| Abstract: | We describe a molecularly defined duplication kit for the X chromosome of Drosophila melanogaster. A set of 408 overlapping P[acman] BAC clones was used to create small duplications (average length 88 kb) covering the 22-Mb sequenced portion of the chromosome. The BAC clones were inserted into an attP docking site on chromosome 3L using ΦC31 integrase, allowing direct comparison of different transgenes. The insertions complement 92% of the essential and viable mutations and deletions tested, demonstrating that almost all Drosophila genes are compact and that the current annotations of the genome are reasonably accurate. Moreover, almost all genes are tolerated at twice the normal dosage. Finally, we more precisely mapped two regions at which duplications cause diplo-lethality in males. This collection comprises the first molecularly defined duplication set to cover a whole chromosome in a multicellular organism. The work presented removes a long-standing barrier to genetic analysis of the Drosophila X chromosome, will greatly facilitate functional assays of X-linked genes in vivo, and provides a model for functional analyses of entire chromosomes in other species.THE X chromosome of Drosophila melanogaster contains ∼2300 protein-coding genes or ∼15% of such genes in the genome. It contains 22 Mb of euchromatic DNA (Adams et al. 2000). About one-third of these genes are predicted to be mutable to a phenotype that can be scored, e.g., lethality, sterility, or abnormal behavior (Peter et al. 2002). However, most molecularly recognized X-linked genes have not been associated with mutations or studied in any detail (http://flybase.org) (Drysdale 2008). Indeed, one hallmark of the X chromosome in D. melanogaster and many other species is that it is haploid in males. In addition, the presence of one copy of the X in an otherwise diploid animal leads to the phenomenon of dosage compensation, a process that essentially doubles the expression of X-linked genes in Drosophila males (Gelbart and Kuroda 2009).The presence of a single X chromosome in males facilitates screens for behavioral or visible mutant phenotypes in the hemizygous male progeny of a single-generation cross. For this reason, the X chromosome has been well saturated for viable mutations. However, many of these mutations have not been mapped since existing methods are tedious. Moreover, mutations in essential genes and genes required for male fertility cannot be propagated and genetically characterized unless they are complemented with a duplication maintained in the male. Hence, the X chromosome has been significantly less studied than the autosomes for mutations in essential and male fertility genes. For many of those mutations, the genes associated with these phenotypes have been elusive due to the lack of appropriate genetic reagents. Thus, X-linked genes in critical developmental and regulatory pathways are underrepresented in reported analyses as compared to similar classes of genes on the autosomes.Mutations in essential and male fertility genes on the X chromosome can be mapped using a variety of techniques. One approach is to rely on recombination in females and perform meiotic mapping against visible markers (Lindsley and Zimm 1992), P-element insertions (Zhai et al. 2003), or SNPs (Berger et al. 2001; Hoskins et al. 2001; Martin et al. 2001; Nairz et al. 2002; Chen et al. 2008), all of which are labor-intensive strategies or require specialized infrastructure. An alternative is complementation mapping using deficiencies, which requires only a single cross. This approach is possible for viable mutations but not for X-linked lethal and sterile mutations since those cannot be propagated through males. Instead, complementation rescue tests need to be carried out using a segregating duplication, e.g., an X chromosome fragment on the Y chromosome [Dp(1;Y)], an autosome [Dp(1;A)], or a free duplication [Dp(1;f)] (Lindsley and Zimm 1992). Currently, duplications that encompass ∼90% of the X chromosome are available. Only three cytological regions at 13A–13F (∼1 Mb), 16D7–16F4 (∼0.3 Mb), and 18A–18F (∼0.8 Mb) are not covered. Unfortunately, these duplications are typically very large (∼1–1.5 Mb) (http://flybase.org/) (Drysdale 2008), limiting their utility for fine mapping. Moreover, most available duplications were isolated following X-ray mutagenesis, and their breakpoints are poorly defined.Hence, a complete set of small molecularly defined duplications of the X chromosome would be extremely useful for identifying mutations in essential and male fertility genes and for fine-scale mapping of any mutation, including recessive viable mutations. In addition to promoting new genetic screens, a duplication set would allow one to map and assess the numerous, poorly characterized X-linked lethal mutants. Moreover, if molecularly defined genomic DNA clones are used to create the duplication set, then epitope tagging using recombineering would permit determination of expression patterns of genes included in the duplications (Venken et al. 2008, 2009; Ejsmont et al. 2009). Finally, such defined duplications would allow one to carry out structure–function analyses of genes through recombineering by introducing point mutations and small deletions into a gene of interest at unprecedented speed (Sharan et al. 2009).Previously, we created the P[acman] (P/ΦC31 artificial chromosome for manipulation) transgenesis platform (Venken and Bellen 2005, 2007; Venken et al. 2006) for retrieval and manipulation of large DNA fragments in a conditionally amplifiable BAC (Wild et al. 2002). Genomic clones inserted into this vector can be subjected to recombineering (Sharan et al. 2009) and used for transformation of these fragments (up to at least 146 kb) into the genome of flies that carry a defined attP docking site using the ΦC31 integrase system (Groth et al. 2004; Venken et al. 2006; Bischof et al. 2007; Markstein et al. 2008). In a next step, we constructed two genomic BAC libraries, one with an average insert size of 21 kb (CHORI-322) and another with an average insert size of 83 kb (CHORI-321) (Venken et al. 2009). These BAC libraries were end-sequenced and mapped onto the genome sequence and are publicly available (http://pacmanfly.org) and distributed (http://bacpac.chori.org/). Here we bring these resources to a next level: BAC TransgeneOmics (Poser et al. 2008) of an entire chromosome in vivo. The 8.2-fold coverage of the X chromosome in mapped clones from the CHORI-321 library allowed us to select a tiled path of overlapping BACs containing almost all of the annotated genes on this chromosome. Here we describe the creation of the first set of molecularly defined duplications covering an entire chromosome of a multicellular organism, and we illustrate its utility for X-chromosome genetics in several experimental paradigms. |
