We describe a second-generation deficiency kit for Drosophila melanogaster composed of molecularly mapped deletions on an isogenic background, covering 77% of the Release 5.1 genome. Using a previously reported collection of FRT-bearing P-element insertions, we have generated 655 new deletions and verified a set of 209 deletion-bearing fly stocks. In addition to deletions, we demonstrate how the P elements may also be used to generate a set of custom inversions and duplications, particularly useful for balancing difficult regions of the genome carrying haplo-insufficient loci. We describe a simple computational resource that facilitates selection of appropriate elements for generating custom deletions. Finally, we provide a computational resource that facilitates selection of other mapped FRT-bearing elements that, when combined with the DrosDel collection, can theoretically generate over half a million precisely mapped deletions.T HE availability of chromosomal deletion collections is of considerable benefit to the Drosophila research community for gene mapping, the phenotypic characterization of alleles, and genomewide genetic interaction screens. A core deficiency kit, composed of 270 genetically heterogeneous deletions covering 92% of the genome, has been built up over many years by the Bloomington Drosophila Stock Center (BDSC; http:/ / flystocks.bio.indiana.edu/Browse/df-dp/dfkit-info.htm). Continuing efforts by the Bloomington Center are currently focused on expanding genome coverage by recovering deletions in the vicinity of haplo-insufficient regions (K. Cook, personal communication). Despite the considerable utility of this collection, it does, by its very nature, suffer from a number of limitations. These include a heterogeneous genetic background, the presence of uncharacterized second-site mutations, and, for most deletions, molecularly undefined breakpoints. More recently, two groups have taken advantage of two key technologies: large collections of transposon insertions precisely mapped to the Drosophila genome sequence and site-specific recombination, to develop tools for producing custom chromosomal deletions in homogeneous genetic backgrounds that are mapped to the genome sequence with single-base-pair resolution (Parks et al. 2004;Ryder et al. 2004;Thibault et al. 2004).Sequence data from this article have been deposited with the EMBL/ GenBank data libraries under accession nos. AJ545047-AJ547612 and AJ622065-AJ622812. In both cases, the new deletion collections are generated using FLP-mediated recombination between pairs of transposon-borne FRT sites, a method originally developed in Drosophila by Golic and Golic (1996). In one case (Parks et al. 2004), a set of .29,000 P-element and piggyBac insertions (Thibault et al. 2004) were used to generate 519 deletions covering 56% of the euchromatic genome (the Exelixis collection). The high number of starting insertions used by this group allows fine-scale coverage of the genome with relatively small deletions; the average size of the exist...
The endocycle is a commonly observed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. How the cell cycle machinery is modified to transform a mitotic cycle into endocycle has long been a matter of interest. In both plants and animals, the transition from the mitotic cycle to the endocycle requires Fzr/Cdh1, a positive regulator of the Anaphase-Promoting Complex/Cyclosome (APC/C). However, because many of its targets are transcriptionally downregulated upon entry into the endocycle, it remains unclear whether the APC/C functions beyond the mitotic/endocycle boundary. Here, we report that APC/CFzr/Cdh1 activity is required to promote the G/S oscillation of the Drosophila endocycle. We demonstrate that compromising APC/C activity, after cells have entered the endocycle, inhibits DNA replication and results in the accumulation of multiple APC/C targets, including the mitotic cyclins and Geminin. Notably, our data suggest that the activity of APC/CFzr/Cdh1 during the endocycle is not continuous but is cyclic,as demonstrated by the APC/C-dependent oscillation of the pre-replication complex component Orc1. Taken together, our data suggest a model in which the cyclic activity of APC/CFzr/Cdh1 during the Drosophilaendocycle is driven by the periodic inhibition of Fzr/Cdh1 by Cyclin E/Cdk2. We propose that, as is observed in mitotic cycles, during endocycles,APC/CFzr/Cdh1 functions to reduce the levels of the mitotic cyclins and Geminin in order to facilitate the relicensing of DNA replication origins and cell cycle progression.
Recognition of polyubiquitylated substrates by the proteasome is a highly regulated process that requires polyubiquitin receptors. We show here that the concentrations of the proteasomal and extraproteasomal polyubiquitin receptors change in a developmentally regulated fashion. The stoichiometry of the proteasomal p54/Rpn10 polyubiquitin receptor subunit, relative to that of other regulatory particle (RP) subunits falls suddenly at the end of embryogenesis, remains low throughout the larval stages, starts to increase again in the late third instar larvae and remains high in the pupae, adults and embryos. A similar developmentally regulated fluctuation was observed in the concentrations of the Rad23 and Dsk2 extraproteasomal polyubiquitin receptors. Depletion of the polyubiquitin receptors at the end of embryogenesis is due to the emergence of a developmentally regulated selective proteolytic activity. To follow the fate of subunit p54/Rpn10 in vivo, transgenic Drosophila melanogaster lines encoding the N-terminal half (NTH), the C-terminal half (CTH) or the full-length p54/Rpn10 subunit were established in the inducible Gal4-UAS system. The daughterless-Gal4-driven whole-body expression of the full-length subunit or its NTH did not produce any detectable phenotypic changes, and the transgenic products were incorporated into the 26S proteasome. The transgene-encoded CTH was not incorporated into the 26S proteasome, caused third instar larval lethality and was found to be multi-ubiquitylated. This modification, however, did not appear to be a degradation signal because the half-life of the CTH was over 48 hours. Accumulation of the CTH disturbed the developmentally regulated changes in subunit composition of the RP and the emergence of the selective proteolytic activity responsible for the depletion of the polyubiquitin receptors. Build-up of subunit p54/Rpn10 in the RP had already started in 84-hour-old larvae and reached the full complement characteristic of the non-larval developmental stages at the middle of the third instar larval stage, just before these larvae perished. Similar shifts were observed in the concentrations of the Rad23 and Dsk2 polyubiquitin receptors. The postsynthetic modification of CTH might be essential for this developmental regulation, or it might regulate an essential extraproteasomal function(s) of subunit p54/Rpn10 that is disturbed by the expression of an excess of CTH.
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