A human telomeric protein

Science

Volumn 270, Issue 5242, 1995, Pages 1663-1667

  Chong, Laura ^b   Van Steensel, Bas ^b   Broccoli, Dominique ^b   Erdjument Bromage, Hediye ^b   Hanish, John ^b   Tempst, Paul ^b   De Lange, Titia ^b  

^a ROCKEFELLER UNIVERSITY   (United States)

^b NONE

Author keywords

[No Author keywords available]

Indexed keywords

CHROMOSOME PROTEIN; DNA BINDING PROTEIN; NUCLEOPROTEIN;

AMINO TERMINAL SEQUENCE; ARTICLE; CHROMOSOME; HUMAN; HUMAN CELL; HUMAN TISSUE; IMMUNOFLUORESCENCE TEST; MOLECULAR CLONING; PRIORITY JOURNAL; PROTEIN ANALYSIS;

EID: 0029586089     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.270.5242.1663     Document Type: Article

References (61)

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7. T. de Lange, Semin. Cell Biol., in press.
8. 27. Human TRF eluted from this column at 0.5 M KCl (Fig. 1). The active fraction was supplied with β-casein and fractionated again by batch elution on nonspecific and specific (TTAGGG) DNA-containing resins. The final sample was precipitated with 20% trichloroacetic acid and 0.015% deoxycholate, fractionated on SDS-PAGE, and transferred to nitrocellulose. The sequenced hTRF preparation was composed of two polypeptides of ∼60 kD; the larger protein was present at subpicomolar amounts and did not yield sequence information. Its relation to hTRF remains to be determined.
9. L. Chong and T. de Lange, data not shown.
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11. Peptides were generated from the nitrocellulose-bound hTRF protein by in situ tryptic digestion [H. Erdjument-Bromage et al., Protein Sci. 3, 2435 (1994)] and fractionated by reversed-phase high-performance liquid chromatography [C. Elicone et al., J. Chromatogr. 676, 121 (1994)], and selected peak fractions were analyzed by a combination of automated Edman degradation and laser-desorption mass spectrometry [P. Tempst, S. Geromanos, C. Elicone, H. Erdjument-Bromage, Methods 6, 248 (1994)]. Peptide sequences were compared to entries in various sequence databases with the use of the National Center for Biotechnology Information (NCBI) BLAST program (S. F. Altschul et al., J. Mol. Biol. 215, 403 (1990)], Average isotopic masses of predicted peptides were summed from the cDNA-derived sequence with the use of ProComp software (version 1.2; P. C. Andrews, University of Michigan, Ann Arbor, MI).
12. Peptides were generated from the nitrocellulose-bound hTRF protein by in situ tryptic digestion [H. Erdjument-Bromage et al., Protein Sci. 3, 2435 (1994)] and fractionated by reversed-phase high-performance liquid chromatography [C. Elicone et al., J. Chromatogr. 676, 121 (1994)], and selected peak fractions were analyzed by a combination of automated Edman degradation and laser-desorption mass spectrometry [P. Tempst, S. Geromanos, C. Elicone, H. Erdjument-Bromage, Methods 6, 248 (1994)]. Peptide sequences were compared to entries in various sequence databases with the use of the National Center for Biotechnology Information (NCBI) BLAST program (S. F. Altschul et al., J. Mol. Biol. 215, 403 (1990)], Average isotopic masses of predicted peptides were summed from the cDNA-derived sequence with the use of ProComp software (version 1.2; P. C. Andrews, University of Michigan, Ann Arbor, MI).
13. Peptides were generated from the nitrocellulose-bound hTRF protein by in situ tryptic digestion [H. Erdjument-Bromage et al., Protein Sci. 3, 2435 (1994)] and fractionated by reversed-phase high-performance liquid chromatography [C. Elicone et al., J. Chromatogr. 676, 121 (1994)], and selected peak fractions were analyzed by a combination of automated Edman degradation and laser-desorption mass spectrometry [P. Tempst, S. Geromanos, C. Elicone, H. Erdjument-Bromage, Methods 6, 248 (1994)]. Peptide sequences were compared to entries in various sequence databases with the use of the National Center for Biotechnology Information (NCBI) BLAST program (S. F. Altschul et al., J. Mol. Biol. 215, 403 (1990)], Average isotopic masses of predicted peptides were summed from the cDNA-derived sequence with the use of ProComp software (version 1.2; P. C. Andrews, University of Michigan, Ann Arbor, MI).
14. Peptides were generated from the nitrocellulose-bound hTRF protein by in situ tryptic digestion [H. Erdjument-Bromage et al., Protein Sci. 3, 2435 (1994)] and fractionated by reversed-phase high-performance liquid chromatography [C. Elicone et al., J. Chromatogr. 676, 121 (1994)], and selected peak fractions were analyzed by a combination of automated Edman degradation and laser-desorption mass spectrometry [P. Tempst, S. Geromanos, C. Elicone, H. Erdjument-Bromage, Methods 6, 248 (1994)]. Peptide sequences were compared to entries in various sequence databases with the use of the National Center for Biotechnology Information (NCBI) BLAST program (S. F. Altschul et al., J. Mol. Biol. 215, 403 (1990)], Average isotopic masses of predicted peptides were summed from the cDNA-derived sequence with the use of ProComp software (version 1.2; P. C. Andrews, University of Michigan, Ann Arbor, MI).
15. TRF cDNAs were isolated from a HeLa cDNA library (Stratagene) by screening with a 33-nucleotide DNA probe that represents the region of homology of T29 peptide sequence to an anonymous cDNA in Gen. Bank (accession number Z19923). Three overlapping cDNAs, together spanning 2684 bp, were sequenced (GenBank accession number U40705). The sequence contains a single long ORF of 439 amino acids starting with an AACATGG initiating codon, which is favorable for translation initiation [M. Kozak, Nucleic Acids Res. 15, 8125 (1987)]. The 3′ end of the TRF cDNA contains an AATAAA polyadenylate [poly(A)] addition signal 18 bp upstream of a poly(A) stretch. The TRF ORF amino acid sequence was compared to proteins in the nonredundant database with the use of the program BLASTP (10). The nuclear targeting signals are of the nucleoplasmin type [J. Robbins, S. M. Dilworth, R. A. Laskey, C. Dingwall, Cell 64, 615 (1991)]. Alignment to the Myb proteins was achieved with the program Clustalw 1.5; a SeqVu 1.0.1 display and Rao rules for conservative amino acid changes were used.
16. TRF cDNAs were isolated from a HeLa cDNA library (Stratagene) by screening with a 33-nucleotide DNA probe that represents the region of homology of T29 peptide sequence to an anonymous cDNA in Gen. Bank (accession number Z19923). Three overlapping cDNAs, together spanning 2684 bp, were sequenced (GenBank accession number U40705). The sequence contains a single long ORF of 439 amino acids starting with an AACATGG initiating codon, which is favorable for translation initiation [M. Kozak, Nucleic Acids Res. 15, 8125 (1987)]. The 3′ end of the TRF cDNA contains an AATAAA polyadenylate [poly(A)] addition signal 18 bp upstream of a poly(A) stretch. The TRF ORF amino acid sequence was compared to proteins in the nonredundant database with the use of the program BLASTP (10). The nuclear targeting signals are of the nucleoplasmin type [J. Robbins, S. M. Dilworth, R. A. Laskey, C. Dingwall, Cell 64, 615 (1991)]. Alignment to the Myb proteins was achieved with the program Clustalw 1.5; a SeqVu 1.0.1 display and Rao rules for conservative amino acid changes were used.
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28. 2, and sedimented onto Alcian Blue-treated cover slips for 15 s at 3000 rpm in a Sorvall RT6000B tabletop centrifuge. Chromosome spreads were immediately fixed in 2% formaldehyde in phosphate-buffered saline, permeabilized in 0.5% Nonidet P-40 in phosphate-buffered saline, and labeled with mouse mAb 12CA5 followed by FITC-labeled goat antibody to mouse IgG (Jackson ImmunoResearch Labs). DNA was stained with DAPI (0.2 μg/ml). Images of interphase nuclei were obtained with a Zeiss Axioplan microscope equipped with a Kodak DCS-200 digital camera. Images were noise-filtered with a 3 × 3 median filter, corrected for background with nontransfected cells as a reference, and merged to obtain triple labeling images using Adobe Photoshop. Chromosome spreads were photographed on Kodak Gold II 400 ISO film and converted to digital images with a Nikon slide scanner, after which FITC and DAPI images were corrected for background and superimposed.
29. 2, and sedimented onto Alcian Blue-treated cover slips for 15 s at 3000 rpm in a Sorvall RT6000B tabletop centrifuge. Chromosome spreads were immediately fixed in 2% formaldehyde in phosphate-buffered saline, permeabilized in 0.5% Nonidet P-40 in phosphate-buffered saline, and labeled with mouse mAb 12CA5 followed by FITC-labeled goat antibody to mouse IgG (Jackson ImmunoResearch Labs). DNA was stained with DAPI (0.2 μg/ml). Images of interphase nuclei were obtained with a Zeiss Axioplan microscope equipped with a Kodak DCS-200 digital camera. Images were noise-filtered with a 3 × 3 median filter, corrected for background with nontransfected cells as a reference, and merged to obtain triple labeling images using Adobe Photoshop. Chromosome spreads were photographed on Kodak Gold II 400 ISO film and converted to digital images with a Nikon slide scanner, after which FITC and DAPI images were corrected for background and superimposed.
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60. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
61. We thank B. Stillman and S. Bell for information about ORC1 and N. Andrews, R. Benezra, A. Blanchi, R. Bose, R. Damell, J. Feng, R. Lang, M. Lui, A. Lustig, N. Segil, O. Sibon, S. Smith, and J. Young for their contributions and advice. Supported by grants from the Lucille P. Markey Charitable Trust and NIH (GM49046) to T.d.L, grants from the National Cancer Institute (P30 CA08748-29) and NSF (BIR-9420123) to P.T., and a Irma T. Hirschl Career Scientist Award to P.T. B.v.S. is the recipient of a Human Frontier Science Program postdoctoral fellowship.