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...
TFIID is a multiprotein complex composed of the TATA binding protein (TBP) and TBP-associated factors (TAF II s). The binding of TFIID to the promoter is the first step of RNA polymerase II preinitiation complex assembly on protein-coding genes. Yeast (y) and human (h) TFIID complexes contain 10 to 13 TAF II Initiation of transcription of protein-encoding genes by RNA polymerase II requires transcription factor TFIID, which is comprised of the TATA-binding protein (TBP) and 10 to 12 TBP-associated factors (TAF II s) (2,40,42). TFIID directs preinitiation complex assembly on both TATA-containing and TATA-less promoters. To date, most of the TFIID components from Saccharomyces cerevisiae and humans have been identified, partially characterized, and shown to be well conserved during evolution (2, 25, 42). However, despite intensive biochemical analysis and genetic studies of Drosophila melanogaster TFIID (dTFIID) (8,22,43,47) several human and yeast TAF II s have no known Drosophila homologues.The different TAF II compositions of the distinct TFIID complexes appear to play key roles in the functional specificity of these complexes. A series of TAF II s, designated core TAF II s, may be present in all TFIID complexes, whereas other TAF II s are only found in defined TFIID subpopulations, often detected in substoichiometric amounts compared to TBP and core TAF II s (2-4, 6, 9, 15, 17). Recently, a novel human multiprotein complex has been characterized which contains neither TBP nor TBP-like factor but is composed of several TAF II s and a number of other polypeptides (5, 45). This complex, called TBP-free TAF II -containing complex (TFTC), contains the GCN5 histone acetyltransferase (HAT) activity, is able to direct preinitiation complex formation and initiation of transcription in in vitro transcription assays, and can mediate transcriptional activation by 45). (26). The finding that coactivators of transcription contribute to HAT activity further strengthens the idea that histone acetylation and deacetylation can regulate gene activation (24, 46). Recent analyses have particularly shown that GCN5 not only displays a HAT activity but also is required for correct expression of various genes in yeast by catalyzing promoter-specific histone acetylation (7, 48) and chromatin remodelling (14). All TBPfree TAF II -HAT complexes, including SAGA, TFTC, PCAF/ GCN5, and STAGA, contain a HAT belonging to the GCN5 family and can acetylate histone H3 in mononucleosomes (5,13,26,30,45). These data suggest that TAF II -GCN5-HAT complexes form transcriptional adapters able to interact with chromatin templates and to potentiate transcriptional activation. Differences in the polypeptide composition of the different TBP-free TAF II -HAT complexes (5) suggest that like TFIID, different subpopulations of TAF II -GCN5-HAT complexes may exist in the cell and may confer a broad range of regulatory capabilities in polymerase II transcription.Human TAF II 30 (hTAF II 30) is present in about 50% of the hTFIID complexes (17). hTAF II 30 in...
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