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

Joshi SS, Cheong H, Meller VH - Fly (Austin) (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences; Wayne State University; Detroit, MI USA.

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 2nd 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.

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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).
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Figure 2: 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).

Mentions: FLP-mediated recombination between P{RS5r} and P{RS3r} elements restores the mini-white marker, which segregates with the 2nd 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 3rd 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.


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

Joshi SS, Cheong H, Meller VH - Fly (Austin) (2014)

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).
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Figure 2: 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).
Mentions: FLP-mediated recombination between P{RS5r} and P{RS3r} elements restores the mini-white marker, which segregates with the 2nd 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 3rd 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.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences; Wayne State University; Detroit, MI USA.

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 2nd 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.

Show MeSH
Related in: MedlinePlus