Academic journal article Genetics

Preferential Breakpoints in the Recovery of Broken Dicentric Chromosomes in Drosophila Melanogaster

Academic journal article Genetics

Preferential Breakpoints in the Recovery of Broken Dicentric Chromosomes in Drosophila Melanogaster

Article excerpt

DROSOPHILA is noted for a combination of facile genetics and high-resolution cytology that allows the recovery and characterization of a variety of chromosome rearrangements. Simple structural changes such as duplications, deficiencies, inversions, and translocations are readily produced through a variety of classical and modern techniques. More complex rearrangements such as balancers, compound chromosomes, and ring chromosomes also have been generated. Ring chromosomes historically have been of interest for a number of unique properties, such as a propensity for loss during early embryonicmitoses and dominant zygotic lethality (Leigh 1976; Ashburner et al. 2005). Despite their "instability," ring chromosome stocks can be remarkably stable, with very few reported instances of spontaneous opening into linear chromosomes.

In this work, we produced a number of linearized ring chromosomes. One goal was to determine whether there are preferred sites of breakage for dicentric chromosomes. Although we have previously shown that dicentric Y, 3, and 4 chromosomes can break and heal in the male germline and be recovered in offspring, these chromosomes allowed little opportunity to determine whether there were preferred sites of breakage (Ahmad and Golic 1998; Titen and Golic 2008, 2010; Titen et al. 2014). In the case of the Y chromosome, low resolution of mitotic cytology and the required role of the Y in male fertility limit the chromosomes that can be recovered. On chromosome 3, aneuploidy limits the range of broken chromosomes that can be recovered, and because chromosome 4 is so small, there is no opportunity to recover a wide range of breakpoints.

We designed a scheme that would allow us to determine whether dicentric chromosomes break at preferred sites. Dicentric chromosomes with two bridging arms were produced by FLP recombinase mediated sister chromatid exchange in a Ring-X chromosome (Figure 1). If the two arms break at similar sites and then heal, a nearly euploid linear X chromosome may be recovered. We expected the vast majority of linear X chromosomes to be broken in heterochromatin because the breakpoints simply need to be in the same block of heterochromatin on each arm but, in molecular terms, may be megabases apart. We expected euchromatic breaks to be rare because the sites of breakage on each arm would need to be at nearly the same site. However, half the recovered linear chromosomes were broken in euchromatin, and the breakpoints were clustered at a limited number of euchromatic sites. This result is most easily explained by a limited number of preferred regions for breakage.

Although the X chromosome is a frequent target of experimental structural manipulation, one form of the X that has not been recovered is a metacentric single X. The Drosophila melanogaster X is normally acrocentric, with all euchromatin and most heterochromatin lying to the leftof the centromere and only a very short heterochromatic arm located to the right. A number of X chromosomes with inversions that encompass varying portions of the leftarm have been generated, but to our knowledge, a single X with its centromere in a medial position has not been generated. Among the linear chromosomes that we recovered are many with the centromere located approximately in the middle of the chromosome.

Materials and Methods

The ring chromosomes we used for these experiments were generated as described previously (Golic and Golic 2010). In addition to an entire X chromosome, they also carry a portion of the Y. They are maintained in stock as R(1;Y)/Y males by C(1)DX/Y females. To look for evidence of somatic breakage of the dicentric, FLP was expressed by heat-shock induction of the 70FLP transgene in females with a normal X and a ring chromosome that carried an insertion of the FRT-bearing element P{.whs.} (Golic and Lindquist 1989; Golic et al. 1997).

The ring chromosomes that we generated tended to be quite filicidal. …

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