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Volumn 288, Issue 5474, 2000, Pages 2205-2208

A metalloprotease disintegrin that controls cell migration in Caenorhabditis elegans

Author keywords

[No Author keywords available]

Indexed keywords

DISINTEGRIN;

EID: 0034705575     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.288.5474.2205     Document Type: Article
Times cited : (101)

References (24)
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    • Mutants were isolated by ethylmethane sulfonate mutagenesis. The mig-17 alleles k135, k147, k167, and k176 were obtained by visual screening of mutants with ventral white-patch phenotypes.
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    • 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. X indicates any residue.
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    • To construct the mig-17::GFP fusion, a 3645-base pair (bp) Sma I-Afl III mig-17 fragment from the cosmid F57B7 was fused to a Bam HI-Spe I DNA fragment of the GFP plus unc-54 3′ untranslated region from pPD95.75 using appropriate synthetic oligonucleotides. This construct contained 1141 bp of the mig-17 upstream sequence and the 2533-bp mig-77 coding region and all introns. For constructions of unc-54p::mig-17::GFP and lag-2p::mig-17::GFP, the mig-17::GFP coding sequence was placed downstream of the unc-54 promoter from pPD52.102 and the lag-2 promoter from pRB1, respectively. Cosmid clones were co-injected at 10 to 50 μg/ml together with the pMK107 plasmid (100 μg/ml), which contains the EF1α promoter fused to GFP. Plasmid constructs were injected at 100 μg/ml with 50 μg/ml pBR322 either with or without 50 μg/ml unc-119(+) plasmid, pDP#MM016B [M. Maduro and D. Pilgrim, Genetics 141, 977 (1995)].
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    • Sonicated worm lysates were incubated with anti-GFP monoclonal antibody (Clontech) in a buffer containing 50 mM tris-HCl (pH 8.0), 100 mM NaCl, 10% glycerol, 1% Triton X-100, 5 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml pepstatin at 4°C for 3 hours. Samples were precipitated with protein A-Sepharose beads (Pharmacia). Immunoprecipitates were subjected to immunoblotting with anti-GFP antibody.
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    • In mig-17(k174); gon-1(q518) double mutants, DTCs did not migrate at all. In mig-17(k174)/mig-17(k174); gon-1(q518)/+ animals, DTC migration defects of mig-17(k174) mutants were not enhanced.
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    • note
    • We thank A. Coulson, A. Fire, J. Kimble, Y. Kohara, T. Stiernagle, and the Caenorhabditis Genetics Center for materials, and A. Antebi, E. Kipreos, M. Lamphier, and I. Mori for critical reading of the manuscript. Supported by special grants from PRESTO to K.N., and CREST, Uehara Memorial, and Advanced Research on Cancer from the Ministry of Education, Culture and Science of Japan to K.M.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.