Cell killing by the Drosophila gene reaper

Science

Volumn 271, Issue 5250, 1996, Pages 805-807

  White, Kristin ^a   Tahaoglu, Elvan ^a   Steller, Hermann ^b,c  

^a MASSACHUSETTS GENERAL HOSPITAL   (United States)

^b HOWARD HUGHES MEDICAL INSTITUTE   (United States)

^c MASSACHUSETTS INSTITUTE OF TECHNOLOGY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

COMPLEMENTARY DNA; VIRUS PROTEIN;

ANIMAL CELL; ANIMAL TISSUE; APOPTOSIS; ARTICLE; BACULOVIRUS; CELL KILLING; DEVELOPMENTAL GENETICS; DROSOPHILA; EMBRYO; NONHUMAN; OPEN READING FRAME; PRIORITY JOURNAL; PROMOTER REGION; RETINA DEVELOPMENT; TRANSGENE;

ANIMALIA; UNIDENTIFIED BACULOVIRUS;

EID: 0030021282     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5250.805     Document Type: Article

References (29)

1. H Steller, Science 267, 1445 (1995)
2. K White and H Steller, Trends Cell Biol 5, 74 (1995)
3. K White et al , Science 264, 677 (1994)
4. P Golstein, D. Marguet, V Depraetere, Cell 81, 185 (1995); J. L. Cleveland and J. N. Ihle, ibid., p 479.
5. P Golstein, D. Marguet, V Depraetere, Cell 81, 185 (1995); J. L. Cleveland and J. N. Ihle, ibid., p 479.
6. S Nagata, Philos. Trans. R. Soc. London Ser B 345, 281 (1994).
7. M. P. Boldin et al , J. Biol. Chem 270, 387 (1995).
8. The hsrpr transgene was made by inserting a 0.9-kb Eco RI fragment containing the 13B2 rpr cDNA (3) into a pCaSpeR vector that contained the hsp70 promoter (11) Two independent lines containing a single insertion of the transgene were tested for their ability to induce apoptosis.
9. Embryos were collected from flies of the genotypes w, hsrpr46/+; H99/+ and from the cross w hsrpr53/Y; H99/+ X w, H99/TM3, for 6 hours at 22°C and aged for 16 hours at 18°C Embryos were heat-shocked on egg collection plates submerged in a water bath at 39°C, allowed to recover at 25°C for 1 hour, and stained with acridine orange as described [J M Abrams, K. White, L. I. Fessier, H. Steller, Development 117, 29 (1993)] Although levels of rpr expression were not directly assessed, expression of the hsp70 promoter is considered to be moderately strong but within the range of strongly expressed endogenous genes.
10. Three heat shock cDNA constructs were tested previously for their ability to rapidly induce apoptosis after heat shock (11) The effects of these heat shock constructs were examined in both wild-type and H99 backgrounds. No excessive cell death was seen in the wild-type background (for example, Fig 1A), although these constructs do induce abnormalities in embryonic morphology upon heat shock In addition, these transgenes do not induce apoptosis in homozygous H99 embryos.
11. The transformation constructs that contained the rpr ORF (hsrprORF) were generated by polymerase chain reaction (PCR), with the rpr 13B2 cDNA as a template The 5′ primer for the wild-type version contained an R1 site and rpr sequence from 20 bases upstream of the AUG triplet to 11 bases downstream. The mutant form (hsrprOPFmut) contained the insertion of an A residue directly after the AUG triplet. resulting in a predicted translation product of 67 amino acids. The 3′ primer contained an Xba I site and extended from 10 bases downstream of the rpr stop codon to 18 bases within the coding region. A single base change was included downstream from the ORF to act as a stop codon for the mutant form. The PCR products were cut with Eco RI and Xba I and inserted into pCaSpeR-hs cut with Eco RI-Xba I [V. Pirrotta, in Vectors: A Survey of Molecular Cloning Vectors and Their Uses, R. L Rodriguez and D. T. Denhart, Eds. (Butterworths, Boston, 1988), pp. 437-456). Embryos from flies of the genotypes w; hsdisco C/+, w; hsrprORFmut37A/+, hsrprORF71/+, and w, hsrprORF57/+; H99/+ X w; H99/TM3 were treated and stained as described (8).
12. M. E. Grether, J. M Abrams, J. Agapite, K. White, H. Steller, Genes Dev. 9, 1694 (1995).
13. B. A. Hay, T. Wolff, G M. Rubin, Development 210, 2121 (1994).
14. An Eco RI fragment containing the 13B2 rpr cDNA was cloned into the Eco RI site of the pGMR vector (72), and the orientation was confirmed by restriction analysis. Transformants with single insertions on the second and third chromosomes were crossed to generate stocks with one, two, three, or four insertions The phenotype of these stocks was generally independent of insertion site
15. Y. Gavrieli, Y Sherman, S. A Ben-Sasson, J. Cell Biol. 119, 493 (1992).
16. R. J Clern and L. K Miller, in Apoptosis II, L D Tomei and F. O Cope, Eds. (Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY, 1994), pp. 89-110; S. Rabizadeh, D. J. LaCount, P. D. Friesen, D. E. Bredesen, J. Neurochem. 61, 2318 (1993); A Sugimoto, P. D. Fnesen, J H Rothman, EMBO J 13, 2023 (1994)
17. R. J Clern and L. K Miller, in Apoptosis II, L D Tomei and F. O Cope, Eds. (Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY, 1994), pp. 89-110; S. Rabizadeh, D. J. LaCount, P. D. Friesen, D. E. Bredesen, J. Neurochem. 61, 2318 (1993); A Sugimoto, P. D. Fnesen, J H Rothman, EMBO J 13, 2023 (1994)
18. R. J Clern and L. K Miller, in Apoptosis II, L D Tomei and F. O Cope, Eds. (Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY, 1994), pp. 89-110; S. Rabizadeh, D. J. LaCount, P. D. Friesen, D. E. Bredesen, J. Neurochem. 61, 2318 (1993); A Sugimoto, P. D. Fnesen, J H Rothman, EMBO J 13, 2023 (1994)
19. D. Xue and H. R Horvitz, Nature 377, 248 (1995); N J Bump et al., Science 269, 1885 (1995).
20. D. Xue and H. R Horvitz, Nature 377, 248 (1995); N J Bump et al., Science 269, 1885 (1995).
21. Flies carrying four pGMRrpr insertions were crossed with flies homozygous for pGMRp35 (12), and the phenotype of the progeny was scored.
22. Embryos were collected from crosses of yw67c23 to w;hsrpr46/+, w;hsrpr53/+, w;hsrprORF57/+. w;hsrpr0RF71/+, w;hsrprORFmut37/+, and w;hsrprORFmut79/+. Embryos were aged at 18° or 22°C until the developmental stage indicated in Fig. 3 and heat-shocked in a water bath for 1 hour at 39°C. The flies were then allowed to develop to adulthood, and w+ (transgenic) and w siblings were counted soon after hatching.
23. M. Ashburner, Drosophila: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)
24. Young adult progeny from the crosses described above were heat-shocked in a water bath for 1 hour at 39°C, and then placed in vials and scored by counting the w and w+ (transgenic) survivors every other day. Flies were placed on fresh food after scoring.
25. H. Y Song, J. D Dunbar, D B Donner, J Biol. Chem 269, 22492 (1994).
26. C Newcomb-Carboy, C.-L Wei, H. Steller, unpublished results
27. Young adult flies were prepared for scanning electron microscopy as described (11)
28. Eye discs were dissected from climbing third instar larvae from yw67c23 and pGMRrpr 4 copy stocks, fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min, and washed in PBS for 15 min. The tissue was dehydrated in methanol for 30 min and rehydrated through 75% methanol and 25% PBT (PBS with 0.1% Tween 20), 50% methanol and 50% PBT, and 25% methanol and 75% PBT for 5 min each, washed in PBT, and treated with 10 μg/ml of protemase K for 5 min. The tissue was then fixed for 20 min in 4% paraformaldehyde, washed, treated with 2 1 ethanol:acetic acid for 10 min at -20°C, washed, and equilibrated for 1 hour in equilibration buffer from the Apoptag fluorescein kit (Oncor). The tissue was incubated in the working strength terminal transferase mixture with the addition of 0 3% Triton X-100 for 3 hours at 37°C, washed in stop buffer for 2 hours at 37°C (both from the Apoptag kit), blocked in PBS with 2 mg/ml of bovine serum albumin, 0 3% Triton X-100, and 5% goat serum, and incubated overnight with preabsorbed fluorescein isothiocynate-conjugated antidigoxygenin antibody (Oncor), diluted 1:1 with blocking solution Final washes were done in PBS with 0.3% TritonX-100, and the samples were visualized on a Bio-Rad confocal microscope.
29. We thank S. Abdalah for the TUNEL protocol, E Seling for assistance with the scanning electron microscope, and members of the White lab for their comments on the manuscript K.W is supported by a grant from Shiseido of Japan to Massachusetts General Hospital/Harvard Medical School, H.S. is an Associate Investigator with the Howard Hughes Medical Institute