Of 60 ecdysteroid compounds tested, only those which were active in other insect test systems elicited the response, and the concentrations required were approximately proportional to the concentrations active in other in vitro systems. It is not elicited by cyclic nucleotides, vertebrate growth factors, or a variety of other non-ecdysteroid reagents. The response occurs substantially normally in the absence of serum, during growth in suspension, and in over-crowded cultures. A halfmaximal response is elicited by approximately 10–8M 20-hydroxyecdysone the response is saturated by 10–7M 20-hydroxyecdysone, which causes detectable elongation within a few hours, and a maximal response after 2–3 days. The assay was used to characterize the conditions required for the response. We have devised a quantitative assay for this morphological response, using the subline Kc-H. Nearly half of the fs lines turned out to be replicates of noninduced fs mutant loci that preexisted in the EMS-treated files.Systematizing these fs genes according to their phenotypic and morphological defects focuses attention on their relevance to five major aspects of oogenesis: (1) the developmental organization of the ovary, (2) the synthesis and deposition of yolk material, (3) the formation of the chorion, (4) the control of egg-laying, and (5) the construction of the internal milieu of the ovarian oocyte for normal embryogenesis.The results of this study demonstrate that a systematic collection and characterization of fs mutants can provide the genetic tools needed to study the complex interactions which proceed undetected during normal oogenesis.Ĭells of the line Kc, derived fromDrosophila melanogaster embryos, extend long processes when exposed to ecdysteroid hormones. By the use of phenotype and complementation tests, the 98 fs lines were resolved into 19 fs genes on the 2nd chromosome and 17 fs genes on the 3rd chromosome.
The protocol can easily be adapted to different affinity-tagged or endogenous protein complexes of interest.Ī newly derived collection of 98 autosomal recessive female-sterile (fs) lines induced by ethyl methanesulfonate (EMS) in Drosophila melanogaster has been studied genetically and cytologically. The procedure can be subdivided into the following steps: acquisition of sufficient starting material, complex stabilization by crosslinking (optional), purification of the protein complex by immunoprecipitation, separation of the isolated material on a polyacrylamide gel, sample preparation for mass spectrometry, and sample analysis. Here, we present a basic method for the purification of the endogenous Par-6/aPKC protein complex, which plays a central role in orchestrating asymmetric cell divisions in the developing nervous system of Drosophila. Therefore, Drosophila now offers the advantages of both genetic and biochemical approaches. Protein biochemical methods have lagged behind for quite some time but meanwhile have reached a state where protein interaction networks can be elucidated at a similar speed and accuracy as genetic interactions. Drosophila melanogaster is one of the best characterized model systems for genetic analysis.
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