COMPONENTES

Polytene chrom osom es w ere prepared from the salivary glands of well-fed stage three larvae from w ild-type O regon R Drosophila as described in M aterials and M ethods. These w ere squashed and fixed before probing w ith a Biotinylated probe corresponding to full-length drICE GRP as described in M aterials and M ethods. The chrom osomes w ere w ashed, reprobed w ith a H R P-conjugated second-layer reagent, rew ash ed a n d the b o u n d probe detected as described in M aterials and M ethods. The chrom osom es w ere then inspected using a microscope and the results show n in Fig 8.1.

The drIC E chrom osom al location is in chrom osom e 3, in b a n d 99C1,2. U nfortunately, there are no deletions over this region in any of the stock centres and there are no strains containing P-elem ents integrated in this region either. Isolation of a drICE null strain is therefore m ore difficult than anticipated since the first step is to carry out an X-ray screen for deletions in chrom osom e 3 over the 99C region. This is being carried out at the m om ent by Dr. A ndreas Bergmann in the laboratory of Professor H erm ann Steller at MIT in Boston.

00

Fig 8.1 dr/CE is at 99C1.

Polytene chromosomes were prepared ; as described in Materials and Methods and hybridised to a biotinylated drICE cDNA probe. The in situ was developed as described in Materials and Methods and the results viewed by microscopic inspection.

8.3 drICE expression in D r o s o p h i l a developm ent

The m odel em erging from results in previous chapters is that drICE appears to be required for the execution of cell death in at least certain Drosophila cell types. drICE m ay therefore be an essential com ponent of the caspase- co n tain in g ap o p to tic m achinery in Drosophila. Since cell d eath occurs (A bram s et al., 1993) or can be induced at alm ost all developm ental stages from early em bryonic th ro u g h larval and p u p a l stages to the a d u lt fly (W hite et al., 1996), it w ould follow that drICE should be expressed at all these stages if it is indeed required for the execution of apoptosis in vivo. I therefore looked at dr ICE m RNA expression at different dev elo p m en tal stages, giving the result show n in Fig 8.2.

drICE expression is highest in 2-6 ho u r em bryos, d ro p p in g by 12-24 h o u r em bryos to a stable, low , detectable level th ro u g h o u t all su b seq u e n t d evelopm ental stages. drICE mRNA is therefore p resen t th ro u g h o u t all Drosophila developm ental stages. The message is the same size at all stages and only a single band can be detected.

Em bryogenesis <Û C\J c\j CO CN CN

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Fig 8.2 Expression of drICE mRNA at different Drosophila

developmental stages.

10 |ig of total RNA from the developmental stages shown were run on a 0.8% agarose gel containing 1.85% formaldehyde and ethidium bromide and blotted overnight onto Hybond-N+. The blot was probed with a pzp]- labelled drICE cDNA at 65°C in Church and washed at 65°C in 0.2XSSC before exposure against a Kodak XAR-5 film for 48hr. at -70°C. The rRNAs were visualised by UV fluorescence.

8.4 Sum m ary and D iscussion

In this chapter, I have described prelim inary attem pts to extend studies on the in v o lv em en t and the req u irem en t for drlC E for the execution of apoptosis in Drosophila to the whole organism.

1 have show n th at drICE is expressed at all developm ental stages from 2 h ours after egg laying (AEL) through to the ad u lt fly. The expression in em bryonic developm ent is particularly interesting since this is the stage for w hich m ost characterisation of cell d eath has been carried out. D uring Drosophila em bryogenesis, the first developm ental cell deaths are seen at aro u n d 7 hours AEL and the last at around 18 ho u rs AEL (Abrams et ah, 1993). H ow ever, it is possible to induce cell death, w hether by X-irradiation or by induction of reaper or grim expression, both before 7 hours and after 18 hours (Chen et ah, 1996b; W hite et a l, 1996). This im plies th at the cell death m achinery is present throughout em bryogenesis, and it is the trigger th at is develop m entally regulated. The m achinery should be p resent at all stages at w hich it is possible to induce cell deaths and this is the case for drlCE. The fact that drlCE is m ost highly expressed prior to the onset of any p ro g ram m ed cell d eath s u n d erlin es the m odel th a t caspase activity is principally post-translationally regulated and not regulated at the level of expression. drICE is therefore expressed at all stages at which apoptosis can be induced and its expression level does not correlate w ith the am ount of developm ental cell death taking place. The fact that drlCE is present at all times w here death can be induced m eans that in vivo as in vitro, drlCE could in principle be responsible for all cell deaths.

To exam ine further w hether drlCE is required for apoptosis in Drosophila, 1 determ ined the drICE chrom osomal location as a first step tow ards isolation of a drICE hom ozygous null fly strain. drICE is localised on C hrom osom e 3 at 99C1,2. H ow ever, there are neither pre-existing deletions spanning this region nor P-elem ents integrated in 99C, and so deletion of drICE requires a long term project to create chrom osom al deletions spanning 99C. This is ongoing in collaboration w ith Dr. A ndreas Bergm ann in the laboratory of Professor H erm ann Steller at MIT.

Chapter 9

D iscussion and Future Work