A strategy for generation and balancing of autosome: Y chromosome translocations

Abstract

We describe a method for generation and maintenance of translocations that move large autosomal segments onto the Y chromosome. Using this strategy we produced (2;Y) translocations that relocate between 1.5 and 4.8 Mb of the 2^nd chromosome.. All translocations were easily balanced over a male-specific lethal 1 (msl-1) mutant chromosome. Both halves of the translocation carry visible markers, as well as P-element ends that enable molecular confirmation. Halves of these translocations can be separated to produce offspring with duplications and with lethal second chromosome deficiencies . Such large deficiencies are otherwise tedious to generate and maintain.

Introduction

Translocations are traditionally generated by randomly induced chromosome breakage. Use of the site-specific FLIP recombinase (FLP), which recombines FLP recombination target (FRT) sites, was pioneered by Golic and Lindquist,¹ and has been widely used for generation of deficiencies with defined end points.^1-3 This method also enables recombination between non-homologous chromosomes, but at a lower efficiency.⁴ Translocations that move autosomal material onto the sex chromosomes allow predictable transmission of material now linked to the X or Y chromosome, making these particularly useful. We obtained insertions of FRT in G9a, situated at 1A1 on the tip of the X chromosome, for the purpose of generating X;A translocations. However, one of these insertions segregates with the Y chromosome, and in fact is inserted in a duplication of the X on the Y chromosome (Dp(1;Y)y⁺). The duplicated region comprises 1A1–1B, and includes y⁺. Using this Y-linked FRT site as one endpoint, we generated large translocations between the Y and 2^nd chromosomes with an efficiency similar to that for production of autosome to X translocations.⁴ Both halves of these translocations carry visible markers, enabling maintenance by selection. Full translocations were easily balanced over the male-specific lethal 1 (msl-1) mutation for maintenance without selection.

Results and Discussion

We obtained insertions of specialized P-elements, P{RS3} and P{RS5}, for the purpose of generating translocations as described in Golic and Golic⁵ (see Table 1 in Materials and Methods for stock descriptions). These elements have FRT sites flanking the 5′ exon or 3′ exon, respectively, of the mini-white gene. Flies carrying intact elements have dark red eyes. FLP-mediated recombination between the flanking FRT sites deletes one exon to generate reduced P{RS3r} and P{RS5r} elements, which retain a single FRT site and the 3′ (RS3r) or the 5′ (RS5r) exons of the mini-white gene. Flies with reduced elements have white eyes.

Table 1. Full genotype and stock numbers of Drosophila strains used in this study.

Description	Stock #, supplier	Genotype	Flybase Id #
P{RS5} on Y	43649 (BDSC) 125639 (DGRC)	Dp(1;Y)y[+], P{w[+mW.Scer\FRT.hs] = RS5}G9a[5-HA-2731] y[+]/w[1118]	FBab0010476; FBti0059095
P{RS3} at 22A2	124255 (DGRC)	w[1118]; P{RS3}CG18131[CB-6892–3]	FBti0059228
P{RS3} at 25B1	124291 (DGRC)	w[1118]; P{RS3}CG3036[UM-8026–3]	FBti0029115
P{RS3} at 56C6	124216 (DGRC)	w[1118]; P{RS3}CB-6775–3	FBti0058933
P{RS3} at 57B1	123609 (DGRC)	w[1118]; P{RS3}CB-5407–3	FBti0028557
3rd chromosome FLP	8705 (BDSC)	w[*]; wg[Sp-1]/CyO; P{w[+mC] = ovo-FLP.R}F1A	FBti0072900
X-linked FLP	8727 (BDSC)	P{w[+mC] = ovo-FLP.R}M1A, w[*]	FBti0072898
msl-1		y w; msl11/CyO

We used P{RS3} elements with insertion sites at cytological positions 22A2 and 25B1 on 2L, and 56C6 and 57B1 on 2R from the Drosophila Genetic Resource Center (DGRC, Kyoto, Japan). Two P{RS5} elements inserted in G9a at cytological position 1A1 on the tip of the X chromosome were also obtained. Although these elements were believed to be close to one another, the mini-white marker of one segregated with the Y chromosome (DGRC Stock # 125639). PCR confirmed that this stock had a P- element in G9a, as well as a copy of G9a lacking the insertion. This is consistent with insertion on Dp(1;Y)y⁺, the Y chromosome present in the strain used to generate the original insertions, which carries 1A1–1B on the Y chromosome (personal communication; Cook, K). This insertion has been renamed Dp(1;Y)y⁺, P{w[+mW.Scer\FRT.hs] = RS5}G9a[5-HA-2731] y⁺/w¹¹¹⁸ and is now available from the Bloomington Drosophila Stock Center (BDSC # 3649). We performed matings to determine whether the Y-linked FRT site could recombine with autosomal sites. The Golic laboratory has demonstrated that the recovery of recombinants between non-homologous chromosomes is dramatically enhanced by a third FRT site.⁴ In accord with these findings, we were unable to obtain 2;Y translocations with 2 FRT sites (approximately 348 vials screened). We then generated flies carrying the Y-linked P{RS5r} element and 2 second chromosome P{RS3r} elements, one on 2L and one on 2R. Both P{RS3r} elements were oriented to enable production of 2;Y translocations (Fig. 1). Cytological and molecular analysis was then used to identify the 2^nd chromosome break point. The number of vials scored for each set of endpoints and the translocations recovered are presented in Table 2.

Figure 1. Mating scheme used to generate T(2;Y) translocations. The 3^rd chromosome FLP source, [w⁺ovo-FLP], was balanced over TM3-Ser¹ to enable translocations to be positively identified. In practice, the eye color of translocations is sufficiently dark as to be readily distinguished in the [w⁺ ovo-FLP] background.

Table 2. Recovery of translocations in this study.

2nd chromosome FRT sites	# Vials scored	Translocations recovered (#)	Rate of recovery of translocations
22A2 and 56C6	140	22A2 (3)	2.14%
22A2 and 57B1	107	0	-
25B1 and 56B6	105	0	-
25B1 and 57B1	238	57B1 (1), 25B1 (1)	0.84%
All	590	5	0.85%

All dysgenic males carried a Y-linked FRT site and the indicated FRT sites on 2L and 2R. The rate of recovery of translocations was (# independently recovered translocations/ # vials scored) × 100.

FLP-mediated recombination between P{RS5r} and P{RS3r} elements restores the mini-white marker, which segregates with the 2^nd chromosome endpoint. Regeneration of w⁺ made it possible to rapidly screen for recombinants, as only recombinant flies had deep red eyes. Our mating utilized a 3^rd chromosome source of ovoFLP in an element that itself produced a deep orange eye color. A Serrate balancer, TM3-Ser¹, was introduced to distinguish recombinants from flies carrying the FLP source. However, this proved unnecessary, as the reconstituted w⁺ marker was distinctive, even in flies retaining the FLP chromosome. Use of an X-linked FLP source (BDSC # 8727) would obviate the need to distinguish male offspring with light and dark eyes. The Y-linked half of the translocation can be followed by the y⁺ marker, which continues to segregate with the Y chromosome. Both halves of the translocation are thus marked, making it possible to follow them independently in a yw background. Furthermore, each half of the translocation can be molecularly confirmed by amplification with genomic primers flanking the insertion sites and outward facing primers in the P{RS5} and P{RS3} elements (Fig. 2). Outward facing primers within each P-element and in flanking genomic sequence were used to confirm the second chromosome break point. A schematic of translocation between the Y-linked 1A1 insertion and the 22A2 insertion on 2L is depicted in Figure 2. Flies with both ends of the translocation were w⁺y⁺, and, when used as template for PCR amplification, produced amplicons from proximal and distal P-element ends for the 1A1 and 22A2 insertions (Fig. 2B). Males with 2 wild type second chromosomes, and thus carrying half of the translocation as a duplication on the Y chromosome, were w y⁺. As expected, these males lacked PCR products from the distal end of the insertion in G9a and the proximal end of the second chromosome insertion (Fig. 2B). No adult w⁺y females were recovered from these stocks, indicating that these deficiencies are completely lethal.

Figure 2. Molecular characterization of T(2;Y)22A2. (A) The insertion of P{RS5r} in G9a is in Dp(X;Y)y⁺, containing 1A1–1B. The relative position of the y⁺ marker is depicted with respect to P{RS5r} (triangle) and the junction between X and Y material (zigzag). Primers that amplify proximal and distal ends of all insertions were used; for simplicity, only those that amplify distal 1A1 (D 1A1) and proximal P{RS3r} at 22A2 (P 22A2) are depicted. Upon translocation, w⁺ is reconstituted on the second chromosome. The template amplified by distal 1A1 primers is now at the tip of the second chromosome. Separation of the translocation halves to produce Dp(2;Y) 22A2 removes the distal 1A1 and proximal 22A2 templates, but proximal 1A1 and distal 22A2 templates (primers not shown) are retained. (B) Amplicons produced by a fly carrying the complete T(2;Y)22A2 translocation (top). All four P-element ends are detected. Amplicons from a Dp(2;Y)22A2 fly identify only the proximal end of the Y-linked 1A1 insertion and the distal end of the 22A2 insertion (bottom).

We recovered 5 independent (2;Y) translocations with endpoints at 22A2 (3), 25B1 (1), and 57B1 (1). Translocations were named by the 2^nd chromosome break point, for example, T(2;Y) 25B1. The rate of recovery of translocations was 0.85% (# independently recovered translocations/ # vials scored) × 100. This is comparable to translocation rates previously reported (0.33 to 0.97%).⁴

Both halves of translocations are clearly visible on polytene squashes (Fig. 3A and B). All 3 translocations can be separated to create viable (Dp(2;Y)22A2) or subviable (Dp(2;Y)25B1 and Dp(2;Y)57B1) duplications. Duplications can sometimes be observed pairing with the wild type 2^nd chromosomes (Fig. 3C). Translocation moves an X telomere from the Y chromosome to the tip of the truncated 2^nd chromosome. This can be seen in a polytene preparation from the T(2;Y)57B1 translocation (Fig. 3D). Importantly, both translocation and duplication males retain all fertility factors on the Y chromosome, and are thus fertile.

Figure 3. Cytological characterization of 2;Y translocations. (A) Polytene preparations from a male larva with the T(2;Y) 22A2 translocation display one second chromosome truncated at 22A2 (arrow). (B) The other half of the translocation is seen protruding from the chromocenter. (C) A male with Dp(2;Y)22A2 shows pairing of the duplication with both wild type homologs (black arrow). A visible connection between the duplication and the chromocenter is marked by a white arrowhead. (D) Translocation moves the X telomere from the Y chromosome to the second. This is marked by a white arrow in a T(2;Y)57B1 male. A white arrowhead marks the 2L telomere on the normal homolog.

The w⁺ and y⁺ markers associated with each half of these translocations make it possible to maintain them by selection in a yw background. However, Dp(2;Y)22A2 is viable, indicating that smaller translocations will be at a risk for loss without selection. Translocations were initially balanced by mating exceptional XXY females to a CyO (In(2LR)O) second chromosome. While possible to create stocks with both sexes carrying balanced translocations, these are plagued by low fertility. To enable easier maintenance, translocations were balanced over an msl-1 mutation (Fig. 4). Females homozygous for msl-1 are fully viable and fertile, however msl-1 is male lethal.⁶ The msl-1 gene, situated at 36F on 2L, is proximal to or on the opposite arm from translocation break points, and thus is present on the translocated second chromosome but absent from separated duplications. Stocks balanced by this method are vigorous and easily maintained. To control the risk of recombination posed by rare XXY females, duplicate stocks are maintained and periodically inspected. The location of other male-specific lethal genes, such as mle (42A6) and msl-3 (65E4), suggests that all (2;Y) translocations and (3;Y) translocations on 3R, or 3L distal to 65E4, could be balanced by a similar strategy.

Figure 4. Balancing T(2;Y) over msl-1. Translocations were generated in a yw background to enable detection of y⁺ and w⁺ markers. yw; msl-1 females are mated to males carrying a full translocation (y⁺w⁺). Sons carrying the full translocation are mated back to yw; msl-1 females to establish a stock that can be maintained indefinitely without constant selection.

Materials and Methods

Flies were maintained at 25 °C on standard cornmeal agar food. All fly strains used in this study are presented in Table 1.

Matings to generate translocations were set with 5 males and approximately 20 yw females per vial. A generalized overview of the mating scheme used to produce T(2:Y) is presented in Figure 1.

The primers used for the molecular characterization of translocations are: Proximal 1A1: Plac1(+)- CCAAGGCTGC ACCCAAGGCT CTGCTCCCAC and 1A1_R1- CGT TCTGCCGGCA GCGAAGTCGA CTG; Distal 1A1: Pry4(+) – TAATCAACAA TCATATCGCT GTCTCAC TCAG and 1A1_F1 - GAAGTGCGTG CGTGTGCGTG CGTC; Proximal 2L at 22A2: Pry4(+) - TAATCAACAAT CATATCGCTG TCTCAC and 22A2_R1 – GTGCTGCCCA TCATCCTTAT GCC and Distal 2L at 22A2: Plac1(+) – CCAAGGCTGC ACCCAAGGCT CTGCTCCCAC and 22A2 _F1 – CCGTCGCCTG CTGTGTACCGC.

10.4161/fly.27814