DCP-1, a Drosophila cell death protease essential for development

Science

Volumn 275, Issue 5299, 1997, Pages 536-540

  Song, Zhiwei ^a   McCall, Kimberly ^a   Steller, Hermann ^a  

^a HOWARD HUGHES MEDICAL INSTITUTE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

PROTEINASE;

ARTICLE; CAENORHABDITIS ELEGANS; CELL DEATH; CELL KILLING; DROSOPHILA; GENE EXPRESSION; NONHUMAN; PRIORITY JOURNAL; PROTEIN EXPRESSION;

AMINO ACID SEQUENCE; ANIMALS; APOPTOSIS; CAENORHABDITIS ELEGANS PROTEINS; CASPASES; CLONING, MOLECULAR; CYSTEINE ENDOPEPTIDASES; DNA FRAGMENTATION; DNA TRANSPOSABLE ELEMENTS; DROSOPHILA; DROSOPHILA PROTEINS; EMBRYO, NONMAMMALIAN; GENE DELETION; GENES, INSECT; HELA CELLS; HELMINTH PROTEINS; HUMANS; MOLECULAR SEQUENCE DATA; MUTATION; RNA, MESSENGER; SEQUENCE HOMOLOGY, AMINO ACID;

EID: 0031023782     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5299.536     Document Type: Article

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48. 35S-labeled hPARP was added and incubated at 37°C for 30 min. In both experiments, CED-3 protein was used as a control. PARP cleavage was analyzed with 10% SDS-PAGE, and p35 cleavage was analyzed with 15% SDS-PAGE.
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50. We basically followed the method of M. Miura et al., Cell 75, 653 (1993). The full-length version of DCP-1 was generated by PCR with the upstream primer (5′-GCGGAGTCGACGATGACCGACGAGTGCGTA-3′) and the downstream primer (5′-CGGATCCGTCGACGCGCCAGCCTTATTGCCGTT-3′) that both have a Sal I site. After treatment with Sal I, the PCR product was cloned into the Sal I-treated, dephosphorylated mammalian expression vector pactβgal (provided by J. Yuan). Clones with the correct orientation were identified by sequencing. Cells were transiently transfected with 1 μg of DNA and 8 μl of lipofectamine reagent (Gibco-BRL), following the manufacturer's instructions.
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52. 2, 5 mM EGTA, 1 mM DTT, 2 mM ATP, 10 mM creatine phosphate, creatine kinase (50 μg/ml), and bovine serum albumin (0.2 mg/ml)] until more than 95% of the cells were broken but nuclei remained intact when analyzed under a microscope. A 30-μl sample of this homogenate was used for each assay. To the homogenate was added 1 μl (0.1 μg) of proteinase K or 1 μl of partially purified DCP-1. For inhibition of DCP-1, 0.1 mM Ac-DEVD-CHO was added with DCP-1. After 3 hours of incubation at 37°C, DNA was extracted and analyzed by agarose gel electrophoresis.
53. 2, 5 mM EGTA, 1 mM DTT, 2 mM ATP, 10 mM creatine phosphate, creatine kinase (50 μg/ml), and bovine serum albumin (0.2 mg/ml)] until more than 95% of the cells were broken but nuclei remained intact when analyzed under a microscope. A 30-μl sample of this homogenate was used for each assay. To the homogenate was added 1 μl (0.1 μg) of proteinase K or 1 μl of partially purified DCP-1. For inhibition of DCP-1, 0.1 mM Ac-DEVD-CHO was added with DCP-1. After 3 hours of incubation at 37°C, DNA was extracted and analyzed by agarose gel electrophoresis.
54. 2, 5 mM EGTA, 1 mM DTT, 2 mM ATP, 10 mM creatine phosphate, creatine kinase (50 μg/ml), and bovine serum albumin (0.2 mg/ml)] until more than 95% of the cells were broken but nuclei remained intact when analyzed under a microscope. A 30-μl sample of this homogenate was used for each assay. To the homogenate was added 1 μl (0.1 μg) of proteinase K or 1 μl of partially purified DCP-1. For inhibition of DCP-1, 0.1 mM Ac-DEVD-CHO was added with DCP-1. After 3 hours of incubation at 37°C, DNA was extracted and analyzed by agarose gel electrophoresis.
55. In situ hybridizations were performed as described (6).
56. 5, provided by the Bloomington Stock Center.
57. P element revertants were generated by standard genetic techniques. Viable and lethal revertants were recovered for both of the P element lines. Southern blot analysis was used to determine the presence of the P elements in the revertant lines.
58. 1862 chromosome appeared to contain additionally an unrelated background mutation that caused dorsal cuticle defects in embryos.
59. TUNEL labeling was carried out as described (9). This technique labels apoptotic nuclei by incorporating biotinylated nucleotides at the end of DNA double-strand breaks [Y. Gavrieli et al., J. Cell. Biol. 119, 493 (1992)]. In addition, antibody staining against the ENGRAILED protein was used to check for the presence of supernumerary cells in the CNS [L. Zhou et al., Curr. Biol. 5, 784 (1995)].
60. TUNEL labeling was carried out as described (9). This technique labels apoptotic nuclei by incorporating biotinylated nucleotides at the end of DNA double-strand breaks [Y. Gavrieli et al., J. Cell. Biol. 119, 493 (1992)]. In addition, antibody staining against the ENGRAILED protein was used to check for the presence of supernumerary cells in the CNS [L. Zhou et al., Curr. Biol. 5, 784 (1995)].
61. Z. Song and H. Steller, unpublished observations; A. Fraser and G. Evan, personal communication.
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66. We thank J. Yuan for providing the pactβgal vector and suggestions; D. Xue for a gift of CED-3 protein and advice; C. Hynds for assistance with figures; B. Reed, K. Matthews, and the Bloomington Stock Center for Drosophila stocks; the Berkeley Drosophila Genome Project for information on the P element strains; and K. Watson and C. Dearolf for useful advice. Supported by the American Cancer Society (K.M.). Z.S. is a postdoctoral associate and H.S. is an associate investigator of the Howard Hughes Medical Institute.