Drosophila mitotic domain boundaries as cell fate boundaries

Science

Volumn 277, Issue 5327, 1997, Pages 825-828

  Cambridge, Sidney B ^a   Davis, Robert L ^b   Minden, Jonathan S ^a  

^a CARNEGIE MELLON UNIVERSITY   (United States)

^b HARVARD MEDICAL SCHOOL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ANIMAL CELL; ARTICLE; CELL DIFFERENTIATION; DROSOPHILA; EMBRYO; EMBRYO DEVELOPMENT; GENE EXPRESSION; GENE EXPRESSION REGULATION; MITOSIS; NONHUMAN; PHOTOACTIVATION; PRIORITY JOURNAL; XENOPUS;

ANIMALS; APOPTOSIS; CELL DIFFERENTIATION; CELL LINEAGE; DROSOPHILA; FUNGAL PROTEINS; GASTRULA; GENE EXPRESSION REGULATION, DEVELOPMENTAL; LIGHT; MITOSIS; TRANS-ACTIVATION (GENETICS); TRANS-ACTIVATORS; TRANSGENES; XENOPUS;

ANIMALIA;

EID: 0030747044     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5327.825     Document Type: Article

References (34)

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4. D. I. Chasman, J. Leatherwood, M. Carey, M. Ptashne, R. D. Kornberg, Mol. Cell. Biol. 9, 4746 (1989). The final preparations were 75 to 80% pure as judged by SDS-polyacrylamide gel electrophoresis. GAL4VP16 was stored at -80°C in storage buffer [50 mM Na-Hepes, 0.2 M NaCl, 1 mM dithiothreitol, 30% glycerol, 0.1 mM EDTA, 10 μM Zn acetate (pH 7.4)].
5. GAL4VP16 was caged according to the following protocol. To one volume of a 1.35 mg/ml solution of the protein in storage buffer (as determined with the Bradford assay) was first added one volume of a 0.1 M sodium carbonate buffer (pH 9.7) and then two volumes of NVOC-CI (Fluka) in 1,4-dioxane. The pH of the buffer was such that the final pH of the reaction was 9.5. The reaction was carried out for 30 min at room temperature and was stopped by the addition of one volume of acidic 0.1 M tris-HCI, which also lowered the pH to 7.4. GAL4VP16 was found to be very stable under the reaction conditions used. The caged protein was washed twice with Centricon C-30 devices (Amicon) in a 20 mM Na-Hepes, 0.2 M NaCl solution (pH 7.3). The unirradiated, caged GAL4VP16 was stable at -80°C for more than 12 months.
6. The extent of caging was determined by fluorescamine labeling of free amines as described [P. Böhlen, S. Stein, W. Dairman, S. Udenfried, Arch. Biochem. Biophys. 155, 213 (1973)]. Parallel samples of ∼30 μg of GAL4VP16 were treated with or without NVOC-CI according to the standard caging protocol but were washed instead with 50 mM sodium phosphate buffer (pH 8). The samples were then brought to a final volume of 1.5 ml containing 1% SDS and were boiled for 3 min. Fluorescamine (0.5 ml, 30 mg/100 ml; Molecular Probes) in 1,4-dioxane was added, and the fluorescence of the sample was analyzed with a fluorimeter (excitation at 390 nm, emission at 485 nm). Results of the caged protein samples were correlated with that of the control sample that was incubated without NVOC-CI.
7. Embryos were prepared for injection as described [J. S. Minden, D. A. Agard, J. W. Sedat, B. M. Alberts, J. Cell Biol. 109, 505 (1989)]. All injections were done during interphase 14. For immunostaining, embryos were fixed and manually devitellinized as described (J. P. Vincent and J. P. O'Farrell, Cell 68, 923 (1992)]. After devitellinization, embryos were washed briefly in methanol (30 s), and antibody staining was carried out by standard protocols as reported [H. M. Bomze and A. J. Lopez, Genetics 136, 965 (1994)]. 5C.2B (anti-UBX) was diluted 1:65, 4CI-I (anti-ANTP) was diluted 1:33, anti-GAL4 (Santa Cruz Biotechnology) was diluted 1:1000, and anti-β-Gal (Sigma) was diluted 1:1500.
8. Embryos were prepared for injection as described [J. S. Minden, D. A. Agard, J. W. Sedat, B. M. Alberts, J. Cell Biol. 109, 505 (1989)]. All injections were done during interphase 14. For immunostaining, embryos were fixed and manually devitellinized as described (J. P. Vincent and J. P. O'Farrell, Cell 68, 923 (1992)]. After devitellinization, embryos were washed briefly in methanol (30 s), and antibody staining was carried out by standard protocols as reported [H. M. Bomze and A. J. Lopez, Genetics 136, 965 (1994)]. 5C.2B (anti-UBX) was diluted 1:65, 4CI-I (anti-ANTP) was diluted 1:33, anti-GAL4 (Santa Cruz Biotechnology) was diluted 1:1000, and anti-β-Gal (Sigma) was diluted 1:1500.
9. Embryos were prepared for injection as described [J. S. Minden, D. A. Agard, J. W. Sedat, B. M. Alberts, J. Cell Biol. 109, 505 (1989)]. All injections were done during interphase 14. For immunostaining, embryos were fixed and manually devitellinized as described (J. P. Vincent and J. P. O'Farrell, Cell 68, 923 (1992)]. After devitellinization, embryos were washed briefly in methanol (30 s), and antibody staining was carried out by standard protocols as reported [H. M. Bomze and A. J. Lopez, Genetics 136, 965 (1994)]. 5C.2B (anti-UBX) was diluted 1:65, 4CI-I (anti-ANTP) was diluted 1:33, anti-GAL4 (Santa Cruz Biotechnology) was diluted 1:1000, and anti-β-Gal (Sigma) was diluted 1:1500.
10. Presumably, this level of modification inhibited GAL4VP16 binding to chromatin, rather than naked DNA, or interfered with specific protein-protein interactions. Anti-GAL4 staining showed that caging did not perturb nuclear localization (12).
11. Ultraviolet doses for photoactivation were quantitated by actinometry as described [C. G. Hatchard and C. A. Parker, Proc. R Soc. London. A235, 518 (1956)] with an assumed quantum yield of 1.26 at 366 nm. Drosophila photoactivations were done with a UPlanFI 60×/1.25 oil-immersion objective on an Olympus IX-70 microscope with an Olympus IX-RFA/Caged attachment. The beam used for photoactivation of single mitotic cells in Drosophila embryos delivered an energy dose of about 8 μJ per second. Thus, a total of 24 μJ were necessary for photoactivation of a single cell. Photoactivation of entire Xenopus embryos required about 82-fold as much energy per area than individual Drosophila cells. In vitro photoactivation with the hand-held UV lamp was less efficient than in the in vivo experiments by a factor of about 8200, which explains the long irradiation times necessary for the gel mobility-shift assay.
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33. The fluorescence intensity units plotted are corrected for background fluorescence. For each time point, the fluorescence value at t = 0 of the respective curve was subtracted. The original uncorrected fluorescence units at t = 0 were 975 (□), 949 (black circle), 857(○), and 1078 (▲).
34. GUbxla stock and anti-UBX antibody was provided by J. Lopez. We thank B. Schmidt and members of the reagent group at the Science and Technology Center for Light Microscope Imaging and Biotechnology for helpful comments and W. McClure, J. Lopez, D. L. Taylor, A. Koretsky, and members of the Minden lab for critical review of the manuscript. Supported by a NSF Training Grant Fellowship, a Lucille Markey Charitable Trust Interdisciplinary Program in Biotechnology Fellowship, and the Friedrich-Naumann-Stiftung (S.B.C.) and by the Medical Foundation (Fleet Bank Trustee) (R.L.D.) and NSF Center for Light Microscope Imaging and Biotechnology grant BIR-8920118 (J.S.M.). J.S.M. is a Lucille Markey Scholar. This work was supported in part by a grant from the Lucille P. Markey Charitable Trust.