.3 1.2 0.7
FIG. 2. Hybridization of the 800-bp ORF from phage 15.4 to an mRNA of approximately 2,100 nucleotides. Total RNAs were separated on a 1.2% agarose-formaldehyde gel, blotted onto nitrocellulose, and hybridized with the 800-bp insert from clone 15.4. Lanes: M, A DNA size markers (BstEII-digested; numbers indicate sizes in kilobases); a, total RNA from AMTa cells; a, total RNA
from MATa cells. observed that the TBF1 gene hybridizes to chromosome XVI (data not shown) and noted that the PEP4 locus maps to the left arm of that chromosome. Appropriate genetic crosses support the conclusion that TBF1 and PEP4 are linked. Strains for genetic mapping along chromosome XVI were derived from strain W303 and contained either an insertion of the HIS3 gene at SIT3, an insertion of the LEU2 gene at PEP4, an insertion of the LEU2 gene at GAL4, an insertion of the LEU2 gene at RAD1, or an integration of the URA3 gene adjacent to TBF1. All constructions were verified by Southern blotting. Standard genetic linkage analysis established that TBF1 was located 12.6 centimorgans centromere proximal to the PEP4 locus on the left arm of chromosome XVI. SIT3 was mapped approximately 32 centimorgans centromere proximal to TBF1 (Table 1). We detected no linkage between TBFI and either GAL4 or RAD1. TBFJ is an essential gene. To assess the requirement for TBF1 protein in vivo, we created an insertion mutation in the TBFJ coding region. The yeast URA3 gene was inserted into the XbaI site as indicated in Fig. 4A. This construction, which interrupts the coding region in the portion of the gene that is necessary for DNA binding, was used to transform two genetically different diploid strains (S288c and W303) to uracil prototrophy. Transformants were analyzed by Southern blots to verify that this construction had replaced one of the wild-type TBFJ alleles (Fig. 4B). Diploids carrying one disrupted allele were then induced to undergo meiosis, and tetrads were dissected. As shown in Fig. 4C, only two spores from each tetrad dissected were viable. None of the colonies arising from these spores was Ura+, indicating that segregants receiving the tbfp::URA3 allele were inviable and that TBFI is essential for cell growth. Microscopic inspection of the inviable spores revealed clumps of -2 to 30 cells
BRIGATI ET AL.
1310
MOL. CELL. BIOL.
GCATGCCTCAACTTAATGACGGTAAGGGCTCCAGCGTATCGTTTTAAAGAAGTATGTCAAGACACTTTCTCCTCCAAGTTGAACAAGCTCAAATTTT
100
GACTATCTGTTCAATAGCGCTATCAAGAAATGTGTTGAAACGGCGAGTTAGTGCAACCAAAGGCCCCTCCGGCATTATTTACAAAACTG
200 300
400
TCCCTCTCATTGTTATTATCGGAAAATCTCTTATGATTTTTCAATTCATTTTACTAATAATATATGTTATAATTCTC
5000
CTCTATATTGCAGGTAAGTGGTAAATTATTATGTATTGAAAAAAGCTTAAAACTGACATTGAAGCCTATATTTGCACGT
6000
CCTATAATTTCCCCCTTCGTAGAGCCGGCAGACTCATCCACTATTTCCTCATGTTTGCCTCCTTTAACATTTAAAAA
7000
ATCACTCATATTGCGTTTGTCCTTATCTTGCAACTTTAGATGTGGTGTCCATCGCGCAGAATCATTTAATTGTTTTCT
800o
Q VP N NNE S L N R FND I I Q S L P A RT ACTCCTTCTT=rATTTTCATGATTGCAATGCCAATATAACAAACTTAACCGTTCATGACTCATCAAACCTGCTGCAGA
9000
R L T I C S L C L L D N I S T Q L L R F L I L N A N S P N I I A V TAGGTGACATAGCTCTTAGTCTCTGACAAATCTCACCAACACTAGGTTCTATCCTAATCCACTCTCCATATTTAGGG
10000
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192 Q AI L Y L D LKXT Q AY TTTGAATCGCAGCTATCTATATCTGATCTTTACA1400
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259 1600
I V QD F NS L TQ S YEV NA QF IREZ L L D Y CN K NHMGL I AAGACGTCAAGTTTTACTTCTGCCCGAGCACGATGGCTCATTTTCAGGRGTGCTGATTTTGAKTAAAAATGGTCTAT
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226
TACTAACTTAATTTCCCATCAATCTGCTGKTAT1500
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126
12000
CTCCTTACGTCTTTCAGACTGGCCCTGTTTAGGTTCCCACTCCCTCCCCCTTAATTGGAGGCACGAGCTTTATATTCT I
26
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292
17000 K
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326
TATGGGCAGAAGGGTCCGGAhATCACCTTATAGACTTGATGTAACGAGTTGATCTCAATACTCTTTCT1800 FHND D Q N QP K NP
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360 1900 392
TCCAAAACCCGCTACACACATCCTTGTGAACGGGTGTGCGGCTCGAGAGCACAGCACTCTGTCCTATTCTCTC20000 N N NGS
S
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426 2100
LKRDKR
459
E N LKN RT
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GGTTGGCCCTCGTGGTCTAGATTCTAGTTTATATGTCCCGGTGGAAA,ATCACGAAAATTT2200 KA K
492
GCCCTAATGGAATTAAATCTTGAGATGGCAACCTTGCTGATATTGATAAATGACGGTACTTGAGAAATTACAGGCAA
23000
A R N WKL Q YLK K
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TCAGAGGATCAATGGCAAATAGGATATATCCATTAAGGACTTTGGCATAGACATGGACATAAGGTTTGTCCTATTAG D
GNX*
GATGGGATGTAGCGCGCTTAAATGAACAGACTTCTAATTTTCTATTGAACCACTCACTATTAAAAATACTTTGATGGGTTGTTAAC
526 24000 559 25000 562 2586
FIG. 3. Nucleotide and predicted amino acid sequences of TBF1. The DNA sequence of an SphI-Hpal fragment containing the complete TBF1 gene is shown, with the predicted amino acid sequence given above in one-letter code.
TABLE 1. Meiotic segregation of TBF1' Genepair
TBFIlxsit3 x pep4 pep4 x sit3 TBFI a
No. PD
Tetrad analysis No. NPD No./IT
D (cM)
32
40
0
71
83
0
28
12.6
28
3
80
44
TBFJ mapping data derived from a cross between AMAT
sit3::HIS3 and
AMATa pep4::LEU2 TBFJ:: URA3. PD, parental ditype; NPD, nonparental ditype; T, tetratype. Linkage values were derived by using the Perkins equation D (in centimorgans [cM]) = 50[Tr asci + 6(NPD asci)J/total asci.
after 3 days of incubation at 30'C. A 2.5-kbp genomic fragment containing the entire TBFJ coding region is able to complement the URA3 insertion mutation when present on a single-copy centromere plasmid (data not shown). TBF1 encodes the predominant (TTAGGG).-binding activity in yeast cells, TBFa. We next sought to identify the TBF1 protein from yeast extracts. Because TBFJ is essential for viability, biochemical analysis of cells lacking the protein is not possible. Consequently, we chose to immunologically tag the protein by incorporating sequences encoding an epitope from influenza virus HAl (13) into the N terminus of the TBFJ coding region (see Materials and Methods). The epitope-tagged protein was functional in vivo because a
YEAST TTAGGG REPEAT-BINDING FACTOR GENE
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SphI
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FIG. 4. Evidence that the TBFI gene is essential for growth. (A) Schematic representation of URA3 insertion into the TBFJ coding region. The TBF1 coding region is indicated by the solid bar. The URA3 gene was inserted at the XbaI site. (B) Southern blot of diploid strain W303 (lane 1) and three Ura+ transformants (lanes 2 to 4). Total genomic DNAs were digested with SphI and PvuII. The blot was hybridized with the 800-bp ORF from the 15.4 phage clone (EcoRI-HpaI fragment). The lower band (2.3 kb) corresponds to the wild-type SphI-PvuII fragment of TBF1, whereas the middle band (3.4 kb) in lanes 2 to 4 corresponds to the URA3 disruption allele. The upper band in all four lanes corresponds to sequences downstream from the PvuII site. Size markers (lane M) are from BstEIIdigested DNA. (C) Tetrad dissection plate of diploid transformant 1. Tetrads were dissected onto YPD agar and incubated at 30°C for 3 days. The dissection plate was then replica plated to synthetic medium lacking uracil. None of the spore colonies grows on media lacking uracil (data not shown).
plasmid containing this altered gene complements a null mutation in TBFI (data not shown). Extracts were prepared from a strain containing the epitope-tagged TBFI gene and fractionated on heparinagarose or phosphocellulose columns. Aliquots from column fractions were assessed for binding activity in an electrophoretic mobility shift assay using a 3TAGGG multimer probe. As shown in Fig. 5A, the predominant TTAGGGbinding activity elutes between 230 and 270 mM ammonium sulfate from a heparin-agarose column. A similar profile was obtained by phosphocellulose chromatography (data not shown). Aliquots from heparin-agarose column fractions were concentrated by precipitation, separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred to nitrocellulose, and probed with an HAl monoclonal antibody. The antibody reacts with a single polypeptide at approximately 65 kDa (the predicted molecular mass of TBF1) that is not present in cells lacking the tagged version
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of the gene. The elution profile of this polypeptide corresponds exactly to that of the TTAGGG-binding activity (compare Fig. 5A and B; in earlier fractions not shown in Fig. 513, the protein level also corresponds closely to that estimated from DNA binding measurements). Moreover, the mobilities of complexes produced with these column fractions are identical to those formed with protein synthesized in vitro from the full-length TBFI gene. Complexes of identical mobility were formed on TTAGGG probes containing either 6, 17, or 50 tandem repeats [Fig. SC; shown is (TTAGGG)17]. Thus, the predominant TTAGGG-binding activity present in these extracts is encoded by the TBFI gene. The data presented above strongly suggest that TBFI encodes a protein described recently by Liu and Tye (18), which they called TBFa. Both proteins were identified by binding to TTAGGG multimers and appear to be the predominant activity in yeast extracts with this property. To confirm the identity of TBF1 and TBFa, we studied the binding of TBF1 to an authentic yeast telomere junction sequence previously shown to contain TBFct-binding sites (18). Two copies of the sequence TTAGGG (separated by 5 bp) occur within this probe and are protected from DNase I digestion in the presence of TBFa. We used a subclone of the same telomere-derived sequences to demonstrate the binding of TBF1 to TTAGGG sequences present on this probe. TBF1 synthesized in vitro formed complexes with the telomere-derived probe that were competed for specifically by unlabeled TTAGGG multimers (Fig. 6A; Fig. 6B, lanes 5 to 7). This result indicates that TBF1 binds specifically to the yeast telomere DNA and suggests that binding involves the T[AGGG motifs within this probe. Binding of proteins in fraction 45 from the heparin-agarose column to the telomere probe gave a more complex pattern of bands than did binding of in vitro-synthesized TBF1 (Fig. 6B). This result was not unexpected, since the fraction is contaminated with RAP1 (data not shown) and there are many binding sites for RAP1 on this telomere junction fragment (Fig. 6A, lane 4). However, one of the bands produced by the heparin-agarose fraction is specifically competed for by TTAGGG-containing competitor DNA (Fig. 6B, lanes 9 and 10), suggesting that it represents a complex containing TBF1 (TBFa), perhaps together with RAP1. Lower-mobility complexes not competed for by TTAGGG may be formed primarily by RAP1, since they are similar in mobility to those produced by in vitro-made RAP1 (Fig. 6A, lane 4). In addition, competition with an unlabeled RAPl-binding site (TEF2) eliminates the slowest-migrating complexes (Fig. 6B, lanes 11 and 12). Taken together, these results indicate that the TBF1-containing heparin-agarose fraction contains the major TTAGGG-binding activity in yeast cells as well as a yeast telomere junction-binding protein and are thus consistent with the idea that TBF1 is identical to the previously described TBFa protein (see below). DISCUSSION We have used a molecular approach to identify and clone a gene (TBFI) encoding a yeast protein that binds to multimers of the sequence TTAGGG. This sequence element, which is the distal repeat in all vertebrate telomeres, occurs at the junction of subtelomeric Y'. and X elements of yeast telomeres. The TBFI gene was identified by probing a yeast expression library in a filter binding assay using a radiolabeled multimer of TIAGGG. We do not know whether a mammalian homolog of TBFI exists. The TTAGGG multi-
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BRIGATI ET AL.
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A --L FT 30 35 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 57 60
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A
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mer probe failed to identify a binding protein from human cDNA libraries, and hybridization experiments with the yeast TBFJ gene have yet to identify a homolog. The existence of a mammalian TTAGGG repeat-binding protein, with an apparent molecular mass of approximately 50 kDa, has recently been demonstrated (33). Determination of the possible relationship of this protein or a Physarum TIAGGG repeat-binding protein (12) to TBF1 may have to await the cloning of their respective genes. It is likely that TBFI encodes the TBFot protein described previously (18). TBFt was identified in phosphocellulosefractionated extracts by its ability to form protein-DNA complexes with a multimer of TTAGGG. In a footprint assay, TBFa was shown to bind specifically to an authentic yeast telomere in a region containing two copies of the sequence TIAGGG separated by 5 bp. TBFo is distinct from RAP1, a protein that binds to sequences within the irregular terminal repeat sequence TG1 3. The assertion that TBF1 encodes the TBFa protein described by Liu and Tye (18) is based on several criteria. First, full-length TBF1 produced in vitro from the cloned gene yields protein-DNA complexes with TTAGGG multimer probes that have mobilities identical to those obtained with crude or partially fractionated yeast extracts. (TBFa is the predominant TTAGGG-binding protein in yeast extracts [18].) To more rigorously confirm the identity of TBF1 and TBFa, we made yeast strains containing a functional TBF1 protein marked by the insertion of an influenza virus antigen near its N terminus. Chromatographic analysis of extracts from these strains shows that the epitope-tagged TBF1 coelutes from heparinagarose columns with the predominant TTAGGG-binding activity. (Coelution was also observed on phosphocellulose columns [data not shown].) TBF1 chromatographic properties are very similar to those described for TBFa with respect to both the salt concentration at which it elutes from phosphocellulose columns and its position relative to other known DNA-binding proteins. In addition, the molecular mass of TBFt (65,000 kDa) is similar to the predicted size of TBF1 (63,000 kDa). Furthermore, TBF1 produced in vitro binds specifically to the yeast telomere probe shown to be
recognized by TBFo. This binding is competed for specifically with TTAGGG-containing competitor DNA as would be expected from the TBFa footprint data (18). Finally, we have recently constructed a strain containing an internal in-frame deletion of TBF1 as its only functional copy of the gene. Extracts prepared from this strain yield complexes
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FIG. 5. Evidence that TBF1 protein is the predominant (TTAG
GG)n-binding activity in yeast extracts. (A) Electrophoretic mobility shift assay of heparin-agaiose column fractions. Extracts were prepared from strain CYS40, which contains an epitope-tagged allele of TBF1 (see Materials and Methods). The probe is (TTAG GG)17. Lanes: -, reaction in the absence of extract; L, unfractionated extract loaded onto the column; FT, flowthrough from the --
column; 30 to 60, column fractions from the portion of the gradient ranging from 200 to 300 mM (NH4)2SO4; C, the complex formed by addition of extract; FP, free probe. (B) Western blot of heparinagarose column fractions. Aliquots from column fractions in panel A were separated on an SDS-10% polyacrylamide gel and transferred to nitrocellulose. The filter was reacted with antibody to the HAl epitope tag and developed with anti-mouse immunoglobulin G conjugated to alkaline phosphatase. Lanes: M, molecular mass standards (indicated in kilodaltons); L, unfractionated extract loaded onto the column; FT, column flowthrough; 45 to 60, column fractions with maximal binding activity for the TTAGGG probe as determined for panel A. (C) In vitro-synthesized TBF1 and heparinagarose fractions produce DNA complexes of identical mobility. The probe used in these electrophoretic mobility shift assays is (TTAGGG)17. Lanes: 1, reaction minus extract; 2, in vitro synthesis reaction minus RNA; 3, in vitro-synthesized TBF1; 4 to 6, fraction 45 from the heparin-agarose column (1, 2, and 5 ,ul of extract, respectively). Arrow indicates the position of the predominant TBF1-DNA complex.
YEAST TTAGGG REPEAT-BINDING FACTOR GENE
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A 12 3 4 5 6
B 1
2 3 4 5 6 7 8 9 tO 11 12
FIG. 6. Complex formation by TBF1 with a yeast telomere junction sequence. (A) Binding of in vitro-synthesized TBF1, RAP1, and fractionated yeast extract to a yeast telomeric junction sequence. The probe is a 180-bp telomere-derived fragment from yeast chromosome III. Lanes: 1, reaction minus extract; 2, in vitro synthesis reaction minus RNA; 3, in vitro-synthesized TBF1; 4, in vitro-synthesized RAP1; 5 and 6, heparin-agarose column fraction 45 (2 and 5 Ill, respectively). (B) Binding competition assays with the telomere junction probe. Lanes: 1, reaction minus extract; 2 to 4, heparin-agarose column fraction 45 (1, 2, and 5 ,ul, respectively); 5 to 7, in vitro-synthesized TBF1 with vector (pUC13) alone as competitor (lane 5) or pUC13-(TTAGGG)17 in onefold (lane 6) or twofold (lane 7) molar excess; 8 to 12, fraction 45 and pUC13 DNA (lane 8), 1x pUC13-(TTAGGG)17 (lane 9), 2x pUC13-(TTAGGG)17 (lane 10), 1x pUC13-TEF2 (lane 11), and 2x pUC13-TEF2 (lane 12). Arrow indicates the complex competed for by 1TAGGG multimers.
with a TTAGGG repeat probe that are of higher mobility than those from an isogenic wild-type strain. This result provides further evidence that TBF1 encodes the major TTAGGG repeat-binding activity (TBFa). Definitive proof of the identity of TBF1 and TBFot will have to await either further purification and characterization of TBF1 or peptide sequence data from purified TBFot. Although TBF1 shows no strong similarity to any protein sequences in currently available data bases, a motif recognition program did reveal a perfect match to the A consensus sequence, (A/G)X4GK(S/T), found in ATP- and GTP-binding proteins, beginning at amino acid 295 in TBF1. However, we were unable to find similarities to the B element shared by a number of ATP-binding proteins (16), nor could we find any other similarities to members of the GTPase family of proteins (7). Determination of the significance, if any, of the A consensus match will have to await further genetic and biochemical analysis of TBF1. Initial experiments, in which in-frame deletions within this region of TBFI have been shown to be inviable, suggest that the region is important for function (2a). The identification of TBF1 as a fusion protein with P-galactosidase and the C terminus of the predicted protein indicates that the TBF1 DNA-binding domain must be contained within its last -250 amino acids. This sequence does not appear to contain any of the known DNA-binding motifs (e.g., helix-turn-helix, Zn finger, or b-ZIP), suggesting that the TBF1 DNA-binding domain may belong to a novel class. Although TBF1 is required for growth, its precise func-
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tion(s) is unclear. The occurrence of TTAGGG sequences within telomere-proximal elements, at least one of which is a TBF1-binding site in vitro, suggests that the protein may play a role in telomere structure and/or function (18). This interpretation must be viewed with some caution. Telomeres that do not contain either Y' or X sequences (in which putative TBF1-binding sites are found) can be generated in yeast cells, indicating that TBF1 binding may not be required for telomere function. However, the DNA sequences of such novel telomeres have not been determined, and it is possible that they contain TTAGGG or related sequences proximal to the TG1_3 terminal repeats. In addition, we have found no evidence in our initial studies of the cloned TBF1 gene for a role at telomeres. Internal in-frame deletion mutations with severe growth defects and temperature-sensitive lethal mutants grown at semipermissive temperatures both fail to display any detectable change in telomere lengths (2a), a phenotype common to several genes involved in telomere structure or maintenance, such as RAP1 (11, 22, 29) and EST1 (21). Whatever function TBF1 fulfills at the telomere, it is not clear why this gene is essential. Telomere integrity is essential for chromosome replication and maintenance, and it is possible that TBF1 contributes to telomere structure in such a way that it is required every cell division. Such a requirement could explain the immediate lethality of spore colonies lacking a functional TBFI gene. However, it is worth noting that mutations in several other genes with either important or essential telomere functions have a pronounced phenotype lag. Spores containing the estl mutation, for example, require approximately 40 generations of growth before lethality is observed (21), and cells containing raplts mutations display a progressive loss of telomere sequences over 25 to 100 generations when grown at semipermissive temperatures
(22). We presently favor the model that TBF1 has other, as yet undefined essential functions. For example, it is conceivable that TBF1 plays an essential role in the regulation of gene expression or chromosome structure. In this regard, it is interesting that two other proteins, RAP1 and CP1, function at essential chromosomal elements (telomeres and centromeres, respectively) yet also play important roles as transcriptional regulatory proteins (2, 8, 9, 23). Crude estimates of the concentration of TBF1 in the cell, based on comparisons of protein-DNA complex yields with those of RAP1, suggest that TBF1 may be a relatively abundant
sequence-specific DNA-binding protein. Perhaps important insights into the function of TBF1 will come from identification of other chromosomal binding sites for the protein. In this regard, it is important to keep in mind that we and others (18) have identified TBF1 (TBFa) by specific searches with TTAGGG probes. A consensus binding site for TBF1, which might indicate the locations of other chromosomal binding sites, has yet to be determined. In addition, study of conditional-lethal mutants of TBFI might provide important clues about its role in the cell. ACKNOWLEDGMENTS We thank L. Van der Ploeg, T. de Lange, M. Carlson, B. Laurent, K. Arndt, C. Roman, P. Sorger, and J. Berman for gifts of strains and plasmid DNAs; T. de Lange, B. Laurent, and members of the Shore laboratory for helpful discussions; and T. de Lange for helpful comments on the manuscript. C.B. thanks F. Campelli and L.
Panzeri (Department of Genetics, Universita Statale, Milan, Italy) for help with experiments done in Genoa. This work was supported by grants from the NIH (GM40094), the American Cancer Society (JFRA-231 and MV-534), the Searle
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Scholars Fund/Chicago Community Trust, and the Irma T. Hirschl Charitable Trust to D.S. and by ACS Institutional Research Grant IRG-177A to the Comprehensive Cancer Center at Columbia University. REFERENCES 1. Ammerer, G., C. P. Hunter, J. H. Rothman, G. C. Saari, L. A. Valls, and T. H. Stevens. 1986. PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors. Mol. Cell. Biol. 6:24902499. 2. Baker, R. E., and D. C. Masison. 1990. Isolation of the gene encoding the Saccharomyces cerevisiae centromere-binding protein CP1. Mol. Cell. Biol. 10:2458-2467. 2a.Balderes, D., S. Kurtz, and D. Shore. Unpublished data. 3. Bankier, A. T., K. M. Weston, and B. G. Barrell. 1987. Random cloning and sequencing by the M13/dideoxynucleotide chain termination method. Methods Enzymol. 155:51-93. 4. Beggs, J. D. 1978. Transformation of yeast by a replicating hybrid plasmid. Nature (London) 285:185-187. 5. Berman, J., C. Y. Tachibana, and B.-K. Tye. 1986. Identification of a telomere-binding activity from yeast. Proc. Natl. Acad. Sci. USA 83:3713-3717. 6. Blackburn, E. H. 1991. Structure and function of telomeres. Nature (London) 350:569-573. 7. Bourne, H. R., D. A. Sanders, and F. McCormiclk 1991. The GTPase superfamily: conserved structure and molecular mechanism. Nature (London) 349:117-127. 8. Buchman, A. R., N. F. Lue, and R. D. Kornberg. 1988. Connections between transcriptional activators, silencers, and telomeres as revealed by functional analysis of a yeast DNA-binding protein. Mol. Cell. Biol. 8:5086-5099. 9. Cai, M., and R. W. Davis. 1990. Yeast centromere binding protein CBF1, of the helix-loop-helix protein family, is required for chromosome stability and methionine prototrophy. Cell 61:437-446. 10. Chan, C. S. M., and B.-K. Tye. 1983. A family of Saccharomyces cerevisiae repetitive autonomously replicating sequences that have very similar genomic environments. J. Mol. Biol. 168:505-523. 11. Conrad, M. N., J. H. Wright, A. J. Wolf, and V. A. Zakian. 1990. RAP1 protein interacts with yeast telomeres in vivo: overproduction alters telomere structure and decreases chromosome stability. Cell 63:739-750. 12. Coren, J. S., E. M. Epstein, and V. M. Vogt. 1991. Characterization of a telomere-binding protein from Physarum polycephalum. Mol. Cell. Biol. 11:2282-2290. 13. Field, J., J.-I. Nikawa, D. Broek, B. MacDonald, L. Rodgers, I. A. Wilson, R. A. Lerner, and M. Wigler. 1988. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol. Cell. Biol. 8:2159-2165. 13a.Goebl, M. Personal communication. 14. Gottschling, D. E., and V. A. Zakian. 1986. Telomere proteins: specific recognition and protection of the natural termini of Oxytricha macronuclear DNA. Cell 47:195-205. 15. Gray, J. T., D. W. Celander, C. M. Price, and T. R. Cech. 1991. Cloning and expression of genes for the Oxytricha telomerebinding protein: specific subunit interactions in the telomeric complex. Cell 67:807-814.
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