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Genetics. Dec 2010; 186(4): 1095–1109.
PMCID: PMC2998296

A New Resource for Characterizing X-Linked Genes in Drosophila melanogaster: Systematic Coverage and Subdivision of the X Chromosome With Nested, Y-Linked Duplications

Abstract

Interchromosomal duplications are especially important for the study of X-linked genes. Males inheriting a mutation in a vital X-linked gene cannot survive unless there is a wild-type copy of the gene duplicated elsewhere in the genome. Rescuing the lethality of an X-linked mutation with a duplication allows the mutation to be used experimentally in complementation tests and other genetic crosses and it maps the mutated gene to a defined chromosomal region. Duplications can also be used to screen for dosage-dependent enhancers and suppressors of mutant phenotypes as a way to identify genes involved in the same biological process. We describe an ongoing project in Drosophila melanogaster to generate comprehensive coverage and extensive breakpoint subdivision of the X chromosome with megabase-scale X segments borne on Y chromosomes. The in vivo method involves the creation of X inversions on attached-XY chromosomes by FLP-FRT site-specific recombination technology followed by irradiation to induce large internal X deletions. The resulting chromosomes consist of the X tip, a medial X segment placed near the tip by an inversion, and a full Y. A nested set of medial duplicated segments is derived from each inversion precursor. We have constructed a set of inversions on attached-XY chromosomes that enable us to isolate nested duplicated segments from all X regions. To date, our screens have provided a minimum of 78% X coverage with duplication breakpoints spaced a median of nine genes apart. These duplication chromosomes will be valuable resources for rescuing and mapping X-linked mutations and identifying dosage-dependent modifiers of mutant phenotypes.

MANY eukaryotes of biomedical and agricultural importance—including humans, other mammals, birds, and Drosophila—are heterogametic. Their sex chromosomes differ drastically in size and genetic composition. In species with X and Y chromosomes, males carry only one copy of each X-linked gene. This poses a serious challenge for experimental geneticists, because males inheriting a mutation in a vital X-linked gene die before they can be used in genetic crosses. In fact, the hemizygosity of X-linked genes in males has been a significant barrier to the functional analysis of many X-linked genes and is largely responsible for the poor genetic characterization of X chromosomes relative to autosomes in most organisms.

The lethality of X-linked mutations can be rescued by providing a wild-type copy of the mutated gene elsewhere in the genome. This can be accomplished with a transgenic construct if the molecular identity of the mutated gene is known. In many cases, however, the mutated gene has not been identified and it is necessary to provide wild-type function with a multigene interchromosomal duplication, i.e., a segment of the X inserted in another chromosome. If the proximal and distal extents of the duplicated segment are known, phenotypic rescue maps the mutated gene to the defined X chromosome region.

Multigene deletions can also be used to map X-linked mutations by complementation, but crosses between individuals carrying deletions and X-linked lethal mutations are impossible without rescuing the lethality of either the deletion or the lethal mutation in males. Projects at the Bloomington Drosophila Stock Center and elsewhere (Parks et al. 2004; Ryder et al. 2007) have generated large collections of deletions with molecularly defined breakpoints in Drosophila melanogaster, but the utility of the X deletions is limited without duplications of the corresponding chromosomal regions.

Duplications are potentially important for gene discovery. Identifying sets of genes involved in the same cellular process is a major focus of functional genomics research and this can be accomplished genetically by identifying dosage-sensitive modifiers of mutant phenotypes. Often, increasing or decreasing the copy number of a gene will enhance or suppress the phenotype associated with mutating another gene involved in the same process. Screening collections of deletions is a popular way to identify interacting genes in Drosophila (for examples, see Seher et al. 2007; Zhao et al. 2008; Aerts et al. 2009; Salzer et al. 2010) and was a major impetus for the assembly of the Bloomington Stock Center “Deficiency Kit,” which provides maximal coverage of the genome with the fewest deletions. Though dosage-sensitive modifiers could also be identified using increased gene dosage, the use of duplications in enhancer and suppressor screens remains largely unexplored. Assembling sets of duplications providing efficient genomic coverage would likely popularize this experimental approach.

The size of duplicated segments determines how duplication chromosomes are used experimentally. Small duplicated segments allow high resolution gene mapping, but they are not suitable for other purposes. Only large duplicated segments are capable of rescuing the lethality of sizable multigene X deletions. Likewise, large duplicated segments provide efficiency in initially localizing mutations and identifying dosage-dependent modifiers. Despite their usefulness, interchromosomal duplications of large segments are among the hardest chromosomal rearrangements to isolate. In Drosophila, many existing duplications were recovered fortuitously as three-breakpoint aberrations following irradiation, but such rearrangements are rare and difficult to identify in screens. Other duplications were methodically constructed from preexisting rearranged chromosomes. This approach works well when it is possible, but it can be used only when progenitor aberrations with appropriate breakpoints are available. Because of these difficulties, the selection of duplication strains generated by Drosophila workers over the past several decades is not satisfactory for many purposes. The duplications are often difficult to use experimentally, their breakpoints are sparsely distributed along the X chromosome and only roughly mapped, and substantial gaps in coverage exist. Obviously, improved duplication resources are needed.

Here we present the methodology and progress of a project at the Bloomington Drosophila Stock Center to construct interchromosomal duplications of large, megabase-scale X segments. Our approach builds on the long history of manipulating Drosophila chromosomes in vivo (Novitski and Childress 1976; Ashburner et al. 2005), but we have eliminated the need for preexisting aberrations by generating progenitor chromosomes using the FLP-FRT system. Indeed, this site-specific recombination system has had an enormous impact on the ability of fly geneticists to engineer many kinds of novel chromosomes (Golic and Golic 1996; Parks et al. 2004; Ryder et al. 2007). We will demonstrate how we have combined FLP-mediated recombination and other chromosome manipulation techniques to produce Y-linked duplications of large X segments. As we will show, appending X segments to Y chromosomes rather than autosomes has advantages both for the synthesis and experimental use of X duplications.

To date, we have generated a minimum of 78% X coverage with duplication breakpoints spaced a median of nine genes apart. We anticipate completion of the project within the coming year. Using these duplications, mutations and genetic modifiers can be mapped first to large X intervals using a tiling set of the largest duplicated segments and then to small chromosome intervals using subsets of the duplications. These duplications will also facilitate deletion mapping. The creation of a set of stocks providing complete duplication coverage and extensive breakpoint subdivision of the X chromosome in a consistent genetic background will remove an impediment to investigating the functions of X-linked genes that has frustrated generations of Drosophila geneticists.

MATERIALS AND METHODS

Fly stocks:

FRT-bearing P{RS5} and P{RS3} insertion stocks were obtained from the Szeged Drosophila Stock Centre. The remaining stocks were obtained from the Bloomington Drosophila Stock Center collection or the Drosophila Genetic Resource Center at the Kyoto Institute of Technology.

Genomic coordinates and cytological breakpoints:

All genomic coordinates and gene counts are based on Genome Release 5.16. Except for the directly observed cytological breakpoints in Table 1, all Dp(1;Y) cytological breakpoints were predicted from Release 5 coordinates using FlyBase map conversion tables (http://flybase.org; Tweedie et al. 2009). For assessing duplication coverage, we have artificially set the euchromatin/heterochromatin boundary at sequence coordinate X:22420000, roughly the most proximal extent of the assembled X chromosome genomic contigs in Genome Release 5.16.

TABLE 1
Dp(1;Y) chromosomes derived from C(1;Y)6, In(1)sc260-14

Mutagenesis:

Adult males received 4500-R exposure to 6000 Ci of 137Cs in a Shepard Mark-1 irradiator.

Cytology:

Mitotic chromosomes were prepared and stained with DAPI by standard methods (Fanti and Pimpinelli 2004). Chromosomes were stained 45 min with 0.5 mg/ml chromomycin A3 (Sigma) in PBS pH 7.7 with 5 mm MgCl2 and rinsed in PBS prior to mounting. Polytene chromosomes were analyzed in standard lacto-aceto-orcein preparations (Carpenter 2004).

Comparative genome hybridization microarrays:

Corning CGAP slides spotted with the AROS Drosophila V1.1.1 ~70 nucleotide oligo set from Eurofins MWG Operon were hybridized and analyzed as described in Erickson and Spana (2006).

PCR:

DNA was prepared from single flies as described in Engels et al. (1990) and amplified using Qiagen HotStarTaq master mix. The following amplification regime was used to confirm the presence of P{RS3} and P{RS5} insertions: 95° for 10 min followed by 38 rounds of 95°, 30 sec; 42°, 30 sec; and 72°, 5 min. Primer sequences are given in supporting information, File S1. For mapping of duplication endpoints, DNA from Dp(1;Y) males carrying an X chromosome transposon insertion was amplified as follows: 95° for 15 min followed by 35 rounds of 95°, 30 sec; 53°, 30 sec; and 72°, 60 sec. The transposons and primers in the mapping panel (Table S1) were chosen to be spaced 10 protein-coding genes apart, but the spacing varied occasionally on the basis of the availability of insertions or the presence of large genes.

Genetic crosses:

Extensive details are provided in File S1.

RESULTS

Generating Y-linked duplications of X chromosome segments:

Our goal is to generate comprehensive duplication coverage and extensive breakpoint subdivision of the X chromosome. The approach we have taken is to replace the tips of Y chromosomes with large segments of the X chromosome. These chromosomes are denoted “Dp(1;Y)” to indicate that a segment of the first chromosome (the X) is duplicated on the Y. In crosses, Dp(1;Y) chromosomes behave like normal Y chromosomes. They show typical Y-linked inheritance. While it is convenient for Dp(1;Y) chromosomes to carry dominant marker mutations for following them in crosses, it is not absolutely necessary. The segregation pattern of the Y is usually sufficient to track Dp(1;Y) chromosomes in experiments. This is a distinct advantage over duplications carried on autosomes, where dominant marker mutations are usually essential for following duplicated segments in crosses. Also, in the context of modifier screens, Y linkage provides flexibility with the easiest way to assay interactions of duplicated X segments with recessive mutations on the autosomes.

Y linkage does not, however, restrict the use of Dp(1;Y) chromosomes to males. Because the Y plays no role in Drosophila sex determination and carries only genes necessary for spermatogenesis, Dp(1;Y) chromosomes may be introduced into females where they can be used to rescue the female-specific phenotypes of X-linked mutations, such as ovarian defects caused by female sterile mutations. (Methods for placing Dp(1;Y) chromosomes into females are described in a later section.) While duplicating large X segments can cause lethality, sterility, and other phenotypes associated with excess hyperploidy, sex determination is unaffected by duplications of sizes compatible with the viability of hyperploid flies (Patterson et al. 1937).

Extensive chromosome manipulations were needed to create the progenitor chromosomes used in screens isolating Dp(1;Y) chromosomes. In this section, we will provide a general overview of the steps. For background, we will first describe the recovery of simple Dp(1;Y) chromosomes—those carrying segments from the tip of the X appended to an intact Y. Then we will present the variation on this method that we used. It employs inversions to duplicate segments from the entire X. In subsequent sections, we will describe how we generated the inversions and how we conducted the final Dp(1;Y) screens. In the overview, we will also show how a single progenitor chromosome gives rise to a set of Dp(1;Y) chromosomes with duplicated X segments of different sizes.

Our approach to isolating Dp(1;Y) chromosomes utilizes an attached-XY chromosome, a single chromosome carrying all X- and Y-linked genes. It was generated by a translocation event (Figure 1) involving an X chromosome break in centric heterochromatin and a Y chromosome break near the telomere. Attached-XY chromosomes are denoted “C(1;Y)” to indicate a compound chromosome formed by a first (X) chromosome and a Y. An attached-XY can substitute for a regular X in crosses and, in most situations, its segregation behavior is indistinguishable from a regular X. If a male carries an attached-XY, there is no need for a regular Y, because all Y-linked spermatogenesis genes are provided by the Y portion of the attached-XY.

Figure 1.
Generating an attached-XY chromosome. Irradiating males can produce a break in X centric heterochromatin proximal to all X-linked genes and a break near the Y tip distal to all genes on the Y. Following translocation, the resulting attached-XY chromosome ...

A Dp(1;Y) can be generated from an attached-XY by deleting most of the X chromosome (Figure 2A). If one breakpoint is positioned near the X tip (breakpoint A) and another is positioned in X centric heterochromatin (breakpoint B), the resulting Dp(1;Y) will carry genes from the end of the X and a segment of X heterochromatin appended to the end of the Y. The yellow (y) gene, which is located near the X tip and necessary for normal pigmentation, is key to identifying Dp(1;Y) chromosomes in screens. When males carrying C(1;Y) chromosomes are irradiated and mated to females carrying y1 mutations, most male progeny with normal pigmentation carry a Dp(1;Y) (Figure 2B).

Figure 2.
Generating Dp(1;Y) chromosomes from attached-XY chromosomes. (A) If attached-XY chromosomes are irradiated to introduce a break near the X tip (breakpoint A) and a break in X centric heterochromatin (breakpoint B), most of the X chromosome will be deleted. ...

If multiple Dp(1;Y) chromosomes are isolated from a screen, the X tip segments will form a nested set: all the tip segments share the telomeric end, but the ends generated by the breakpoints (breakpoint A) will differ (Figure 2A). In this way, the X tip region can be subdivided finely with duplication breakpoints and mutations near the tip of the X can be mapped with precision in rescue experiments.

Many of the proximal deletion breakpoints (breakpoint B) will fall in X centric heterochromatin as shown in Figure 2A, but they may also fall in the Y arm or in basal X euchromatin (Figure 3). Y breakpoints result in the deletion of Y-linked spermatogenesis genes and males carrying these Dp(1;Y) chromosomes are sterile. These Dp(1;Y) chromosomes are not recovered in stable stocks when irradiated males are crossed to normal females. Breakpoints in basal euchromatin result in Dp(1;Y) chromosomes with two sets of duplicated genes: one set from the X tip and another from the X base. X centric heterochromatin and the Y arm are much larger targets for irradiation-induced breakpoints than basal X euchromatin, so Dp(1;Y) chromosomes carrying genes from the base of the X are less common than the other two classes. The total number of duplicated genes that a Dp(1;Y) can carry from both the tip and base of the X is limited by hyperploidy effects. Drosophila is generally quite tolerant of hyperploidy and duplications of up to half a chromosome arm have been recovered (Ashburner et al. 2005), but our experience has been that duplications of >10% of X euchromatin are rare and flies carrying extremely large duplications have low viability and fertility.

Figure 3.
Position of the proximal deletion breakpoint. The deletion giving rise to a Dp(1;Y) from an attached-XY can break in X centric heterochromatin (top), in the short arm of the Y (middle) or in X basal euchromatin (bottom). Breaks in X centric heterochromatin ...

The problem with irradiating a regular attached-XY chromosome as described above is that only X-linked genes near the tip or base can be recovered in Dp(1;Y) chromosomes. What about the genes in the middle of the X? Fortunately, the method can be extended by introducing inversions into the X portion of the attached-XY chromosome (Figure 4). If the inversion has one distal breakpoint (breakpoint C) near the X tip and another breakpoint in the middle of the X (breakpoint D), irradiating this “inversion + attached-XY” chromosome can generate Dp(1;Y) chromosomes carrying genes from the middle of the X as well as genes from the X tip. The size of the segment containing medial X genes is determined by the position of the distal deletion breakpoint (breakpoint E).

Figure 4.
Generating Dp(1;Y) chromosomes from inversion + attached-XY chromosomes. To isolate Dp(1;Y) chromosomes carrying genes from the middle of the X, an inversion is introduced into the attached-XY chromosome (breakpoints C and D). The inversion is ...

If multiple Dp(1;Y) chromosomes are isolated from an inversion + attached-XY, the segments from the middle of the X will form a nested set. These nested segments will share a common end corresponding to the inversion breakpoint (breakpoint C), but their other ends will differ by the positions of the distal deletion breakpoints (breakpoint E). All the Dp(1;Y) chromosomes will share a common distal segment extending from the X telomere to the distal inversion breakpoint (breakpoint C). As in screens with regular attached-XY chromosomes, Dp(1;Y) chromosomes derived from inversion + attached-XY chromosomes will also carry genes from the X base if the proximal deletion breakpoint (breakpoint F) falls in basal euchromatin and they will delete Y-linked spermatogenesis genes if the breakpoint falls in the Y arm (similar to Figure 3).

Inversions with different proximal breakpoints (breakpoint D) move different regions to the X tip so that different sets of genes can be recovered in Dp(1;Y) chromosomes. Consequently, if a set of inversion + attached-XY chromosomes existed with proximal inversion breakpoints (breakpoint D) distributed along the X chromosome, it would be possible to generate Dp(1;Y) chromosomes providing duplication coverage of all X regions (Figure 5). The multiple nested sets would also subdivide the entire X with duplication breakpoints for use in high-resolution gene mapping.

Figure 5.
Hypothetical distribution of medial duplicated segments in Dp(1;Y) chromosomes derived from different inversion + attached-XY chromosomes. If the inversions in inversion + attached-XY chromosomes have different proximal breakpoints, nested ...

A preliminary test of the method:

To our knowledge, Dp(1;Y) chromosomes have been derived from inversion + attached-XY chromosomes in only three unpublished screens carried out by Abraham Schalet [screens generating Dp(1;Y)y+lz+, Dp(1;Y)y+na+, and Dp(1;Y)y+g+ (Lindsley and Zimm 1992) and a screen for Dp(1;Y)dx+1 through dx+8 (http://flybase.org/; Tweedie et al. 2009)]. To assess the method in our hands, we isolated Dp(1;Y) chromosomes using the preexisting inversion In(1)sc260-14 (Sutton 1943). Males with In(1)sc260-14 on an attached-XY were irradiated and mated to y1 females. We recovered 39 y+ males from ~93,000 progeny. Ten males were fertile and Dp(1;Y) stocks were established; the remaining 29 sterile males likely carried duplications lacking one or more Y-linked spermatogenesis genes. As shown in Table 1, the Dp(1;Y) chromosomes rescued the phenotypes of a variety of mutations in the 10C to 11D region of the X chromosome.

The largest duplicated medial segment contained seven polytene subdivisions, suggesting we could cover the entire X with Dp(1;Y) chromosomes if we had proximal inversion breakpoints spaced roughly every 5 subdivisions on the 120-subdivision X map. This would allow the largest duplicated segments from every screen to overlap the common end of the next set of segments (as shown in Figure 5) yet avoid intolerable levels of hyperploidy. Only genes lethal to males in two copies will prevent full coverage. To maximize coverage, inversion breakpoints would need to lie close to the distal sides of the two known X-linked diplolethal loci: an unnamed diplolethal in 3F and Haplo-diplo lethal (Hdl) in 12A (Stewart and Merriam 1973; Salz 1992; J. Merriam, personal communication).

Generating inversion + attached-XY chromosomes:

To generate comprehensive duplication coverage of the X chromosome with Dp(1;Y) chromosomes as shown in Figure 5, it was necessary to generate a large set of inversions on attached-XY chromosomes. We wanted the inversions to share the same distal breakpoint (breakpoint C in Figure 4), but to have proximal breakpoints (breakpoint D) distributed along the length of the X. To generate the inversions, we used the FLP-FRT site-specific recombination system (Golic and Golic 1996). As shown in Figure 6, inversions can be recovered upon FLP-induced recombination if the two FRTs are present in opposite orientations on the same chromosome.

Figure 6.
Generating an inversion using the FLP-FRT system. FLP recombinase induces recombination between pairs of FRT sites in an orientation-specific manner. If FRT sites are placed in cis in opposite orientations, FLP recombinase will catalyze the formation ...

To screen for the inversions, we used the FRT-bearing transgenic constructs P{RS3} and P{RS5}, which were specially designed to reconstitute the white (w) gene upon recombination (Figure 7) (Golic and Golic 1996). P{RS3} carries FRTs flanking the 5′ exon of w. Upon FLP-induced recombination, the 5′ w exon is removed. Likewise, P{RS5} carries FRTs flanking the 3′ w exons so that they are removed upon FLP-mediated recombination. In both cases, removal of w exons renders the w gene nonfunctional and flies carrying these rearranged transgenes have white eyes in the absence of other functional copies of w. When these rearranged transgenes are subsequently combined in the presence of FLP recombinase, recombination between the FRTs will reconstitute a functional w gene. In this way, flies carrying chromosomal aberrations can be identified as red-eyed progeny of white-eyed parents.

Figure 7.
Using P{RS3} and P{RS5} insertions to detect FLP recombinase-mediated recombination events. P{RS3} and P{RS5} were designed to allow the detection of recombination between FRT sites by the ...

To isolate inversion + attached-XY chromosomes, we first placed P{RS3} and P{RS5} insertions on the C(1;Y)N12 attached-XY chromosome (Figure 8; Kennison 1981). C(1;Y)N12 is an X chromosome broken in centric heterochromatin combined with a Y chromosome broken distal to the spermatogenesis genes on its short arm. For convenience in following C(1;Y)N12 in crosses, it is marked at the tip of the long arm of the Y with the dominant BS mutation affecting eye shape. We first placed proximal P{RS5} insertions distributed along the X onto C(1;Y)N12 chromosomes by meiotic recombination following the w+ eye color marker on P{RS5} and BS. We then placed a common distal P{RS3} insertion on these chromosomes by meiotic recombination assaying the amplification of PCR products unique to each construct to identify recombinants.

Figure 8.
Generating inversion + attached-XY chromosomes. The construction of inversion + attached-XY chromosomes proceeded in four steps. First, P{RS5} insertions distributed along the X were placed on an attached-XY chromosome ...

To generate inversions, we first exposed the attached-XY chromosomes carrying P{RS3} and P{RS5} insertions to heat shock-induced FLP to remove the 5′ w exon from P{RS3} and the 3′ w exons from P{RS5}. This was an efficient step: typically, one-third of the progeny were white eyed. We then induced inversions by exposing the chromosomes to FLP again. Inversion-bearing progeny were red eyed from reconstitution of w+ at the distal inversion breakpoint. The frequency of w+ flies varied considerably with a range of 1 in 12,600 to 1 in 140 progeny and a median rate of 1 in 1040 progeny. The inversions were verified in polytene chromosome preparations. Figure 9 and Table S2 show the 28 inversions we generated on C(1;Y)N12. Six inversion + attached-XY chromosomes were isolated by a related, but more efficient screening strategy that eliminated PCR screening for the initial recombinant chromosomes carrying both P{RS3} and P{RS5} (File S1).

Figure 9.
Inversion + attached-XY chromosomes. Proximal breakpoints for the 28 inversions generated for Dp(1;Y) screens are shown. They correspond to the positions of P{RS5} insertions. All inversions (except In(1)BSC30 and In(1)BSC31) share ...

The P{RS3} and P{RS5} insertions were isolated in an isogenic background tested for normal development and behavior (Ryder et al. 2004). We substituted all chromosomes used in our crosses into this standard background so that the final Dp(1;Y) stocks will be a high-quality genetic resource suitable for experiments involving background-sensitive phenotypes, such as behavioral phenotypes. This genetic uniformity also increases the utility of these strains in screens for dosage-based enhancement and suppression of mutant phenotypes.

Dp(1;Y) screens and breakpoint mapping:

Using the inversion + attached-XY chromosomes to isolate Dp(1;Y) chromosomes is straightforward, albeit labor intensive. Males carrying an inversion + attached-XY chromosome are irradiated and mated to y1 females. Dp(1;Y)-bearing y+ male progeny are backcrossed to establish stocks. On average, the screens produced one Dp(1;Y) chromosome supporting male fertility every ~23,000 progeny. The Dp(1;Y) chromosomes isolated and characterized to date provide a minimum of 78% coverage of X euchromatin (>17.5 of 22.4 Mb), a minimum of 78% coverage of X euchromatic genes (>1742 of 2231 genes), and extensive genomic subdivision (Table 2, Table S3). The largest stretch of contiguous coverage is 5.6 Mb in the 7B–11D region. The X tip segment shared by all Dp(1;Y) chromosomes accounts for 1.7% (0.3 Mb) of coverage. We have placed 221 Dp(1;Y) chromosomes from these screens into public distribution (http://flystocks.bio.indiana.edu/Browse/dp/BDSC-Dps.php).

TABLE 2
Dp(1;Y) chromosomes recovered

We located the irradiation-induced breakpoints of the duplicated segments on the genome map by two methods. Our primary mapping strategy localizes the ends of duplicated segments between adjacent transposon insertion sites (Figure 10). We designed PCR primers flanking the insertion sites of transposons located within the region to be duplicated (Table S1). With short extension times, PCR fragments are amplified only when there is no transposon between the primer sites. When females carrying insertions are mated to Dp(1;Y)-bearing males and DNA is prepared from their male progeny, PCR fragments are amplified only if the primer sites are present on the Dp(1;Y). In this way, we mapped the ends of duplicated segments to intervals with the target size of 10 protein-coding genes (Table S3). Duplication ends falling in adjacent mapping intervals can lie 0 to ~20 genes apart.

Figure 10.
PCR strategy for mapping the extents of duplicated segments. PCR primers were designed to flank the insertion sites of X-linked transposons. When males carry a Dp(1;Y) and an X with a transposon, a PCR fragment will be amplified from their DNA only if ...

We mapped the breakpoints of a few duplicated segments using comparative genome hybridization (CGH) microarrays. In this technique, genomic DNA samples from wild-type and Dp(1;Y)-bearing males are labeled with different fluorochromes and hybridized to the same genomic microarray. Duplicated segments are identified as contiguous blocks of genes with twofold-increased relative fluorescence (Erickson and Spana 2006). Because the microarrays contain a probe from most annotated genes, duplication endpoints can usually be mapped with two-gene resolution (see Table S3). Due to its expense, we used this method to analyze only a cytologically preselected subset of Dp(1;Y) chromosomes from the In(1)BSC6 screen.

As an example of genomic coverage and subdivision provided by our Dp(1;Y) chromosomes, Figure 11 shows duplicated segments in their uninverted orientation derived from three inversions (In(1)BSC20, In(1)BSC21, and In(1)BSC22). As planned, the nested sets overlap and there is an even distribution of endpoints across the region with a breakpoint in 18 of the 28 PCR mapping intervals targeted by these screens, i.e., between the In(1)BSC19 and In(1)BSC22 proximal breakpoints. In this 490-gene region, the largest region between two breakpoints contains at most 42 genes.

Figure 11.
Duplication coverage and genomic subdivision provided by three Dp(1;Y) screens. Three nested sets of duplicated segments provide full coverage of the region targeted by the In(1)BSC20, In(1)BSC21, and In(1)BSC22 screens. Arrows indicate the positions ...

Current coverage and subdivision of the entire X is depicted in Figure 12. Using the minimal estimates of duplication sizes from completed Dp(1;Y) screens, we have calculated that 96% of the intervals between breakpoints contain ≤30 genes, 89% contain ≤20 genes and 62% contain ≤10 genes. The median interval size is 9 genes or ~107 kb. Dp(1;Y) chromosomes in specific X chromosome regions may be viewed graphically using the GBrowse aberrations viewer on FlyBase (http://flybase.org/cgi-bin/gbrowse/dmelabs/).

Figure 12.
Current duplication coverage and subdivision of the X chromosome. The nested sets of duplicated segments from the 14 completed screens and 5 screens in progress provide 78% X coverage and extensive subdivision. The X tip region common to all Dp(1;Y) chromosomes ...

It was easier to obtain large duplicated medial segments in some regions than others. For example, the In(1)BSC3 screen produced a 165-gene (1.68 Mb) duplicated segment even though fewer progeny were screened than in the In(1)BSC10 screen where a 69-gene (0.84 Mb) segment was the largest recovered (Table 2). All our completed screens were large enough to give duplication endpoints evenly distributed across the desired chromosomal intervals, but screen size correlated poorly to size of the largest duplicated segment (r = −0.23). We attribute these regional differences to the effects of hyperploidy for different sets of genes. Other than previously identified diplolethal genes, there are no clear predictors of permissible duplication size.

The positions of proximal deletion breakpoints giving rise to Dp(1;Y) chromosomes:

As described above, the proximal breakpoint of the deletion that gives rise to a Dp(1;Y) from an inversion + attached-XY (breakpoint F in Figure 4) can fall in basal X euchromatin. Consequently, a Dp(1;Y) chromosome can carry genes from the X base in addition to genes from the middle and tip of the X. To assess how many Dp(1;Y) chromosomes carry basal euchromatic genes, we designed PCR primers flanking the insertion sites of transposons in basal X euchromatin and assayed for duplication of the insertion sites as described previously. We also examined CGH microarray data when available.

We could not detect breakpoints in the euchromatic gene stnA or in the region between it and X centric heterochromatin by our PCR mapping approach (Figure 13), because this region is present on all Dp(1;Y) chromosomes as a segment of X centric heterochromatin and adjacent euchromatin associated with the BS marker on the Y tip (Figure 8; Brosseau and Lindsley 1958). Our microarray analyses showed this region extends distally to the five-gene region between fog and stnA (Figure 13; X:22228492..22384175). Consequently, all Dp(1;Y) chromosomes carry at least one copy of five basal euchromatic genes (stnA, stnB, and three proximal genes). They may also carry euchromatic genes between fog and stnA and heterochromatic genes. No gene probes in the region showed higher than twofold relative fluorescence in the Dp(1;Y) chromosomes analyzed by CGH microarrays.

Figure 13.
Duplications in Dp(1;Y) chromosomes with breakpoints in basal X euchromatin. When the proximal breakpoints of deletions giving rise to Dp(1;Y) chromosomes fall in basal X euchromatin, Dp(1;Y) chromosomes can carry genes from the X base in addition to ...

The proximal deletion breakpoints fell in basal X euchromatin at a relatively high frequency. Of the 193 Dp(1;Y) chromosomes analyzed for duplication of basal genes by PCR, 36 duplicated Cda4 (two genes distal to fog; Figure 13, Table S4). The duplicated segments extend varying distances distally with the largest segment reaching polytene region 19E (X:20631444..20795940). The basal segments carried by these Dp(1;Y) chromosomes provide coverage and breakpoint subdivision of 7.2% of the X euchromatin.

We also wished to verify that proximal deletion breakpoints often fall in the short arm of the Y and that Dp(1;Y) chromosomes arising from these events delete Y-linked spermatogenesis genes. Across all screens, we saw an approximately sixfold higher recovery of sterile vs. fertile y+ males, suggesting that the proximal deletion breakpoints fall more frequently in the short arm of the Y than in X centric heterochromatin or the adjacent basal euchromatin. To show that the sterility can be attributed to the deletion of Y-linked spermatogenesis genes, we rescued the sterility with a redundant Y. In the screen with In(1)BSC11, we substituted homozygous attached-XY (C(1;Y)1, y1) females for the normal X, y1/X, y1 females usually mated to irradiated inversion + attached-XY males. Thirteen of the 17 Dp(1;Y) chromosomes recovered in fertile y+ males with a redundant Y did not support fertility in the absence of an extra Y, demonstrating that the sterility could be rescued by duplicating Y-linked spermatogenesis genes. Because male sterile Dp(1;Y) chromosomes have limited experimental utility, they were discarded and are not counted in Table 2 or listed in Table S3.

Rescue of mutant phenotypes with Dp(1;Y) chromosomes:

To verify that duplicated gene copies are functional and to illustrate how Dp(1;Y) chromosomes can be used to rescue the phenotypes of X-linked mutations, we crossed females bearing a recessive mutation with a lethal or visible phenotype to males carrying a Dp(1;Y) containing a wild-type copy of the mutated gene and examined the phenotypes of male progeny. We tested Dp(1;Y) chromosomes from 13 of the 16 nested sets and Table 3 shows that, as expected, the duplicated genes completely rescued the mutant phenotypes in nearly every case (85 of 90 crosses). In addition, males carrying Dp(1;Y) chromosomes containing the achaete, Notch, or Beadex genes displayed the well-known bristle and wing phenotypes associated with hyperploidy of these genes. These results indicate that duplicated genes are expressed as expected.

TABLE 3
Phenotypic rescue by nested sets of Dp(1;Y) chromosomes

The three cases of nonrescue and the two cases of partial rescue are probably explained as suppression of gene expression by heterochromatic position effects. When euchromatic regions are juxtaposed to heterochromatin by chromosomal rearrangements, the compacted chromatin state can spread into the euchromatin and suppress gene expression. The likelihood that a particular gene will be suppressed depends on the distance the gene lies from heterochromatin, the strength of suppression exerted by the heterochromatic sequences and the inherent susceptibility of the gene to suppression.

Rescue of bristle defects caused by a forked mutation (f1) showed the expected pattern for heterochromatic position effect suppression. Of all the Dp(1;Y) chromosomes derived from In(1)BSC19, the one placing f closest to centric heterochromatin (Dp(1;Y)BSC206) was the only one unable to rescue. Likewise, the wing defects caused by upheld mutations (up1 and up101) were rescued in 14–50% of males by Dp(1;Y)BSC185, a Dp(1;Y) derived from In(1)BSC14 positioning up quite close to heterochromatin. [In fact, it is unlikely longer duplicated segments could be recovered using In(1)BSC14, because the end of the duplicated segment in Dp(1;Y)BSC185 defines the proximal boundary of the 1- to 4-gene interval containing the diplolethal locus in region 12A. For further discussion of the Hdl region, see Venken et al. (2010)].

In contrast, the rescue of the wing phenotypes of miniature (m1) and dusky (dy1) mutations shows that position effects can be idiosyncratic. Of the Dp(1;Y) chromosomes derived from In(1)BSC13 and In(1)BSC26, only Dp(1;Y)BSC51 was unable to rescue the phenotypes even though m and dy are positioned farther from heterochromatin than they are in Dp(1:Y)BSC54, Dp(1;Y)BSC102, and Dp(1;Y)BSC103, which rescued the phenotypes. Dp(1;Y)BSC52 and Dp(1;Y)BSC101, which place m and dy roughly the same distance from heterochromatin as Dp(1;Y)BSC51, also rescued the phenotypes. We attribute the inability of Dp(1;Y)BSC51 to rescue to the presence of heterochromatic sequences near m and dy with unusually strong suppressive effects.

The nonrescue of f, m, or dy phenotypes is probably not explained by the disruption of these genes during irradiation. The probability of mutating a particular gene with 4 kR exposure is ~1 in 5000 (Ashburner et al. 2005). We used a slightly higher dose (4.5 kR), but the likelihood of a duplicated segment carrying a mutated gene is still low. Likewise, nonrescue is not explained by mitotic loss of Dp(1;Y) chromosomes during development, because we have seen no y+ or BS mosaicism. To demonstrate gene expression is suppressed by heterochromatic position effects, it is sometimes possible to restore it with well-established position effect suppressors such as low temperature. Though the f and dy phenotypes were not rescued by Dp(1;Y)BSC51 and Dp(1;Y)BSC206 in flies reared at 18°, the nature of the chromosomal rearrangements in the Dp(1;Y) chromosomes suggests heterochromatic suppression is still the most likely explanation for the lack of rescue.

Using Dp(1;Y) chromosomes to rescue mutant phenotypes in females:

The Y-linked inheritance pattern of Dp(1;Y) chromosomes makes it easy to track duplicated genes in experimental crosses, but it may not be apparent how Dp(1;Y) chromosomes can be used to rescue mutant phenotypes in females where a Y chromosome is not usually present. As we will show, recovering Dp(1;Y) chromosomes in XXY females and using them to rescue the phenotypes of recessive X-linked mutations is straightforward. Such experiments are useful in mapping X-linked mutations with female-specific phenotypes, such as defects in oogenesis.

XXY females arise from primary nondisjunction in both males and females. In females, nondisjunction results in XX and 0 gametes. XX eggs fertilized by Y sperm generate XXY females. In males, nondisjunction results in XY and 0 gametes. XY sperm fertilizing X eggs also generate XXY females. Consequently, any cross of XX females to males with a Dp(1;Y) chromosome can result in XXY females carrying a Dp(1;Y). XXY females themselves produce mostly XY and X eggs, though they can produce XX and Y eggs by secondary nondisjunction (Xiang and Hawley 2006). The dominant BS marker is quite useful in identifying females inheriting a Dp(1;Y)BSC chromosome, but crosses can be adapted to use y+, w+, or any marker present in a duplicated segment.

Rescue of a recessive female sterile (fs) phenotype can be shown by crossing fs/balancer females to Dp(1;Y) males to recover fs/balancer/Dp(1;Y) female progeny arising from nondisjunction in the mother and then crossing these XXY females to fs/Y males to recover fertile fs/fs/Dp(1;Y) females. We have successfully rescued the recessive sterility of an ovarian tumor mutation (otu4) with Dp(1;Y)BSC35 and Dp(1;Y)BSC36 by this approach (see File S1 for details of XXY crosses), but it relies on the relatively low rate of nondisjunction in normal females. Spontaneous nondisjunction occurs at a rate of ~1 in 5000 female meioses (Ashburner et al. 2005), so large crosses must be set up to recover the initial XXY female.

The less labor-intensive approach is to recover XXY females following nondisjunction in males. Most of our Dp(1;Y) chromosomes are maintained in stock with winscy, a homozygous viable X balancer, and XXY females produced by nondisjunctional winscy/Dp(1;Y) sperm are readily obtained. Rescue can be demonstrated in two ways. First, fs/balancer females can be crossed to winscy/Dp(1;Y) males to produce fs/winscy/Dp(1;Y) females. These females can be crossed to fs/Y males to produce fertile fs/fs/Dp(1;Y) females. We have rescued otu4 sterility with Dp(1;Y)BSC35 by this approach as well. Alternatively, winscy/winscy/Dp(1;Y) females can be recovered directly from the stock and crossed to fs/Y males to produce fs/winscy/Dp(1;Y) females, which can then be crossed to fs/Y males to produce fertile fs/fs/Dp(1;Y) females. We have used this method to rescue the recessive lethality and sterility phenotypes of Nl1N-ts1 females with Dp(1;Y)BSC77 and Dp(1;Y)BSC79 and the wing and sterility phenotypes of fu1 with Dp(1;Y)BSC15. We prefer the latter alternative, because all it requires is expansion of the Dp(1;Y) stock until a XXY female is recovered.

Relying on male nondisjunction to recover XXY females is efficient, because nondisjunction in Dp(1;Y)BSC males is elevated. Typically, spontaneous nondisjunction in males occurs at a rate of ~1 in 2000 meioses (Ashburner et al. 2005), but we measured nondisjunction in Dp(1;Y)BSC182 males at ~1 in 200 meioses. This rate is probably typical for all Dp(1;Y)BSC chromosomes, because XXY females and X0 males are commonly seen in the stocks. The reason for elevated nondisjunction is not apparent—it is not a property of all Dp(1;Y) chromosomes (Zimmering and Wu 1964). Nevertheless, it simplifies the use of these Dp(1;Y) chromosomes for rescuing phenotypes in females, because experiments can be initiated with XXY females directly from the stocks.

Regardless of the specific crosses, the first step in using a Dp(1;Y) to rescue a female-specific phenotype is the recovery of a XXY female carrying a Dp(1;Y). She can be used directly in a rescue experiment, or she can be used to establish a stock with a high proportion of XXY females. Because 30–50% of the female progeny of XXY females are themselves XXY, selecting for high numbers of XXY females in subsequent generations is easy.

In conclusion, Y linkage is not a significant barrier to the use of Dp(1;Y) chromosomes to rescue the phenotypes of X-linked mutations in females. Such experiments require only an appreciation of Y inheritance in XXY females—a straightforward variation of normal sex chromosome behavior. As this is the most complicated use of Dp(1;Y) chromosomes most investigators are likely to encounter, we feel the flexibility and ease of use provided by Y-linked duplications in most other experiments outweigh their potential disadvantages in this one situation.

DISCUSSION

We have presented results of an ongoing project to provide coverage of the D. melanogaster X chromosome with nested sets of Y-linked duplications in a genetically uniform background. The project was initiated to address the poor selection of material resources for the genetic analysis of X-linked genes. Construction of all the necessary progenitor inversion + attached-XY chromosomes for comprehensive Dp(1;Y) screens is complete and we have isolated duplications providing at least 78% coverage. These Dp(1;Y) chromosomes also provide extensive breakpoint subdivision of the X with the median interval between breakpoints containing nine genes. This is a far better selection of duplications than existed previously. With the possible exception of two small regions containing diplolethal genes, our continuing efforts should provide complete duplication coverage within the coming year. We anticipate that the full set of Dp(1;Y) chromosomes will comprise ~300 stocks.

The Bloomington Stock Center duplication project is currently one of two large-scale efforts generating X chromosome duplications with molecularly defined breakpoints in Drosophila. The accompanying article by Venken et al. (2010) describes a collection of small, 70–120 kb X segments inserted into a third chromosome target site using the ΦC31 transgenesis system. The size of duplicated segments is the most significant consequence of the different approaches. The largest segment recovered by the transgenesis method to date is ~146 kb (Venken et al. 2006), while the size of segments isolated by our method is limited only by aneuploidy effects. The largest segment we have isolated is 1.68 Mb (165 genes). It remains to be seen whether transgenesis methods can be developed to transform larger duplicated segments, but in vivo chromosome manipulation approaches currently provide the only means of recovering duplicated segments of more than ~10 genes.

The two sets of duplications are complementary resources. We anticipate that mapping mutations and identifying dosage-dependent modifiers will involve three successive steps. First, a gene will be localized with coarse resolution to a large X interval using a tiling set of the largest duplications from our Dp(1;Y) screens. This will be an efficient step, because maximal coverage of the X can be provided with approximately two dozen duplications. Second, the gene will be mapped at medium resolution using duplications within a nested set of Dp(1;Y) chromosomes. Finally, the gene will be mapped with fine resolution using the transgenic duplications. While the resolution provided by Dp(1;Y) breakpoints is equivalent to the resolution provided by the transgenic duplications in some X regions, the average resolution of 3–5 genes provided by the transgenic duplications exceeds the median resolution of nine genes provided by our Dp(1;Y) chromosomes. Mapping to successively smaller intervals using duplications from both projects should prove to be an effective and efficient process.

The creation of X duplications has been accompanied by a large-scale project at the Bloomington Stock Center to improve the selection of chromosomal deletions. We have generated >830 deletions with sequence-mapped endpoints using the FLP-FRT system described by Thibault et al. (2004) and Parks et al. (2004). These and similar deletions isolated by Exelixis (Parks et al. 2004) and the DrosDel Project (Ryder et al. 2007) combine to provide the best genomic deletion coverage and breakpoint subdivision in any multicellular eukaryote. Phenotypic rescue with duplications from the two duplication projects and complementation with molecularly defined deletions from the three deletion projects will enable X-linked genes to be localized with near single gene resolution. The Dp(1;Y) chromosomes will enhance the utility of the deletion collection, because, unlike the transgenic duplications, they are large enough to rescue the lethality of most X deletions.

For all methods of gene rescue, the chromosomal context of duplicated genes is important. Regulatory elements near transgene insertion sites often suppress the expression of transformed genes. While individual genes are not removed from their normal chromosomal sites in the Dp(1;Y) chromosomes and their expression will probably not be affected by novel regulatory elements, the chromosomal rearrangements make heterochromatic position-effect suppression a potential concern. Because we saw evidence of heterochromatic suppression in only a small number of rescue experiments, it should affect the use of the Dp(1;Y) chromosomes in relatively few instances. The diversity in size among the duplicated segments and the substantial overlap of adjacent sets of nested duplicated segments should make it possible to identify Dp(1;Y) chromosomes rescuing most X-linked mutations—even if heterochromatic position effects occasionally necessitate the use of other genetic tools, such as the transgenic duplications, for fine mapping. In fact, the redundancy of coverage and experimental flexibility provided by the Dp(1;Y) chromosomes and the transgenic duplications are beneficial outcomes of two independent projects.

In summary, we have presented an important new research resource that will alleviate longstanding difficulties associated with the analysis of X-linked gene function. We anticipate these Dp(1;Y) chromosomes will be useful for many rescue, mapping, and modifier experiments. Because the isolation of these duplications required complicated progenitor chromosomes and multiple large screens, they would never have resulted from hypothesis-driven research. Their creation required a focused project and targeted resource-development funding. The same is true for many of the material resources that have propelled model organism research in recent years (for examples, see Hayashi et al. 2002; Bellen et al. 2004; Dietzl et al. 2007; Ryder et al. 2007; Ni et al. 2009; Guan et al. 2010). Because research resources to a large extent determine the kinds of experiments that are possible, resource-development projects such as ours are significant in the breadth of their impact. We are confident we have expanded “what is possible” with this new resource and hope it will be used heavily by the research community.

Acknowledgments

We thank John Roote, Ed Ryder, Michael Ashburner, Jim Kennison, Annette Parks, and Kathy Matthews for help and guidance, Kevin Bogart and Millie Winner for assistance in the pilot screen, and Robert Eisman, Stacy Holtzman, Ellen Popodi, and Koen Venken for critical comments on the text. This work was supported by National Center for Research Resources grant RR014106 to K.R.C., Indiana Genomic Initiative (INGEN) funding to T.C.K., and National Science Foundation grant DBI-0841154 to K.R.C. and T.C.K.

Notes

Supporting information is available online at http://www.genetics.org/cgi/content/full/genetics.110.123265/DC1.

Available freely online through the author-supported open access option.

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