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Volumn 285, Issue 5435, 1999, Pages 1917-1919

Constitutive activation of toll-mediated antifungal defense in serpin- deficient Drosophila

Author keywords

[No Author keywords available]

Indexed keywords

GENE PRODUCT; PEPTIDE; SERINE PROTEINASE INHIBITOR;

EID: 0033578917     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5435.1917     Document Type: Article
Times cited : (388)

References (31)
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    • A. Poltorak et al., Science 282, 2085 (1998).
    • (1998) Science , vol.282 , pp. 2085
    • Poltorak, A.1
  • 12
  • 13
    • 0345537391 scopus 로고    scopus 로고
    • in preparation. The sequence data for Spn43Aa, Spn43Ab, and Spn43Ac are available from the European Molecular Biology Laboratory and Gen-Bank under accession numbers AJ245442, AJ245443, and AJ245444, respectively
    • C. Green et al., in preparation. The sequence data for Spn43Aa, Spn43Ab, and Spn43Ac are available from the European Molecular Biology Laboratory and Gen-Bank under accession numbers AJ245442, AJ245443, and AJ245444, respectively.
    • Green, C.1
  • 14
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    • E. Levashina et al., Eur. J. Biochem. 233, 694 (1995); E. Levashina, S. Ohresser, B. Lemaitre, J. L. Imler, J. Mol. Biol. 278, 515 (1998).
    • (1995) Eur. J. Biochem. , vol.233 , pp. 694
    • Levashina, E.1
  • 16
    • 0344675140 scopus 로고    scopus 로고
    • E. Levashina and J. M. Reichhart, data not shown
    • E. Levashina and J. M. Reichhart, data not shown.
  • 20
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    • J. M. Reichhart et al., data not shown
    • J. M. Reichhart et al., data not shown.
  • 27
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    • note
    • A chimeric protein composed of glutathione S-transferase (GST) fused to the Spn43Ac gene product was produced with the use of a GST-Spn43Ac expression vector as follows: A Sac I (filled in)-Xho I 1200-bp fragment of Spn43Ac cDNA was subcloned into the Sma I and Xho I sites of the pGEX2T expression vector (Pharmacia). The GST-Spn43Ac fusion protein was expressed in Escherichia coli strain LE 392 and purified according to Pharmacia standard procedures on a glutathione Sepharose 4B column. Antibodies were obtained by inoculating the recombinant protein (100 to 200 μg for each innoculation) into a rabbit according to standard methods. Hemolymph was extracted from flies with a Nanoject apparatus (Drummond Scientific) and recovered in phosphate-buffered saline. Hemolymph extract (5 μg) was loaded on a 10% SDS-polyacrylamide gel. After SDS-polyacrylamide gel electrophoresis, proteins were blotted onto Hybond ECL nitrocellulose membranes (Amersham). Blots were incubated overnight at 4°C with a 1/5000 dilution of antiserum to GST-Spn43Ac. After washing with tris-buffered saline, the blots were incubated for 1 hour at 37°C with a 1/5000 dilution of horseradish peroxidase-conjugated donkey antibody to rabbit immunoglobulin G (Amersham). The blots were developed with the use of an ECL system (Amersham).
  • 28
    • 0344675133 scopus 로고    scopus 로고
    • note
    • Immune challenge was performed by pricking adult flies with a fine needle dipped into a concentrated culture of E. coli and Micrococcus luteus as described in (1); see also (22). Northern blotting experiments were performed as in (7); for (A) to (C), total RNA was extracted from whole flies; for (D), polyadenylated RNA was prepared as in (1). The cDNA probes used were as follows: drosomycin, diptericin, cecropin A1, rp 49, as described in (1); metchnikowin, as in (14); Spn43Ac, an Eco Rl fragment of ∼750 base pairs (bp) corresponding to the 3′ region of Spn43Ac cONA (13).
  • 29
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    • note
    • 1/CyO backgrounds. The resulting stocks were crossed with each other to yield a heteroallelic nec combination.
  • 30
    • 0345537384 scopus 로고    scopus 로고
    • note
    • Protein extracts from embryos and whole flies were prepared as in (17). Extracts (40 μg) were separated by electrophoresis on a 12.5% SDS-polyacrylamide gel, and immunoblotting was performed as in (26).
  • 31
    • 0344675130 scopus 로고    scopus 로고
    • note
    • We are grateful to R. DeLotto for providing antisera to Spaetzle, and to J. Roote and J. A. Campos-Ortega for fly stocks. The technical assistance of A. Meunier and R. Klock is gratefully acknowledged. Supported by institutional funds from the CNRS and grants from the Human Frontiers Science Program and Rhone-Poulenc Agro. J.-M. R. acknowledges support from the Marie Curie Research Training Program and the NATO Scientific Research Program. Work in M.A.'s laboratory was supported by a Medical Research Council Programme grant. The authors from both laboratories thank the Fondation Les Treilles (Schlumberger), which sponsored a meeting at Les Treilles where this collaboration was initiated.


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