Generating Y-linked X chromosome duplications for region 4D to 5D
Kim Cook, Russell Garton, Adam Brown, Jennifer Deal, Megan Deal, Eric Spana*, Thom Kaufman & Kevin Cook
Bloomington Drosophila Stock Center, Indiana University
*Model System Genomics Unit, Duke University
We have generated a set of Y-linked duplications of X chromosomal segments by irradiating an attached-XY with an inversion in the X chromosome. The progenitor C(1;Y)N12, In(1)BSC6, P{w+mC=3'.RS5+3.3'}BSC6 w1118, BS chromosome has the following form:
1Lt to 1B5|5D1 to 1B5|5D1 to X heterochromatin breakpoint distal of bb|YS to YL|BS
In(1)BSC6 is described in FBab0046071 and its construction was described in FBrf0209647. P{w+mC=3'.RS5+3.3'}BSC6 is associated with the distal inversion breakpoint. 
Large irradiation-induced deletions within this progenitor chromosome with one breakpoint within the inversion near the distal inversion breakpoint and one breakpoint in X heterochromatin or basal X euchromatin produce Dp(1;Y) chromosomes of the form:
1Lt to 1B5|5D1 to medial breakpoint|basal X breakpoint to X het breakpoint distal of bb|YS to YL|BS
The Dp(1;Y) chromosomes share the same distal 1Lt to 1B5 segment containing the yellow (y) gene. The medial segments of these Dp(1;Y) chromosomes share a common end derived from the inversion breakpoint at 5D1, but they extend different distances distally on the standard X map. The Dp(1;Y) chromosomes carry segments of different sizes from the base of the X. The breakpoints typically lie in basal X heterochromatin, but they may lie in basal euchromatin. Dp(1;Y) chromosomes with breakpoints in YS cannot be established in stock, because deletion of YS male fertility genes renders males sterile. The BS-marked tip of YL in C(1;Y)N12 carries a portion of the base of the X distal to bb.  From Comparative Genome Hybridization microarrays of chromosomes derived from C(1;Y)N12, we determined that the distal breakpoint of the basal X segment lies between Release 5 coordinate  X:22228492  in fog and coordinate  X:22384175  in stnA and that genes proximal to stnA are duplicated. We could not determine the proximalmost extent of this segment, but the microarrays indicated that the duplicated segment extends at least to coordinate  X:22416503  in CG13865. We saw no evidence for a large duplicated segment encompassing the BarH1 and BarH2 genes in 16A, but the resolution of the microarrays is not high enough to exclude the possibility of a small duplicated segment from the region.
We exposed C(1;Y)N12, In(1)BSC6, P{w+mC=3'.RS5+3.3'}BSC6 w1118, BS males lacking a free Y chromosome to ~4500 rads in a 137Cs irradiator and mated them to winscy females. Dp(1;Y) chromosomes were recovered in y+ w+ BS males. Stocks were established by crossing these males to winscy females. 21 independent Dp(1;Y) chromosomes were established in stocks from approximately 884,000 progeny screened.  
We maintained the isogenic background of the DrosDel transposon collection in generating the progenitor C(1;Y)N12, In(1)BSC6 chromosome and in all the Dp(1;Y) screen crosses. Only the proximal regions of the X chromosome and the Y chromosome of C(1;Y)N12 were introduced from a different genetic background. Fourth chromosomes were not tracked. 
The distal 1Lt to 1B5 segment shared by these duplications extends proximally to Release 5 coordinate  X:387562 , the insertion site of P{RS3)CB-5805-3 used in the construction of In(1)BSC6.
We mapped the irradiation-induced breakpoints at coarse resolution by polytene cytology and chose a subset of duplication chromosomes for further analysis.  To map the breakpoints at fine resolution and determine which genes are present on the Dp(1;Y) chromosomes, DNA samples from duplication-bearing males were compared to samples from wild type males by Comparative Genomic Hybridization microarrays as described in Erickson and Spana, 2006 (http://flybase.org/reports/FBrf0193934.html ). Corning CGAP slides spotted with the AROS Drosophila V1.1.1 ~70 nucleotide oligo set from Eurofins MWG Operon (www.operon.com) were used for the analysis. Most annotated genes were represented by a single oligo (denoted by a DM number). Sequences present at two copies in duplication-bearing males are detected by higher relative fluorescence when compared to sequences present in one copy in wild type males. 
The distal extents of the basal duplicated segments were determined by the positions of the proximal irradiation-induced breaks. Because genes proximal to fog are duplicated in the portion of the X chromosome appended to the tip of YL, we could not determine with confidence if genes from this region are present in the basal portions of the X proximal to the irradiation-induced breaks. No genes distal to fog are duplicated in the basal segments of Dp(1;Y) chromosomes characterized.  
On the standard X map, the proximal end of all the duplicated segments lies at Release 5 coordinate  X:5882812 , the insertion site of P{RS5}5-SZ-3429 used in the construction of In(1)BSC6.
The distal extents of the medial segments were determined by the positions of the distal irradiation-induced breaks.  The following table shows the Release 5 coordinates of the microarray oligonucleotides flanking the distal duplication breakpoints:
Dp(1;Y)BSC91   X:4665273--4665341    X:4670742--4670810 
Dp(1;Y)BSC92   X:4821917--4821985    X:4849103--4849171 
Dp(1;Y)BSC93   X:4961358--4961426    X:4969249--4969317 
Dp(1;Y)BSC94   X:4969249--4969317    X:5026586--5026654 
Dp(1;Y)BSC95   X:5198244--5198312    X:5201097--5201165 
Dp(1;Y)BSC96   X:5299568--5299636    X:5314625--5314693 
Dp(1;Y)BSC97   X:5523433--5523501    X:5537021--5537089 
Dp(1;Y)BSC98   X:5559109--5559177    X:5565679--5565747 
Dp(1;Y)BSC99   X:5772623--5772691    X:5775863--5775903 
The following table summarizes the breakpoints for the medial duplicated segments in each of the Dp(1;Y) chromosomes.  The distal extents of the medial segments are shown as ranges composed of the genomic coordinates for the distal ends of the two oligonucleotides defining the breakpoint interval.
Dp(1;Y)BSC91   X:4665273--4670742 ;5882812 (4D1-4D1;5D1)
Dp(1;Y)BSC92   X:4821917--4849103 ;5882812 (4D6-4D7;5D1)
Dp(1;Y)BSC93   X:4961358--4969249 ;5882812 (4E2-4E2;5D1)
Dp(1;Y)BSC94   X:4969249--5026586 ;5882812 (4E2-4F1;5D1)
Dp(1;Y)BSC95   X:5198244--5201097 ;5882812 (4F4-4F4;5D1)
Dp(1;Y)BSC96   X:5299568--5314625 ;5882812 (4F9-4F10;5D1)
Dp(1;Y)BSC97   X:5523433--5537021 ;5882812 (5A8-5A9;5D1)
Dp(1;Y)BSC98   X:5559109--5565679 ;5882812 (5A10-5A11;5D1)
Dp(1;Y)BSC99   X:5772623--5775863 ;5882812 (5C6-5C6;5D1)