Tankyrase, a poly(ADP-ribose) polymerase at human telomeres

Science

Volumn 282, Issue 5393, 1998, Pages 1484-1487

  Smith, Susan ^a   Giriat, Izabela ^a   Schmitt, Anja ^a,b   De Lange, Titia ^a  

^a ROCKEFELLER UNIVERSITY   (United States)

^b EUROPEAN MOLECULAR BIOLOGY LABORATORY   (Germany)

Author keywords

[No Author keywords available]

Indexed keywords

ADENOSINE DIPHOSPHATE RIBOSE; ANKYRIN; TANKYRASE; UNCLASSIFIED DRUG;

AMINO TERMINAL SEQUENCE; ANIMAL CELL; ARTICLE; HUMAN; HUMAN CELL; NONHUMAN; NORTHERN BLOTTING; NUCLEOTIDE SEQUENCE; PRIORITY JOURNAL; PROTEIN ANALYSIS; PROTEIN BINDING; PROTEIN DOMAIN; PROTEIN INTERACTION; PROTEIN LOCALIZATION; PROTEIN STRUCTURE; SEQUENCE ANALYSIS; SEQUENCE HOMOLOGY; TELOMERE;

ANIMALIA;

EID: 0032553473     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5393.1484     Document Type: Article

References (33)

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9. A full-length tankyrase cDNA TT20 containing a 4134-nudeotide (nt) insert was isolated as follows. First, two overlapping cDNAs, 32 and 21, encompassing 8901 nt encoding amino acids 235 to 1327 were isolated from a HeLa cDNA library that had been probed with a polymerase chain reaction product (representing amino acids 973 to 1163) made from TR1L-4. The 5′ end sequence was extended by rapid amplification of cDNA ends (RACE) and then used to screen a human testis library (Stratagene, La Jolla, CA). Analysis of two other testis library isolates, TT7 and TT9 (GenBank accession numbers AF082558 and AF082559), indicated that they were similar to TT20 along their length, but each had an insertion of ≃100 nt [TT7, insertion after amino acid 640 (in ANK repeat 14), and TT9, insertion after amino acid 881 (in ANK repeat 21)]. Both insertions contained stop codons resulting in truncated proteins, as confirmed by in vitro translation. It is not known if these truncated proteins are expressed in vivo.
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21. The tankyrase cDNA TR1L-4 hybridized to three mRNAs of ≃6, 8, and 10 kb with the same ubiquitous expression pattern. Testis express two additional transcripts, a 2.5-kb species and an abundant 4.5-kb species. The 4.2-kb TT20 tankyrase cDNA isolated from the testis library is large enough to represent the 4.5-kb transcript, suggesting that this cDNA is nearly full length. The larger transcripts present in most tissues may be due to longer 3′ untranslated regions (UTRs) because sequence analysis of a tankyrase cDNA from a HeLa cell library revealed the same sequence as the full-length 4.5-kb cDNA, but with an additional 5 kb of 3′ UTR. When the most 3′ 1 kb of this cDNA was used as a probe in a Northern blot, it hybridized exclusively to the largest (10-kb) transcript.
22. For immunoblots, HeLa cells were suspended directly in Laemmli loading buffer and loaded at ∼50,000 cells per lane. Crude nuclei were isolated from rat testis (after hypotonic lysis), extracted with 0.4 M KCl, pelleted, and suspended in Laemmli buffer. In vitro-translated tankyrase was generated with a coupled transcription-translation reticulocyte lysate system (Promega). A 1-μg amount of TT20 was incubated with T3 RNA polymerase under standard conditions, and 10% of the reaction was loaded per lane. Protein samples were fractionated on SDS-polyacrylamide gels, transferred to nitrocellulose, and blocked in 5% milk in phosphate-buffered saline (PBS) containing 0.1% Tween-20. Antibody incubations were in 1% milk in PBS containing 0.1% Tween-20. Blots were first incubated with rabbit antibody to tankyrase (4 μg/ml) or rabbit preimmune serum (1:500) and then with horseradish peroxidase-conjugated donkey antibody to rabbit immunoglobulin G (IgG (Amersham) (1:2500). Bound antibody was detected by enhanced chemiluminescence (Amersham). For generation of antibody to tankyrase (antitankyrase), the Ank2 fragment representing amino acids 973 to 1149 of tankyrase was fused to vector pET-22b(+) (Novagen) and expressed in Escherichia coli. The protein was isolated in inclusion bodies and used to immunize one rabbit. The resulting immune serum, rabbit anti-tankyrase 465, was affinity purified against Ank2 protein coupled to CnBr-activated Sepharose (Sigma).
23. Tankyrase protein was detected by protein immunoblot analysis in the following human cell lines: 293, transformed embryonic kidney cells; IMR90 and WI38, primary lung fibroblasts; WI38 VA13/2RA, immortalized lung fibroblasts; GM847, SV-40-immortalized fibroblasts; Daudi and Raji, lymphoma; HT1080, fibrosarcoma; and MCF7, breast adenocarcinoma. Several of these cell lines expressed only the larger set of tankyrase mRNAs (6 to 10 kb), indicating that the 142-kD polypeptide can be expressed from one of these transcripts.
24. 2, and sedimented onto cover slips for 15 s at 3000 rpm in a Sorvall RT6000B centrifuge. Chromosomes were swollen for 15 min in 25% PBS, fixed in 3.7% formaldehyde in 25% PBS for 10 min, and permeabilized with 0.5% NP-40 in 25% PBS for 10 min. Samples were blocked with 1% bovine serum albumin (BSA) in PBS, and then incubated with rabbit anti-tankyrase (1 μg/ml) and a mouse polyclonal serum to full-length baculovirus-derived TRF1 (1:10,000). Primary antibodies were detected with fluorescein isothiocyanate (FITC)-conjugated donkey antibody to rabbit IgG and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated donkey antibody to mouse IgG (1:100) (Jackson Laboratories). DNA was stained with 4,6-diamino-2-phenytindole (DAPI) (0.2 μg/ml). Images were obtained with a Zeiss Axioplan 2 microscope with a Photometrics charge-coupled device camera and then processed and merged with Adobe Photoshop. Immunolocalization analysis of cycling HeLa cells indicates additional subcellular locations for tankyrase (S. Smith and T. de Lange, in preparation).
25. + was omitted. Samples were immunoblotted (14) and probed with 10H, a mouse monoclonal antibody to poly(ADP-ribose) (1:250) [H. Kawamitsu et al., Biochemistry 23, 3771 (1984)] followed by horseradish peroxidase-conjugated sheep antibody to mouse IgG (Amersham).
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33. We thank an anonymous reviewer, T. Meier, and members of the de Lange laboratory for comments on this manuscript; J. Lue for technical assistance; and G. de Murcia for a gift of 10H antibody. T.d.L. is a recipient of the Burroughs Wellcome Fund Scholar Award in Toxicology. S.S. is a Leukemia Society of America Special Fellow. A.S. was a fellow of the German National Science Foundation. Supported by grants from the NIH (GM49046 and CA76027), the Rita Allen Foundation, and the Sandoz Foundation.