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Volumn 285, Issue 5426, 1999, Pages 418-421

Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis

Author keywords

[No Author keywords available]

Indexed keywords

CELL PROTEIN; COMPLEMENTARY DNA; CYCLIN DEPENDENT KINASE; CYCLINE; MUTANT PROTEIN; SECURIN; UNCLASSIFIED DRUG;

EID: 0033575347     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5426.418     Document Type: Article
Times cited : (700)

References (35)
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    • note
    • 6 fusion. A 269-amino acid recombinant protein was expressed in Escherichia coli according to the protocol provided by the manufacture (Novagen). Recombinant protein was purified up to 90% with Ni-nitrilotriacetic acid (NTA) beads (Qiagen). Full-length hSecurin (or PTTG) was isolated with primers 5′-ATGGCTACTCTGATCTATGTTGATAAGG-3′ and 5′-TTAAATATCTATGTCACAGCAAACAGGTG-3′. The PCR product was then cloned into a modified version of pCS2 for in vitro translation and transfection into mammalian cells. The same fragment was also cloned into a modified pET28a vector to express recombinant protein in E. coli. Protein more than 90% pure was obtained by affinity purification with Ni-NTA beads (Qiagen) followed by Resource Q column chromatography. Rabbit polyclonal antibodies to the 269-amino acid hESP1 fragment and full-length hSecurin (or PTTG) were made by Zymed with purified recombinant proteins as antigen. Crude sera were affinity-purified by passing them through affinity AffiGel-10 (BioRad) columns coupled with corresponding antigens. Antibodies were eluted from the affinity-columns by 100 mM glycine (pH 2.5) followed by 100 mM triethylamine (pH 11.5). Affinity-purified antibody to hESP1 (anti-hESP1) cross-reacts with Myc tag. This antibody does not bind to in vitro-translated hSecurin (24). The anti-cyclin B1 used in this study was a rabbit antibody purchased from Santa Cruz Biotechnology.
  • 15
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    • E. Harlow and D. Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988).
    • (1988) Antibodies: A Laboratory Manual
    • Harlow, E.1    Lane, D.2
  • 16
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    • note
    • 2-M (2N), and the percentage for each stage of the cell cyde was calculated.
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    • note
    • 2, 1 mM EDTA. 1 mM dithiothreitol, insulin (0.1 mg/ml), and 10% glycerol (same below); once in 300 mM KCI and 0.5% NP-40 wash buffer; and once in 500 mM KCl and 0.5% NP-40 wash buffer.
  • 25
    • 0344767043 scopus 로고    scopus 로고
    • note
    • Single-letter 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.
  • 27
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    • H. Funabiki et al., EMBO J. 16, 5977 (1997).
    • (1997) EMBO J. , vol.16 , pp. 5977
    • Funabiki, H.1
  • 33
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    • note
    • Extract prepared from nocodazole-arrested HeLa S3 celts was fractionated on a Resource Q column with a KCl concentration gradient from 100 mM to 1 M in the lysis buffer. Human ESP1 was eluted at about 250 mM KCl. Fractions containing hESP1 were pooled and loaded onto a Superdex 200 column.
  • 35
    • 0344767041 scopus 로고    scopus 로고
    • note
    • We thank C. Pfleger and S. Rankin for comments on the manuscript G. Fang, H. Yu, and other members of the Kirschner laboratory for helpful discussion and technical assistance; C. Pfleger and E. Lee for providing modified pCS2 and pET28a vectors; and Q. Zang for mammalian cell transfection. H.Z. is a fellow of The Jane Coffin Childs Memorial Fund for Medical Research. T.J.M. was supported by a Physician Post-doctoral Grant from the Howard Hughes Medical Institute and a Mentored Clinical Scientist Development Award from the National Heart, Lung, and Blood Institute. Supported by grants GM39023 and GM26875 from NIH awarded to M.W.K.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.