Drosophila preparation

All elastic proteins contain distinct domains, of which at least one is made up of elastic repeat sequences, and they all contain cross-links between residues in either the nonelastic or elastic domains [9]. Previously, the Drosophila CGI5920 gene was tentatively identified as one encoding a resUin-like protein [31]. To prepare recombinant resilin, we chose to express the first exon of the Drosophila CG15920 gene [29], which encodes an N-terminal domain in the native protein comprising 17 copies of the putative elastic repeat motif GGRPSDSYGAPGGGN [31]. 

Silica gel plates also have been used for the separation of 16 different eye pigments of Drosophila melanogaster using two-dimensional development in nonpolar solvent systems [55]. Although not very common, two-dimensional development may be nsed in preparative scale on thick-layered plates for further analysis. 

Whereas the structure and function of the 20S proteasome have been elucidated in great detail (for review see Baumeister et al 1998), the 19S regulator is understood only dimly at present. Structural studies are hampered by the low intrinsic stability of this assembly and extensive remodelling, which makes it notoriously difficult to obtain homogeneous preparations. Nevertheless, analyses of yeast. Drosophila and human 26S... 

For MALDI-TOF MS several sample preparations are available with different matrices (26). The choice of the matrix and of the sample preparation should be adapted to the molecular mass of the compounds and to the complexity of the samples to analyze. a-Cyano-4-hydroxycinnamic acid (4HCCA) is preferred for peptides between 1 and 15 kDa, and the sandwich sample preparation can be universally used for molecular mass determination of pure peptides and enzymatic fragments or complex mixtures (e.g., crude hemolymph, enzymatic digests). The procedures reported below are the ones used for the discovery of the Drosophila immune-induced peptides (19,27,30). 

Fig. (5). Typical mass spectra from the Drosophila hemolymph (0.1 pL) collected from a single fly at 6h, 24h post challenge and from a control (unchallenged) fly. MALDI mass spectra were acquired with a sandwich sample preparation and a-cyano-4-hydroxycinnamic acid as matrix. Numbers (1-24) correspond to the Drosophila immune-induced molecules (DIMs).

An 18-mer DNA derived from in vitro selection to bind hemin has been previously reported. In a further report, the authors have prepared the RNA analogue of the aptamer to examine the peroxidative properties of each. Whilst both RNA and DNA aptamers displayed peroxidase activity, the RNA analogue bound with 30-fold weaker affinity. An RNA aptamer selected to inhibit the Drosophila B52 protein binds to B52 and inhibits B52-stimulated pre-mRNA splicing. It has been expressed in cell culture and animals, and binds B52 in vivo, suppressing all phenotypes. 

The preparation of the embryonic cockroach brain neuronal cultures is a complicated procedure which has been described in detail by Beadle and Lees ( ). In brief, the brains were dissected from 23-26 day old embryos of Periplaneta americana and broken up by passage through Pasteur pipettes of decreasing diameter. The cultures were initiated in a medium consisting of 5 parts Schneider s Drosophila medium and 4 parts Eagle s Basal medium, and after 7 days growth were transferred to a... 

Drosophila embryos are protected both by an outer layer called chorion and an impermeable and opaque vitelline membrane. Therefore preparation of whole mount Drosophila embryos for staining with antibodies and/or other fluorescent markers must go through the following steps chorion removal, fixation, vitelline membrane removal, and membrane permeabilization. The next subsection introduces the basic procedures for embryo collection and chorion removal that are common to all protocols described here, as well as the two most common fixation methods with or without methanol (the latter requiring hand devitellinization of embryos). The first one works well for immunostaining, while the second is ideal for F-actin staining with phalloidin. 

Fig. 1. Examples of fluorescence preparations of Drosophila whole mounts using the protocols is described in this chapter. All confocal images were obtained with a LeicaTCS4D confocal microscope, (a) Confocal optical section of a D. melanogaster embryo whole mount at blastoderm stage double stained with phalloidin—rhodamine (red) and DAPI (blue) to allow simultaneous visualization of nuclei and cortical actin around cell membranes. Anterior is to the left.

---